PHYSIOLOGICAL REVIEWS
Vol. 81, No. 3, July 2001
Printed in U.S.A.
Spectrin and Ankyrin-Based Pathways:
Metazoan Inventions for Integrating Cells Into Tissues
VANN BENNETT AND ANTHONY J. BAINES
I. Introduction
A. Overview of the erythrocyte membrane skeleton
II. Genes, Proteins, and Protein Interactions
A. Spectrins
B. Ankyrins
C. Proteins that promote spectrin-actin interactions
III. Physiological Functions
A. Overview
B. Stabilization of cell surface membranes at sites of cell-cell contacts
C. Cell sheet morphogenesis
D. Assembly of voltage-gated Na⫹ channel-rich domains in excitable cells
E. Targeting of Ca2⫹-release channels to the Ca2⫹ compartment of the ER
F. Orientation of mitotic spindles in asymmetric germ cell division in Drosophila
G. A role for protein 4.1 in selective and directed membrane protein accumulation
IV. Clinical Implications
A. Functional channelopathies due to defects in ankyrin-dependent targeting
B. Use-dependent dystrophies of muscle and nerves due to defects in the spectrin-based
transcellular mechanical coupling pathway
C. Abnormal nervous system development due to L1 CAM defects
V. Summary and Perspectives
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
Howard Hughes Medical Institute and Departments of Cell Biology and Biochemistry, Duke University
Medical Center, Durham, North Carolina; and Department of Biosciences, University of Kent,
Canterbury, United Kingdom
1354
1354
1355
1356
1363
1368
1373
1373
1374
1375
1375
1376
1377
1378
1379
1379
1380
1381
1381
Bennett, Vann, and Anthony J. Baines. Spectrin and Ankyrin-Based Pathways: Metazoan Inventions for Integrating Cells Into Tissues. Physiol Rev 81: 1353–1392, 2001.—The spectrin-based membrane skeleton of the humble
mammalian erythrocyte has provided biologists with a set of interacting proteins with diverse roles in organization
and survival of cells in metazoan organisms. This review deals with the molecular physiology of spectrin, ankyrin,
which links spectrin to the anion exchanger, and two spectrin-associated proteins that promote spectrin interactions
with actin: adducin and protein 4.1. The lack of essential functions for these proteins in generic cells grown in culture
and the absence of their genes in the yeast genome have, until recently, limited advances in understanding their roles
outside of erythrocytes. However, completion of the genomes of simple metazoans and application of homologous
recombination in mice now are providing the first glimpses of the full scope of physiological roles for spectrin,
ankyrin, and their associated proteins. These functions now include targeting of ion channels and cell adhesion
molecules to specialized compartments within the plasma membrane and endoplasmic reticulum of striated muscle
and the nervous system, mechanical stabilization at the tissue level based on transcellular protein assemblies,
participation in epithelial morphogenesis, and orientation of mitotic spindles in asymmetric cell divisions. These
studies, in addition to stretching the erythrocyte paradigm beyond recognition, also are revealing novel cellular
pathways essential for metazoan life. Examples are ankyrin-dependent targeting of proteins to excitable membrane
domains in the plasma membrane and the Ca2⫹ homeostasis compartment of the endoplasmic reticulum. Exciting
questions for the future relate to the molecular basis for these pathways and their roles in a clinical context, either
as the basis for disease or more positively as therapeutic targets.
http://physrev.physiology.org
0031-9333/01 $15.00 Copyright © 2001 the American Physiological Society
1353
1354
VANN BENNETT AND ANTHONY J. BAINES
I. INTRODUCTION
A. Overview of the Erythrocyte
Membrane Skeleton
The membrane skeleton of mammalian erythrocytes
was first visualized in electron micrographs of detergentextracted erythrocytes (440). The erythrocyte membrane
skeleton is organized as a polygonal network formed by
five to seven extended spectrin molecules linked to short
actin filaments ⬃40 nm in length (42, 247, 360) (Fig. 1A).
The spectrin-actin network of erythrocytes is coupled to
the membrane bilayer primarily by association of spectrin
with ankyrin, which in turn is bound to the cytoplasmic
domain of the anion exchanger (17, 20, 23, 256, 400, 441).
The anion exchanger is associated into dimers (305),
which associate with separate sites on the membranebinding domain of ankyrin to form pseudo-tetramers (53,
281, 332, 437). Anion exchanger dimers also are associated on their cytoplasmic surface with band 4.2 (442).
Additional membrane connections are provided at the
spectrin-actin junction by a complex between protein 4.1,
p55, a member of the MAGUK family, and glycophorin C
(170, 266, 267) (Fig. 1B).
Several proteins responsible for capping actin and
defining the length of actin filaments as well as stabilizing
spectrin-actin complexes have been localized to spectrinactin junctions by electron microscopy (91, 402). Protein
4.1 stabilizes spectrin-actin complexes (400, 401). Adducin associates with the fast-growing end of actin filaments
in a complex that caps the filament and promotes assembly of spectrin (135, 222, 223, 239) (Fig. 1C). A nonmuscle
isoform of tropomyosin is associated with the sides of
actin filaments (127). Tropomyosin is of the same length
as actin filaments visualized in electron micrographs and
is a candidate to function as a morphometric ruler defining the length of actin filaments in erythrocyte membranes. Tropomodulin caps the slow-growing end of actin
filaments in a ternary complex involving tropomyosin
(124 –126, 421).
Components of the erythrocyte membrane skeleton
have been the subject of recent reviews or papers that
include discussions of tropomodulin (125), protein 4.1
(68, 118), protein 4.2 (62), p55 (56), spectrin (164, 410),
the anion exchanger, and glycophorin C (4, 381). The
contributions of these proteins to mechanical properties
of erythrocyte membranes have also been summarized
(97, 289).
A major function of the spectrin skeleton in erythrocytes is to provide mechanical support for the membrane
bilayer and allow survival of these cells in the circulation.
The essential nature of the spectrin-skeleton in red cell
biology was first demonstrated in mutant mice with deficiencies in ␣- and ␤-spectrin and ankyrin (33, 152). Numerous mutations have subsequently been catalogued in
humans with hereditary hemolytic anemias. Defects in
lateral associations of the spectrin-actin network result in
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
Emergence of metazoans from their unicellular ancestors required solutions to a new set of problems imposed by function of cells in the context of tissues and in
motile organisms of immense size compared with individual cells. These requirements of communal life include
ability of cells to develop micron-scale spatial organization of cell surfaces and of intracellular compartments to
optimize cell-cell interactions and intercellular signaling.
In addition, cells incorporated into an actively moving
organism must deal with enormous mechanical stresses
at the plasma membrane-cytoskeleton interface compared with free-living cells. Understanding the molecular
basis for such metazoan adaptations represents an interdisciplinary challenge involving biochemistry, cell biology, physiology, and molecular medicine. This review
focuses on the molecular physiology of spectrin and the
spectrin-associated proteins ankyrin, adducin, and protein 4.1. These proteins were first discovered as components of the membrane skeleton of human erythrocytes
(see below) and are required for survival of erythrocytes
in the circulation. The erythrocyte membrane proteins are
members of closely related families that are associated
with membranes in simple metazoans, including Caenorhabditis elegans and Drosophila melanogaster, and are
expressed in most vertebrate tissues. Spectrin, ankyrin,
adducin, and protein 4.1 are modular proteins that are not
present in their assembled state in the completed Saccharomyces cerevisea or Arabidopsis thaliana genomes and
so far have not appeared in Zea mays genomic sequences.
These proteins therefore are likely to have evolved early
in evolution of metazoans, following divergence of plants
and fungi, and represent candidates for roles in specialized activities of multicellular animals.
Recent discoveries based on studies involving C. elegans and D. melanogaster as well as gene knock-outs in
mice will be reviewed that demonstrate functions of spectrin- and ankyrin-based protein assemblies in diverse
roles that are all related to multicellular life. Functions
that will be described include morphogenesis of epithelial
tissues, targeting of ion channels and cell adhesion molecules to specialized regions in myelinated axons, and
sorting of Ca2⫹ homeostasis proteins to the Ca2⫹ compartment of the endoplasmic reticulum (ER) of striated
muscle. The clinical implications of these observations
are only beginning to be appreciated and are discussed.
Elucidation of physiological roles of spectrin and ankyrinassociated proteins is based on a strong foundation at a
molecular level. The review includes current information
regarding gene family members, atomic structures, oligomeric state, and protein interactions. The human erythrocyte remains the best understood in terms of its membrane skeleton, and the review begins with a brief
summary of this system.
Volume 81
July 2001
SPECTRIN AND ANKYRIN-BASED PATHWAYS
1355
abnormally shaped cells in elliptocytosis and poikilocytosis and include loss of spectrin dimer-tetramer interactions (395) and deficiency of protein 4.1 (67, 120, 383).
Defects in membrane associations result in loss of unsupported phospholipid bilayer and spherocytosis. Molecular
defects include spectrin deficiency from a variety of
causes (1, 2, 52, 94, 112). A substantial literature has
documented naturally occurring mutations/deficiencies of
skeletal proteins resulting in hereditary hemolytic anemias in humans and mice (reviewed in Refs. 87, 166, 396).
An emerging area of interest is mutations resulting from
targeted gene knock-outs in mice resulting in hemolytic
anemias that may foreshadow human disorders. An ex-
ample is the ␤-adducin null mouse, which exhibits spherocytosis (143).
II. GENES, PROTEINS, AND
PROTEIN INTERACTIONS
Solution of the organization of the spectrin-based
membrane skeleton of the human erythrocyte membrane
has provided the biochemical equivalent of a high-resolution genetic pathway of interacting membrane structural
proteins. The discovery that other tissues express isoforms of ankyrin (18) and spectrin (20, 40, 146, 151, 237,
343) suggested that the erythrocyte membrane skeleton
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
FIG. 1. Organization of the erythrocyte membrane skeleton. A: electron microscopy of the membrane skeleton.
Spectrin tetramers are cross-linked at nodes (junction points) by short filaments of F-actin and 4.1/adducin. Approximately six spectrins interact with each node. At the central region of the spectrin tetramers are bound ankyrin/anion
exchanger complexes. [From Liu et al. (247).] B: membrane-cytoskeleton connections in erythrocytes. Ankyrin is linked
through its ank-repeat region to dimers of the anion exchanger (gray). Each ankyrin is capable of cross-linking two anion
exchanger dimers. The 15th helical repeat of ␤-spectrin (yellow) interacts with ankyrin. At the NH2-terminal region of
␤-spectrin is a binding site for 4.1 (red). 4.1 forms a ternary complex with the transmembrane protein glycophorin C and
the membrane-associated guanylate kinase p55 (green). C: the spectrin-actin junction. Short filaments of F-actin
containing 14 –16 monomers tether spectrin at the nodes shown in A. The negative (⫺) end of the filaments is blocked
by tropomodulin, and nonmuscle tropomyosin lies along the filament. ␤-Spectrin binds actin via its NH2-terminal CH
domains (CH1 and CH2) and the first two triple helical repeats (Rpt 1 and Rpt 2). Approximately six spectrins would fit
a short actin filament if the spectrins were arranged along the side of the filament as indicated in this figure. This is
consistent with the six spectrins per node indicated in A. Spectrin-actin interaction is promoted by protein 4.1 and adducin.
Adducin has a high affinity for the positive (⫹) end of the filaments and possibly recruits other proteins to the positive end.
1356
VANN BENNETT AND ANTHONY J. BAINES
had a broad relevance for other cell types. However,
although the basic structural principles established in
erythrocytes are likely to apply in other tissues, the organization, protein interactions, and functions of spectrinbased structures are considerably more diverse in other
cells. Nevertheless, understanding the physiological roles
of these proteins begins with their structure and biochemistry. This section focuses on genes, alternatively spliced
variants, protein structure, and protein interactions of
generally expressed forms of spectrin, and the proteins
that interact with spectrin: ankyrin, protein 4.1, and adducin.
Spectrins are extended, flexible molecules ⬃200 –260
nm in length and 3– 6 nm across with actin-binding domains at each end (20, 146, 362, 400). Spectrins are comprised of ␣- and ␤-subunits, which are both related to
␣-actinin (43, 105, 319, 387, 408). The ␣- and ␤-subunits
are associated laterally to form antiparallel heterodimers,
and heterodimers are assembled head-head to form heterotetramers (Fig. 2, Table 1).
Metazoan spectrins exhibit 50 – 60% sequence similarity along the length of the predicted polypeptide chains,
when compared between Drosophila, C. elegans, and vertebrates, with some regions with 70 – 80% identity. Candidates for prototypic spectrins, possibly comprised of a
single subunit, have also been characterized biochemically and by electron microscopy in Dictyostelium (16)
and Acanthamoeba (336). However, sequence information is not yet available for these presumed spectrin ancestors. Spectrin subunits are absent from completed S.
cerevisiae and Arabidopsis thaliana genomes, although
individual domains of spectrin are represented. Similarly,
no spectrin sequence has yet appeared in genome of Zea
mays. Polypeptides cross-reacting with spectrin have
been reported in higher plants (118, 284, 364) as well as
green algae (176) and Chlamydomonas (252). However,
these immunoreactive forms of spectrin have not been
characterized in terms of primary sequence or visualized
by electron microscopy. Definition of the full scope of the
spectrin family awaits completion of genome sequencing.
However, the available data demonstrate that spectrins
are ancient proteins present in their modern form in
simple metazoans.
The spectrin repertoire of the completed C. elegans
and D. melanogaster genomes includes one ␣-subunit
(105), one ␤-subunit (41, 159, 292), and one ␤-H subunit
(106, 276, 387). Currently characterized spectrins in humans include two ␣-subunits (␣1, ␣2) (353, 419), four
␤-subunits (␤1, ␤2, ␤3, ␤4) (24, 187, 261, 286, 310, 372, 427,
428), and a ␤-H subunit (also referred to as ␤5) (371)
(Table 1). ␤-Spectrins also include isoforms that have not
been characterized at a molecular level. ␤NM is a ␤-type
subunit identified at neuromuscular junctions based on
immunoreactivity (31). ␤Golgi-Spectrin has been discovered by Beck et al. (9) to be an immunoreactive form of
␤-spectrin associated with Golgi structures. ␤Golgi shares
epitopes with ␤1-erythrocyte spectrin, but establishing the
relationship of ␤Golgi spectrin with other ␤-spectrins will
require molecular characterization. Recently, ␤3-spectrin
has been proposed to function as the Golgi spectrin (372).
However, the pattern of expression and cellular localization of ␤3-spectrin do not support a general role in Golgi
function (310).
Alternative splicing provides additional diversity
among ␣- and ␤-spectrins. ␤ I, ␤ II, and ␤ IV spectrins are
all differentially spliced. ␤ I, ␤ II, and ␤ IV spectrins have
COOH-terminal regions that are subject to differential
mRNA splicing to generate “short” or “long” COOH-terminal regions (24, 168, 427). The ␤ III polypeptides described to date have a long COOH-terminal region that
includes a pleckstrin homology (PH) domain (310, 372).
One nomenclature refers to ␤-spectrin spliceoforms by
the order of their discovery (429): short ␤ I is ␤ I ␴ I, and
long ␤ I is ␤ I ␴ II. However, this system has become
confusing as new family members have been discovered:
long ␤ IV is ␤ IV ␴ 1 and short ␤ IV is ␤ IV ␴ 4, while long
␤ II is ␤ II ␴ 1 and short ␤ II is ␤ II ␴ 2. We will refer
instead to spliceoforms by molecular weight or in some
way to help describe their domain composition.
The long COOH-terminal regions of ␤-spectrins have
a PH domain (see below) linked by an apparently unstructured region of ⬃100 amino acid residues to the last
(partial) triple helical repeat. The polypeptide chain terminates 50 – 60 residues after the PH domain. Short
COOH-terminal isoforms do not contain a PH domain.
About halfway through the linker region after the last
(partial) triple helical repeat, the sequences diverge and
terminate after 22–28 residues. In both ␤ I and ␤ II spectrins, the short COOH-terminal region contains multiple
Ser or Thr residues that are potential substrate sites for
casein kinase II. In the case of the short ␤ I COOHterminal region (i.e., in erythrocyte ␤-spectrin), at least
six of these residues are substrates for casein kinase II
(163, 323). As described more fully in section IIC, the PH
domain probably represents a major ligand-binding site in
␤-spectrins. Thus differential splicing modulates both the
interactive and regulatory properties of the ␤-spectrins.
␤ II is subject to further differential splicing. A splice
variant termed ELF1 represents a truncated ␤-spectrin,
consisting of little more than a calponin homology domain
(the CH1 domain) and the COOH-terminal region of the
short ␤ II (287).
Combinatorial association of ␣-spectrins with various ␤- and ␤-H subunits yields ␣/␤ and ␣/␤-H heterotetramers with distinct functions and patterns of expression
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
A. Spectrins
Volume 81
July 2001
SPECTRIN AND ANKYRIN-BASED PATHWAYS
1357
(Fig. 2, Table 1). Human ␣1/␤1-spectrins were first characterized in mammalian erythrocytes, and also are expressed in striated muscle and a subset of neurons in the
central nervous system (227, 300, 345). Avian erythrocytes, in contrast, have ␣2/␤2-spectrin (343). ␣2/␤2, ␣2/␤3,
and ␣2/␤4 represent the major forms of spectrin in nonerythroid vertebrate tissues. Terminal web spectrin comprised of ␣2 and a presumed ␤-H spectrin are localized in
apical domains of epithelial tissues such as small intestine, while ␣2/␤2-spectrins are associated with basolateral
domains (147).
1. Domains
␣-Spectrins contain 22 domains with the following
features: domains 1–9 and 11–21 are comprised of triple
helical repeats also found in ␤ and ␤-H spectrins (see
below); domain 10 is an SH3 (src homology domain 3)
motif; the COOH-terminal domain 22 is related to calmodulin (393) (Figs. 2 and 3). Domain 11 of vertebrate ␣2subunits contains a 35-residue extension with the cleavage site for Ca2⫹-activated protease and a calmodulinbinding site (162, 235). D. melanogaster spectrin contains
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
FIG. 2. The domain structure of spectrin polypeptides and proteins. The ␣- and ␤-subunits are composed of discrete
folding units or domains. Triple helical repeating units make up the length of each spectrin subunit. At the NH2 termini
of ␤-spectrins are tandem calponin homology domains, which bind actin. High-affinity interaction with actin requires the
first two triple helices too (indicated as atypical repeats). At the COOH termini of the subunits are differentially spliced
“tails”: these can either contain pleckstrin homology domains or short regions of sequence rich on phosphorylation sites.
The high molecular mass ␤-H spectrins have additional triple helical repeats plus an internal SH3 domain. ␣-Spectrins
contain COOH-terminal EF hands in a “calmodulin-like” structure. Interactions between ␣- and ␤-subunits lead to the
formation of tetramers. Lateral associations between ␣ and ␤ occur near the NH2 terminus of ␤ and the COOH terminus
of ␣, such that the subunits lie anti-parallel to each other, and the EF hands are close to the CH domains. At the NH2
terminus of ␣, the first helical repeat actually only comprises one helix; the most COOH terminal in ␤ comprises two
helices. These interact “head to tail” to form an intact triple helix bringing two laterally associated ␣␤-dimers together
to form an (␣␤)2-tetramer. Tetramers can form between ␣ and ␤-H in the same way.
1358
TABLE
VANN BENNETT AND ANTHONY J. BAINES
1. Spectrin genes, subunits, and heterotetramers
Drosophila
melanogaster
Caenorhabditis elegans
Subunit
Gene
Subunit
Gene
Homo sapiens
Subunit
Gene
Chromosome
SPTA1
SPTAN1
SPTB
SPTBN1
SPTBN2
betaIV
BSPECV
1q21
9q33-q34
14q23-q24.2
2p21
11q13
19q13.13
15
Spectrin subunits
␣
spc-1
␣
␤
unc-70/bgs-1 ␤
␤-H
sma-1
␤-H
I(3)dre3 ␣1
␣2
␤-Spec
␤1
␤-G/␤2
␤3
␤4
karst
␤-H/␤5
␣/␤
␣/␤-H
␣/␤-H
␣2/␤-H
␣1/␤1;
?␣1/␤2;
?␣1/␤3;
?␣1/␤4;
␣2/␤2;
␣2/␤3;
␣2/␤4
a predicted calmodulin-binding site at a different position
than vertebrates (105).
␤-Spectrins contain 19 domains beginning with a
highly conserved NH2-terminal actin-binding domain comprised of two adjacent calponin homology domains (6,
46), followed by 17 consecutive triple-helical repeat domains and ending with a COOH-terminal domain which
includes a PH domain (Figs. 2 and 3). Ankyrin-binding
sites are at a site in the midregion of the tetramer (75, 400)
and have been assigned to repeat number 15 of ␤1-spectrin (210). Repeat 17 is a partial helical repeat that pairs
with the COOH terminus of ␣-spectrins to form a noncovalent triple-helical structure (see below).
␤-H spectrins of D. melanogaster (106, 387), C. elegans (276), and humans (371) have NH2-terminal actinbinding domains and COOH-terminal domains closely related to ␤-spectrins but contain 30 triple-helical repeats
instead of 17. ␤-H spectrins of Drosophila and C. elegans
also have an SH3 domain inserted in the fifth triple helical
domain. ␤-H spectrins lack an ankyrin-binding site (276,
371, 387).
A) TRIPLE-HELICAL DOMAINS. Atomic resolution of the
structure of triple-helical repeat domains obtained by Xray crystallography (100, 155, 434) as well as NMR (319)
provides a striking confirmation of the structure originally
predicted by Speicher and Marchesi (367). Triple helical
repeats are comprised of two parallel and one antiparallel
␣-helices, which are stabilized by interactions between
hydrophobic residues spaced in a heptad repeat pattern
found in other examples of paired helical structures. Tandem triple helical repeats of spectrin and ␣-actinin are
comprised of antiparallel ␣-helices connected by an extended ␣-helix (100, 155) (Fig. 3A). Comparison of hydro-
dynamic properties of single and multiple domains suggests that serial repeats are flexible and configured such
that the average end-end length is reduced compared with
values predicted for rigid rods (403). One possible source
of flexibility is bending of the ␣-helix interconnecting
domains. A novel mechanism for shortening end-end distances by rearrangement of helices has been proposed by
Grum et al. (155) based on alternative structures observed
by crystallography. An extended series of triple helical
repeats may also exhibit a superhelical twist (155, 274).
Spectrin repeats have recently been demonstrated by
atomic force microscopy to reversibly unfold and refold
when subjected to forces in the range of 35 pN (347).
Spectrin and other proteins (see below) with triple helical
domains therefore have the potential to function as molecular springs that can store energy and dampen deformations resulting from mechanical stress. The potential
role of this elastic behavior in spectrin function is discussed below.
B) ACTIN-BINDING DOMAIN. The NH2-terminal actin-binding domains of the ␤-spectrins are comprised of a pair of
calponin homology (CH) domains (47). These are similar
in sequence to a region of the smooth muscle actinbinding protein calponin; such tandem pairs occur in
other proteins that have lateral associations with actin
filaments, including dystrophin, utrophin, ␣-actinin, and
fimbrin (Fig. 3B). Related calponin-based actin-binding
domains have been recently found in the plakin family of
proteins involved in connection of actin filaments with
intermediate filaments and microtubules (435, 436), and
in cortexillins, which bundle actin filaments (116).
X-ray crystallography has resolved the atomic structures of the NH2-terminal (45) and COOH-terminal (6) CH
domains of ␤-spectrin, and of tandem CH domains of
fimbrin (149) and utrophin (291) (Fig. 3B). The tandem
pairs of CH domains of fimbrin and utrophin interact with
actin, and their binding to actin filaments has been resolved at atomic resolution (149, 161, 208, 291). The
␤-spectrin CH domains are also likely to interact with
actin. The actin binding activity of ␤-spectrin is restricted
to the ␤-chains (44, 238). A minimal actin-binding fragment of erythrocyte spectrin has been produced by limited trypsin digestion and derives from residues 47–186
(207). These residues represent the NH2-terminal CH domain (known as CH1), which in utrophin and fimbrin is
the highest affinity actin binding site (161, 291).
Several considerations suggest that the site of contact with F-actin involves the junction between CH domains, with the first domain providing most of the interactions (6, 161). The second CH domain may contribute
by enhancing the affinity for F-actin and/or have a regulatory role (6) (see below). Ironically, the single calponin
domain of calponin itself lacks actin-binding activity
(145), suggesting the likely possibility that CH domains
have additional unresolved functions.
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
Spectrin heterotetramers
␣/␤
Volume 81
July 2001
SPECTRIN AND ANKYRIN-BASED PATHWAYS
1359
C) PH DOMAIN. ␤-Spectrins contain a PH domain located in the COOH-terminal segement, which is deleted in
certain alternatively spliced isoforms (see above). These
domains extend out from spectrin rods in the midregion
of spectrin tetramers and are placed within 10 nm of each
other (see Fig. 2). PH domains are ⬃100-residue folding
units first resolved in pleckstrin, which is a major protein
kinase C substrate in platelets, and subsequently been
found in many proteins (340). A unifying feature of proteins with PH domains is a role in signaling and proximity
to plasma membranes. Ligands for PH domains may include polyphosphatidylinositol lipids as well as proteins.
The three-dimensional structures of the PH domains
of mouse (190, 262) and D. melanogaster ␤-spectrins
(447) reveal similar folds to PH domains of other proteins
(340) (Fig. 3C). PH domains include a seven-stranded
␤-sheet arranged as a ␤-barrel with a COOH-terminal
␣-helix. Solution of PH domain structure from other proteins indicates that while the overall folding of PH domains are conserved, variations in loop lengths and composition provide substantial variability in potential
interaction surfaces (447). Consistent with structural predictions, binding activities of PH domains of spectrin and
other proteins are distinct both with respect to interactions with various phosphatidylinositol lipids and to proteins (340).
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
FIG. 3. Spectrin domains. A: triple helical repeats. Ribbon representation of two
triple helical repeats of chicken ␣-spectrin
(from the PDB file 1CUN; Ref. 155). B:
calponin homology domains. The figure
shows a ribbon representation of the tandem calponin homology domains of utrophin (taken from the PDB file 1QAG; Ref.
291). The equivalent sequence in human
␤-G spectrin is 68% identical and is likely
to adopt a similar overall conformation,
based on the known structures of individual spectrin CH domains (6, 46). Three
actin binding sequences (ABS) have been
mapped in utrophin and are shown in red
(ABS1), purple (ABS2), and cyan (ABS3).
ABS1 and ABS2 are in the first calponin
homology domain (CH1); ABS3 is in the
second (CH2). ABS1 and ABS2 are highly
conserved between utrophin and ␤-G spectrin (80 and 68% identical, respectively)
and probably present the primary site of
interaction with filamentous actin. ABS3 is
less conserved in sequence between spectrin and utrophin (25% identical), even
though the helical conformation of this sequence is preserved in spectrin. C: pleckstrin homology domain. The pleckstrin homology domain of ␤-spectrins represents a
major binding site for protein and lipid
ligands. This figure shows the interaction
of the PH domain of mouse ␤-G spectrin
with inositol 1,4,5-trisphosphate (IP3)
(shown as the small molecule in ball and
stick, bottom right). The PH domain
makes contact with the 4- and 5-phosphate
groups through salt bridges and hydrogen
bonds; the 1-phosphate group (on the right
of the IP3 molecule) is mostly solvent exposed, and the inositol ring has virtually no
interactions with the protein. Residues
shown likely to interact with the ligand are
(from left) Tyr-69, Lys-8, Arg-21, and Ser22. (from the PDB file 1BTN; Ref. 190). D:
SH3 domain. SH3 domains are thought to
interact with their targets via hydrophobic
interactions with proline-rich sequences.
The spectrin SH3 has a “stripe” of hydrophobic residues along one surface that are
thought to mediate interactions with targets such as e3B1 (456). The figure shows
the residues of this stripe (from left to
right: Phe-52, Trp-41, Pro-54, Tyr-57, Tyr13) (from the PDB file 1SHG; Ref. 298).
1360
VANN BENNETT AND ANTHONY J. BAINES
2. Subunit interactions
Spectrin heterotetramers are assembled through the
following interactions between ␣- and ␤-subunits: 1) a
lateral and antiparallel association between ␤-subunits
and ␣-subunits; 2) head-head association between laterally associated heterodimers by linkage between partial
triple-helical repeats at the COOH-terminal end of the
␤-subunits and the NH2-terminal end of ␣-subunits (see
Fig. 2). Structural requirements for lateral association
between ␣- and ␤-spectrins have been analyzed for erythrocyte (14, 368, 403) and D. melanogaster spectrin (409,
411). The minimal domains required for ␣-␤ complexes
are the first two triple-helical domains of ␤-spectrin and
the last two triple helical domains of ␣-spectrin (368). The
first two triple helical domains of ␤-spectrin and last two
of ␣-spectrin are closely related to the four triple helical
domains of ␣-actinin, which also associate laterally in an
antiparallel orientation (100). The atomic structure of
tandem ␣-actinin domains reveals a dimer formed stabi-
lized by electrostatic interactions provided by complementary surfaces (100). Association between four triple
helical domains of ␣1- and ␤1-spectrins is of high affinity
with a dissociation constant (KD) of 10 nM (403). Further
stabilization of ␣-␤ complexes is provided by interaction
between the calmodulin-related domain of ␣-spectrin and
the calponin homology domains of ␤-spectrin (409, 411).
The lateral association of ␣- and ␤-spectrin subunits
is highly conserved and occurs between D. melanogaster
and vertebrate spectrins (43). The high affinity of ␣- and
␤-subunits implies that spectrin will not exist as independent ␣- or ␤-subunits but is an obligatory heterodimer or
tetramer. Reports of ␤-spectrin unaccompanied by an
␣-subunit in striated muscle and at neuromuscular junctions (31, 338) suggest the possiblity of a heretofore unrecognized ␣-subunit or an immunoreactive but otherwise
highly diverged ␤-spectrin.
Head-to-head contacts between ␣- and ␤-spectrin
subunits are believed to occur through contacts resembling pairing between helices in triple helical bundles (89,
211, 221, 366, 395, 433). The NH2 terminus of ␣-spectrin
provides one helical segment, and the COOH-terminal
repeat of ␤-spectrin provides two antiparallel helices,
with ␣-␤ pairing resulting in a noncovalent triple helical
segment. Flanking residues on ␣-spectrin also contribute
to ␣-␤ association (55).
Defects in spectrin tetramer formation have been
established in erythrocytes of patients with hereditary
elliptocytosis (315, 395). Mutations in ␣- and ␤-spectrin
defined in these patients would be predicted to disrupt
helical pairing predicted from biochemical studies (89,
396, 434). These mutations include substitution of prolines, which would be expected to disrupt an ␣-helix, as
well as mutations in residues predicted to provide contacts between helices. Human mutations in the partial
helical domain of ␣-spectrin have been introduced into D.
melanogaster ␣-spectrin and result in a temperature-sensitive phenotype (89) (see below).
3. Spectrin superfamily
Proteins that contain NH2-terminal CH domains,
COOH-terminal calmodulin-related domains, and intervening triple helical domains comprise the spectrin superfamily. Currently recognized proteins with these combined features include ␣-actinins and dystrophins. In
addition, trabeculin/macrophin/MACF of vertebrates
(236) and Kakapo of D. melanogaster (154) are newly
recognized members of the plakin family that have CH
domains, 23–29 triple helical domains, and a calmodulinrelated domain, as well as domains related to plectin and
the plakin family (Fig. 4). The COOH termini of these
proteins contain a microtubule-binding domain (206, 236).
These proteins thus can interact with actin filaments,
cadherins, or integrins through the plakin domain and
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
D) CALMODULIN-RELATED DOMAIN. ␣-Spectrins contain EFhand motifs located at the NH2 terminus of that are juxtaposed to the actin-binding domain on the adjacent
␤-subunit. The EF-hand domain of ␣-spectrin shares
structural homology with calmodulin and also exhibits a
Ca2⫹-dependent conformational change (392, 393). An
important distinction between spectrin and calmodulin is
that spectrin only contains the two NH2-terminal EF
hands and lacks the two COOH-terminal EF hands
present in calmodulin. Intact human erythrocyte spectrin
or recombinant ␣ I or ␣ II EF hands bind Ca2⫹ selectively
with a stoichiometry corresponding to the number of EF
hands but with an unphysiological affinity in the range of
hundreds of micromolar (8, 257, 258). However, intact
horse spectrin molecules have been reported to bind Ca2⫹
at many sites of micromolar affinity, which presumably
are not related to EF hands (414). Fowler and Taylor
(123) reported that low micromolar levels of Ca2⫹ influenced human erythrocyte spectrin-actin interactions, although spectrin-actin and spectrin-4.1-actin interactions
were described as Ca2⫹ insensitive by Ohanian et al.
(307). Clearly much remains to be understood about the
significance of Ca2⫹ binding by spectrins.
E) SH3 DOMAIN. SH3 domains, initially observed in the
Src protein tyrosine kinase, are present in many proteins
involved in cell signaling and mediate interactions with
proline-rich stretches in a variety of target proteins (321).
SH3 domains are inserted in ␣-spectrins (419) and in
invertebrate ␤-H spectrins (106, 276) (Fig. 2). The structure of the ␣-spectrin SH3 domain has been resolved at an
atomic level by X-ray crystallography (298) and NMR (30,
352) (Fig. 3D). The three-dimensional structure of spectrin SH3 domains is a compact ␤-barrel and exhibits the
same overall fold as other SH3 domains.
Volume 81
July 2001
SPECTRIN AND ANKYRIN-BASED PATHWAYS
1361
microtubules. Spectrin-like repeats also are present in
eight to nine tandem copies in unc 73/kalirin/Trio, which
also contain a dbl/pleckstrin homology domain (83). Trio/
Unc 73 is required for axon pathfinding in C. elegans and
D. melanogaster (259, 375).
Triple-helical repeats of ␣-actinin are most similar to
the first repeats of ␤-spectrin (repeats 1 and 2) and the
last repeats of ␣-spectrin (domains 20 and 21) (43). ␣-Actinin thus contains within a single polypeptide the COOHterminal domain of ␣-spectrin (calmodulin-related domain and last two triple helical repeats) and the NH2terminal domain of ␤-spectrin (CH domains and first two
triple helical repeats) (Fig. 4). These considerations have
led to suggestions that ␣- and ␤-spectrins evolved from a
homodimeric ␣-actinin-like precursor polypeptide (41,
319, 387, 408). One possible evolutionary scenario is that
␣-actinin became elongated through insertion of seven
repeats by an unspecified mechanism, followed by duplication events resulting in a giant ␣-actinin-like protein.
Insertion of a transcriptional promoter within a repeat has
been proposed to provide the basis for the modern split
repeat at the ␣-␤ tetramerization site and the origin of
separate ␣- and ␤-spectrin genes (387, 408).
4. Spectrin-protein interactions
A) SPECTRIN-ACTIN INTERACTION. The site of contact of
F-actin with the actin-binding domains of ␣-actinin (273),
fimbrin (161), and utrophin (291) has been mapped to
actin subdomains 1 and 2 as well as subdomain 1 of the
adjacent actin monomer by image analysis of electron
micrographs. These conclusions are supported by genetic
mapping of actin contact sites with fimbrin in yeast (175,
177). The fimbrin actin-binding domain induces a confor-
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
FIG. 4. Representatives of the spectrin superfamily. Members of the spectrin superfamily have in common multiple
triple helical repeats, a pair of calponin homology domains at the NH2-terminal of one subunit, and a calmodulin-like
domain in the COOH-terminal region of one subunit. Specific functions that distinguish each member of the family are
conferred by variations in the number of triple helical repeats, and the presence of varying additional ligand-binding
domains that include SH3 and PH domains in spectrin, plakin, and GAR22 domains in trabeculin/kakapo and a
cysteine-rich domain in dystrophin. In addition, individual helices can contain specific binding activities, for example, the
ankyrin binding activity of the 15th repeat in ␤-spectrins.
1362
VANN BENNETT AND ANTHONY J. BAINES
regulated by calmodulin and is localized in the NH2-terminal region. The other ␤-spectrin site is located in the
COOH-terminal domain, which includes the PH domain.
Association of the spectrin PH domain with membranes has been demonstrated in living cells using green
fluorescent protein-tagged ␤-spectrin PH domain (415).
One class of spectrin-PH domain interactions is likely to
involve PI lipids, since the PH domain of ␤1-spectrin
associates with sites in brain membranes stripped of peripheral proteins that are blocked by inositol 1,4,5trisphosphate (IP3) and presumably represent phosphatidylinositol sites (416).
C) SPECTRIN-ION CHANNEL INTERACTIONS. Candidates for
spectrin-binding proteins in brain synaptosomes include
the NR2 and NR1 subunits of the NMDA receptor (422).
Spectrin binds to the NR2B subunit at sites distinct from
those of ␣-actinin-2 and members of the PSD95/SAP90
family. The spectrin-NR2B interactions are inhibited by
Ca2⫹ and fyn-mediated NR2B phosphorylation, but not by
Ca2⫹/calmodulin or by calmodulin kinase II-mediated
phosphorylation of NR2B. The spectrin-NR1 interactions
are unaffected by Ca2⫹ but inhibited by Ca2⫹/calmodulin
and by phosphorylation of NR1 by protein kinases A and
C. The NR1 subunit thus is a candidate to interact with the
Ca2⫹/calmodulin-regulated site on ␤-spectrin (80, 374).
Spectrin associates via the ␣-spectrin SH3 domain
with the ␣-subunit of the amiloride-sensitive Na⫹ channel,
EnNaC (349, 457), and with the Na⫹/H⫹ exchanger, NHE2
(59). The amiloride-sensitive Na⫹ channel/spectrin complexes also contain the protein Apx, which is an apically
localized protein identified in Xenopus (457). Association
with ␣-spectrin may be responsible for apical targeting of
NHE2, since deletion of the spectrin-binding loop results
in basolateral localization of the channel (59). ␣/␤H
(␤TW) is the most likely form of spectrin to associate with
the apically targeted NHE2 and amiloride-sensitive Na⫹
channels since ␣/␤-spectrin generally is localized to the
basolateral domains of epithelial cells (438).
The cGMP-gated cation channel of rod photoreceptor
plasma membranes copurifies with a 240-kDa polypeptide
subsequently identified as spectrin based on immunoreactivity (290). Direct linkage between spectrin and the
cation channel is likely since antibodies against each
protein coimmunoprecipitates the other from relatively
pure preparations of the channel. The 240-kDa polypeptide was localized to the inner surface of the rod outer
segment plasma membrane and excluded from the disk
membranes, as would be expected for an association of
this protein with the cGMP-gated cation channel in vivo.
D) SPECTRIN AS A MEMBRANE ADAPTOR FOR CYTOPLASMIC PROTEINS. Cytoplasmic ligands for spectrin include a protein
termed HsSH3bp1 that binds to tyrosine kinases and binds
to spectrin through association with the ␣-spectrin SH3
domain (432, 456). HsSH3bp1 is associated with macropinosomes and may couple spectrin to these intracellular
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
mational change in F-actin (161), suggesting the possibility that ␤-spectrin could also perturb F-actin structure.
Comparison of actin-binding activities of the ␤-spectrin actin-binding domain alone and with triple-helical
repeats suggests that the first triple helical repeat also
participates in spectrin-actin complexes (238) (Fig. 1C).
These results support a model where ␤-spectrin actually
lies along the actin filament, thus engaging in contacts
involving two actin monomers (238). Interestingly, similar
studies of dystrophin-actin interactions have concluded
that lateral associations occur between actin and dystrophin triple helical domains (in this case repeat number 11)
(351).
There is evidence that different ␤-spectrins exhibit
varying modes of interaction with actin. It is interesting to
note that a very small ␤ II-spectrin splice variant ELF1 (a
“mini-spectrin”) contains a single CH domain that is similar to the CH1 domain of other ␤-spectrins. Since ␤1spectrin CH1, isolated as a tryptic fragment, binds actin
avidly in vitro (207), it is likely that ELF1 is a true actinbinding protein, even though some other proteins with
single CH domains do not necessarily bind actin (6).
Intriguingly, the CH1 tryptic fragment of ␤ I-spectrin appears to have a higher affinity interaction with actin than
might have been expected: it inhibits interaction of whole
erythrocyte spectrin with actin with a half-maximal effect
at 5 ␮M, while the intact erythrocyte dimer binds to actin
with a KD of 250 ␮M (309). This indicates a higher affinity
interaction than native erythrocyte ␣ I/␤ I-spectrin dimer.
One possibility might be that ␣ I-spectrin has a suppressive effect on the interaction of ␤1 CH domains with actin.
As we note below, the weak interaction of erythrocyte
spectrin with actin is enhanced by proteins 4.1 and adducin.
The tandem CH domains in ␤-spectrins may have
additional roles in actin binding. CH1, the NH2-terminal
CH domain, probably provides the primary interaction
with actin (see Ref. 291). CH2, the COOH-terminal of the
pair, binds actin only weakly (46). In utrophin, CH2 provides a specificity for actin subtypes: utrophin binds ␤-actin (cytoskeletal) with higher affinity than ␣-actin (sarcomeric). The CH domains of ␤-spectrin therefore have the
potential to target spectrin to particular isoforms of actin,
as is the case with utrophin (426). In this context, it is
interesting to note that erythrocyte membranes contain
only ␤-actin, rather than the ␤-␥ mixture typical of most
cytoskeletal systems (333).
B) SPECTRIN-MEMBRANE INTERACTIONS. Spectrins are coupled to membranes by multiple pathways including direct
association with membrane-spanning proteins, interaction with phospholipids, and through interactions with
ankyrins (see below). Binding of spectrin to ankyrinindependent protein sites has been measured in brain
membranes (80, 251, 373, 374). ␤-Spectrin associates with
membranes through two distinct classes of sites. One is
Volume 81
July 2001
SPECTRIN AND ANKYRIN-BASED PATHWAYS
B. Ankyrins
Ankyrin in human erythrocytes provides a high-affinity link between the cytoplasmic domain of the anion
exchanger and the spectrin/actin network (Fig. 1). The
ankyrin family has a general role as an adapter between a
variety of integral membrane proteins and the spectrin
skeleton. The C. elegans genome contains a single ankyrin
gene, (312), whereas the D. melanogaster genome has
two ankyrin genes, Dank1 and Dank2 (35, 110). The
ankyrin gene family of mammals currently includes three
members: ankyrin-R (R for restricted; also termed
ankyrin1) (229, 260), first characterized in erythrocytes,
(227); ankyrin-B (B for broadly expressed, also termed
ankyrin 2) first characterized in brain (313); and ankyrinG (G for general or giant; also termed ankyrin 3) independently discovered in searches for components of the node
of Ranvier (219) and for epithelial ankyrins (93, 327, 385).
Ankyrins are expressed in most tissues, and in many
cases all three ankyrins are found in the same cell type.
Diseases of humans attributed to ankyrins include
hereditary spherocytosis, which can result from decreased expression and/or mutated forms of ankyrin-R
(112). In mice, a similar disorder (the nb/nb mutation)
results in a nearly complete deficiency of 210-kDa iso-
forms of ankyrin-R in erythrocytes, neurons, and striated
muscle (452) with a phenotype of severe anemia (33) and
degeneration of a subset of Purkinje cell neurons accompanied by cerebellar dysfunction (326). Ankyrin mutations in C. elegans result in the unc44 phenotype, which
includes abnormal axon guidance and uncoordinated
movements (312).
1. Domains
Ankyrins are modular proteins comprised of three
domains conserved among family members as well as
specialized domains found in alternatively spliced isoforms (see Fig. 5). Conserved domains are an NH2-terminal membrane-binding domain, a 62-kDa spectrin-binding
domain, and a 12-kDa death domain. The membranebinding domains are comprised of 24 copies of a 33residue repeat known as the ANK repeat that is involved
in protein recognition in many types of proteins (34, 359)
(Fig. 6). The 24 ANK repeats form 4 subdomains, each
comprised of the basic folding unit of 6 repeats (280). The
role of six-repeat subdomains as protein binding sites is
discussed below. Death domains were first reported in
proteins such as Fas and the tumor necrosis factor receptor that participate in apoptosis pathways (394). These
domains can associate with related death domains in
other proteins. The protein interactions of the ankyrin
death domain could involve self-association and/or interactions with other proteins and are not yet resolved. The
death domain is followed by a regulatory domain subject
to alternative splicing that in the case of ankyrin-R modulates both binding of the anion exchanger and of spectrin
(81, 157). A regulatory function for this domain has not
been demonstrated for ankyrin-B or ankyrin-G. However,
the COOH-terminal domains are the most divergent
among ankyrin family members and are likely to have
distinct functions that are yet to be defined.
2. Diversity due to alternative splicing
Ankyrin genes are expressed and processed in a complex tissue-dependent and developmentally regulated
fashion, giving rise to a large number of isoforms that
utilize different combinations of functional domains. For
example, alternative splicing generates ankyrin-G isoforms missing all or portions of their membrane-binding
domain (93, 178, 219, 327). Alternate exon usage is also
used to generate an unusual isoform of ankyrin-R in striated muscle. This isoform lacks both the membrane-binding and spectrin-binding domains and contains only the
COOH-terminal 26 kDa plus a hydrophobic stretch (452).
The functions of these small ankyrins are likely to involve
intracellular as opposed to plasma membrane roles, since
these truncated ankyrins are associated with Golgi, lysosomal, and sarcoplasmic reticulum membranes (11, 88,
92, 452).
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
organelles (432). Spectrin PH domains are likely to associate with proteins in addition to their interactions with PI
lipids (above). An interesting example is RACK1, a protein
kinase C-anchoring protein, which associates selectively
with the ␤-spectrin PH domain and activated protein kinase C to form a ternary complex (348).
A search for annexin VI-binding proteins revealed
spectrin as one of ⬃14 membrane-associated proteins
that associate with annexin VI in blot overlays (420).
Annexin VI has subsequently been implicated in directing
proteolytic degradation of spectrin during receptor-mediated endocytosis (204). The interpretation of these experiments was that annexin VI promoted proteolysis of spectrin, which resulted in relaxation of restraints of budding
of a subset of coated pits. Annexin VI, according to this
model, utilizes spectrin as an adaptor to direct and possibly activate a protease.
E) SPECTRIN AS A REGULATOR PROTEIN. Spectrin, at nanomolar concentrations, inhibits phospholipase D as well as
phospholipases C and A2 in in vitro assays (254, 255). The
basis for inhibition was most likely not a direct interaction of spectrin with these enzymes, but rather competition for their phospholipid substrates (255). Spectrin in
lymphocytes associates with CD45 (192, 248), which is a
member of a family of membrane-spanning glycoproteins
with protein-tyrosine phosphatase activity. Spectrin binds
to CD45 with a nanomolar affinity and upregulates the
tyrosine phosphatase activity of CD45 (248).
1363
1364
VANN BENNETT AND ANTHONY J. BAINES
Volume 81
Ankyrin-B and ankyrin-G spliceoforms also include
polypeptides with insertions of up to 2,500 amino acid
residues and molecular mass of up to 480 kDa, which are
abundantly expressed in axons (49, 219, 224, 225) (see
below). The inserted sequences in both genes are placed
between the spectrin-binding and death domains (see Fig.
5). Ankyrin-G polypeptides of 270 and 480 kDa contain
inserted sequences beginning with a serine/threonine-rich
stretch of ⬃400 residues, that is followed by sequence
with similarity to that of the inserted sequence of 440-kDa
ankyrin-B. The 270-kDa ankyrin-G lacks a 190-kDa stretch
of this sequence resulting from the use of an alternate
splice donor site.
Much of the 220-kDa inserted sequence of 440-kDa
ankyrin-B has the configuration of an extended random
coil based on physical properties of expressed polypeptides (49). Because the alternatively spliced insert of
ankyrin-G has a highly polar hydrophilicity profile similar
to that of ankyrin-B, it is likely that the 480-kDa ankyrin-G
also contains an extended stretch of random coil inserted
between its spectrin-binding and death domains. The
properties of the inserted sequences suggest a structural
model for 440-kDa ankyrin-B and 480-kDa ankyrin-G
where the membrane-associated head domain is separated from the death domain by an extended filamentous
tail domain encoded by the inserted sequence (see Fig. 5).
The length of the tail domain could be up to 0.5 ␮m if fully
extended, which is a distance that, in principle, could be
resolved in the light microscope.
Ankyrin isoforms possessing the tail domain are
highly localized to axons, whereas isoforms lacking this
domain are localized to the neuron cell body (49, 224).
Possible functions of the tail domains include axonal
targeting motifs, deregulation of ankyrin membrane-binding domains by physical separation from the regulatory
domain, and long-range connections between molecules
interacting with the death domain/COOH-terminal domains and membrane-binding domains. Given the potential distance spanned by the inserted sequence, these
putative ankyrin-binding molecules could be in distinct
membrane domains or even different cellular compartments.
3. Ankyrin-binding membrane proteins
A defining feature of the ankyrin family is their ability
to interact through the membrane-binding domain with
structurally diverse proteins with apparently unrelated
primary sequences. Currently identified ion channels/
pumps that associate with ankyrin and colocalize in cells
include anion exchanger isoforms (AE1, AE2, AE3) (22,
23, 196, 293), the Na⫹-K⫹-ATPase (216, 297, 304), the
voltage-dependent Na⫹ channel (263, 269), and the Na⫹/
Ca2⫹ exchanger (240). IP3 receptor and ryanodine receptor Ca2⫹-release channels also associate with ankyrins
(36, 37, 199). Evidence for an in vivo interaction between
ankyrin-B and Ca2⫹ release channels is based on altered
targeting of these proteins in striated muscle of ankyrinB-deficient mice (397) (see below). Finally, ankyrin also
associates with cell adhesion molecules (CAMs) including
CD44 (203, 249) and the L1 CAM family (L1/neurofascin/
NrCAM/CHL1/NgCAM) (76, 79, 107, 182, 450). Evidence
for physiologically important interactions between
ankyrins and L1 CAMs include downregulation of L1 in
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
FIG. 5. Summary of human ankyrin genes and
their alternatively spliced variants. Canonical
ankyrins are of 190 –220 kDa and have membranebinding, spectrin-binding, and death domains. Giant
ankyrins of 270 – 480 kDa have insertions of up to
2,400 amino acid residues between the spectrinbinding and death domains and are abundant in
axons. Small ankyrins of 26 –120 kDa are missing
various domains found in canonical ankyrins and
are associated with intracellular organelles.
July 2001
SPECTRIN AND ANKYRIN-BASED PATHWAYS
1365
axons of ankyrin-B (⫺/⫺) mice (358), and reduction of
Dank2 in cell bodies of neurons of neuroglian mutant flies
(35).
The ankyrin membrane-binding domain also interacts with cytoplasmic proteins including tubulin (19, 75,
82) and clathrin (283). Ankyrin associates with microtubules with a relatively low affinity in the micromolar
range, and the physiological significance of this interaction remains to be determined. However, ankyrin association with clathrin occurs with a KD in the nanomolar
range and is specific for the fourth subdomain of ankyrin
and the terminal knob domain of clathrin. Evidence that
ankyrin-clathrin interactions are functionally important is
that microinjection of the fourth subdomain inhibits receptor-mediated endocytosis of low-density lipoprotein
(283).
The basis for diversity of binding partners for the
ankyrin membrane-binding domains is due to special
properties of the ankyrin repeats, a motif which mediates
protein recognition for a large number of proteins including the transcription factors GABP-␤, Nf␬/I␬B, and SwI 6,
p53 binding protein and p16 cyclin inhibitor (reviewed in
Ref. 359). Atomic structures for these proteins reveal that
ankyrin repeats are folded into a series of antiparallel
␣-helices connected by loops that are arranged perpendicular to the helices (Fig. 6). Residues located at the tips
of the loops are the most variable between repeats and
contain recognition sites for diverse proteins. The four
repeat subdomains of ankyrins each have distinct binding
properties, and these subdomains also cooperate with
each other to generate a further level of diversity (281,
282). An additional feature of ankyrin is that binding
interactions can occur simultaneously with different
membrane proteins, resulting in multiprotein complexes
(281, 282).
Sites of ankyrin interactions have been evaluated for
the erythrocyte anion exchanger (AE1) (78, 96, 424), the
Na⫹-K⫹-ATPase (197, 453), and the L1 CAM family (181,
450). A general conclusion from these studies is ankyrin is
capable of structurally distinct interactions with diverse
proteins. This conclusion is reinforced by the fact that
ankyrin has independent binding sites for AE1 and L1
CAMs (281, 282). Another conclusion from analysis of
ankyrin-binding sites of the Na⫹-K⫹-ATPase (450) and L1
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
FIG. 6. Schematic view of ankyrin domains
and their protein interactions (top) and atomic
structure of Ank repeats (bottom). The NH2-terminal membrane-binding domain of ankyrins is
composed of 24 Ank repeats folded into 6-repeat
subdomains. These domains associate with a
variety of membrane proteins as well as tubulin
and clathrin. The bottom panel shows a stack of
six ankyrin repeats in the protein I-␬B (PDB file
1NFI; Ref. 193). The two images (a and b) show
the same structures rotated by 90°. The sites of
protein interactions are likely to be the loops of
Ank repeats, based on structures of complexes
of I-␬B and several other Ank repeat proteins
with their protein partners (see Ref. 359).
1366
VANN BENNETT AND ANTHONY J. BAINES
of L1 CAM family members is provided by divergence
between extracellular domains encoded by different
genes as well as multiple alternatively spliced variants of
each gene (as many as 50 estimated in the case of neurofascin) (165). Consistent with the diverse extracellular
domains, a range of functions has been attributed to the
L1 CAM family, including axon fasciculation, axonal guidance, neurite extension, a role in long-term potentiation,
synaptogenesis, and myelination (39).
Analysis of LAD-1, the single L1 CAM gene in C.
elegans, has provided a global view of the pattern of
expression in a metazoan and suggests a general expression at sites of cell-cell contact early in embryonic development and in essentially all cell types (Chen and Bennett, unpublished data). LAD-1 binds to GFP-tagged worm
ankyrin (unc44) in cultured cells and colocalizes with
ankyrin in most cell types. These findings suggest that
LAD-1 is the major receptor for ankyrin (unc-44) in the
worms. Given that ankyrin is multivalent with respect to
neurofascin and the anion exchanger (282), it is possible
that ankyrin forms heterocomplexes with LAD-1 and various ion channels (see Fig. 7 for an example of vertebrate
L1 CAM/Na⫹ channel complexes).
An extrapolation from the results in C. elegans is that
additional members and/or alternatively spliced forms of
the L1 CAM family in vertebrates are expressed in all
tissues and very early in development (the two-cell stage),
and function as coreceptors for ankyrin. Precise colocalization of L1 CAMs, ankyrin, and voltage-gated Na⫹ channels has been demonstrated at nodes of Ranvier and axon
initial segments in the vertebrate nervous system (77,
228). It will be of interest to determine if other ion channels reported to associate with ankyrin also are colocalized with CAMs and if these have homology to L1 CAMs.
Another variation on the theme of ion channels and CAMs
may be provided by the Na⫹-K⫹-ATPase which has an
␣-subunit, responsible for ion permeation, associated
with a ␤-subunit which has properties of a CAM (148).
Residues critical for ankyrin-binding activity of L1
CAM cytoplasmic domains are localized in a 26-residue
stretch which includes a sequence, EDGSFIGQY, which is
highly conserved in all family members from C. elegans to
mammals (one exception is CHL1 which has the sequence
EDGSFIGAY) (136, 182, 451; Chen and Bennett, unpublished data). Mutations of the FIGQY tyrosine in the cytoplasmic domain of neurofascin (Y81H/A/E) greatly impair neurofascin-ankyrin interactions (451). Mutation of
human L1 at the equivalent tyrosine (Y1229H) is responsible for certain cases of mental retardation (212). Mutations F77A and E73Q greatly diminish ankyrin-binding
activity, whereas mutation D74N and a triple mutation of
D57N/D58N/D62N result in less loss of ankyrin-binding
activity. These results provide evidence for a highly specific interaction between ankyrin and L1 CAM members.
The basis for specificity of association between var-
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
CAMs (450) is that ankyrin-binding sites of these proteins
share a conformation as a random coil even though they
lack obvious sequence similarity. An ankyrin-membrane
protein complex has not yet been resolved at an atomic
level, but these considerations suggest a model where
random coils of at least certain ankyrin-binding proteins
associate with loops of ankyrin repeats (see above) (450).
Given that ankyrin repeats fold into subdomains, it is
likely that loops of ankyrin repeats in different subdomains also could collaborate to create a “binding pocket.”
Cytoplasmic domains of the erythrocyte anion exchanger (5) and neurofascin (450) are both dimers in
solution. Considered together with the finding that
ankyrin has two binding sites for neurofascin as well as
for two sites for the anion exchanger, the existence of
dimers implies that these proteins and ankyrin are capable of forming oligomeric complexes. One predicted configuration of the anion exchanger/ankyrin complex at low
ratios of ankyrin to the anion axchanger is as a pseudoanion exchanger tetramer with two dimers independently
associated with distinct sites on the ankyrin membranebinding domain (281). Evidence that such an arrangement
occurs in erythrocyte membranes is provided by observations that the anion exchanger behaves as a mixture of
dimers and tetramers in native membranes and only as a
dimer in ankyrin-deficient membranes (439). One could
also imagine that at higher ankyrin-to-membrane protein
ratios it would be possible to form linear arrays of dimeric
membrane proteins cross-linked by the multivalent
ankyrin membrane-binding domain. These complexes
could be further immobilized by coupling to the spectrinbased membrane skeleton through the spectrin-binding
domain of ankyrin. Ability to form such large immobilized
complexes between ankyrin and neurofascin could be
important for the assembly of specialized membrane domains such as axon initial segments and nodes of Ranvier
where these proteins are localized (see below).
A) L1 CAMS AS GENERAL CORECEPTORS FOR ANKYRINS. The
major class of ankyrin-binding proteins in mammalian
brain is a group of CAMs in the Ig/FnIII superfamily
known as L1 CAMs. These molecules together comprise
over 1% of the membrane protein in adult brain tissue and
are abundantly expressed on axons in the developing
nervous system (reviewed in Ref. 181). The L1 CAM family of cell adhesion molecules in the vertebrate nervous
system is comprised of L1, NgCAM, NrCAM, CHL1, and
neurofascin, in D. melanogaster by neuroglian (181), and
in C. elegans by LAD1 (L. Chen and V. Bennett, unpublished data). L1 CAMs possess variable extracellular domains comprised of six Ig and three to five fibronectin
type III domains, along with a relatively conserved cytoplasmic domain (181). Extracellular domains of L1 CAMs
participate in homophilic interactions as well as a variety
of interactions with soluble proteins and other CAMs (39,
121, 350). Extensive diversity in extracellular interactions
Volume 81
July 2001
SPECTRIN AND ANKYRIN-BASED PATHWAYS
1367
ious L1 CAMs and different ankyrin family members in the
context of cells has yet to be determined. However, the
ability of neurofascin to participate in high-affinity interactions with both ankyrin-R (282) and ankyrin-B (Chen
and Bennett, unpublished data) and the ability of LAD-1 to
associate with mammalian ankyrin (Chen and Bennett,
unpublished data) suggest that these interactions most
likely are not restricted by intrinsic affinity. This raises
the interesting question as to how different ankyrins in
the same cell are segregated with their respective binding
partner and how these interactions are regulated.
B) L1 CAMS AND PHOSPHOTYROSINE-BASED SIGNALING. The association between ankyrin and neurofascin is abolished
by phosphorylation of the FIGQY tyrosine, which as noted
above is conserved among all L1 CAM family members
(136). Phosphorylation of the FIGQY tyrosine inhibits
activity of neurofascin in mediating cell adhesion and cell
segregation in cultured cells (398). Inhibitory effects of
FIGQY phosphorylation on cell-cell interactions of neurofacscin were proposed to result from loss of ankyrinmediated cross-linking of neurofascin (398).
Antibodies specific for the phosphotyrosine form of
the FIGQY peptide resolve members of the L1 CAM family
at strategic sites in the nervous system of vertebrates
including paranodes of nodes of Ranvier, the AChR domain of the neuromuscular junction, and in zones enriched in migrating neurons (S. Jenkins, A. Sen, N. Cramarcy, R. Sealock, and V. Bennett, unpublished data).
Phospho-FIGQY tyrosine forms of LAD-1 are localized in
the pharynx and in the vulva of C. elegans in a complementary pattern to the distribution of ankyrin, indicating
the conservation of this phosphorylation as well as the
potential for its extraneuronal expression (Chen and Bennett, unpublished data). These considerations suggest
that L1 CAMs participate in vivo in a phosphotyrosinebased signaling pathway, with one consequence being
loss of ankyrin binding. The extracellular ligand(s), kinase(s), and phosphatase(s) involved in determining the
phosphorylation state of L1 CAMs remain to be defined.
FIGQY-phosphorylated L1 CAMs, by analogy with SH2
and PTB systems, also may associate with yet to be
identified phosphotyrosine-dependent adaptors.
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
FIG. 7. Schematic model for organization of 480/
270-kDa ankyrin-G and ankyrin-associated proteins
(spectrin, L1 CAMs, voltage-gated Na⫹ channel) at
axon initial segments and nodes of Ranvier. 480/270kDa ankyrin-G (219), 186-kDa neurofascin and NrCAM
(77), and ␣/␤ IV spectrin (24) are colocalized with
voltage-gated Na⫹ channels at nodes of Ranvier and
axon initial segments. Ankyrins are multivalent in in
vitro assays (281, 282) and are proposed in this model
to interact with both the channel and adhesion molecules. Neurofascin and possibly NrCAM are palmitoylated at a cysteine residue in the membrane-spanning
domain (342), which could contribute to a phospholipid microdomain. The extended tail domain of 480kDa ankyrin-G could extend up to 0.5 ␮m into the
axoplasm and is shown interacting with a vesicle in a
speculative model.
1368
VANN BENNETT AND ANTHONY J. BAINES
C. Proteins That Promote Spectrin-Actin
Interactions
1. Protein 4.1
A) PROTEIN 4.1 IN ERYTHROCYTES. Protein 4.1 purified
from mammalian erythrocytes associates with ␤-spectrin
at a site in the NH2-terminal region (12, 399, 400), and it
also interacts with F-actin (12, 63, 296). Protein 4.1 can
promote a spectrin-4.1-F-actin ternary complex (401). In
the presence of 4.1, strong spectrin-actin interaction is
observed: the association constant for the ternary complex is 1 ⫻ 1012 M⫺2 (309). The spectrin-4.1 complex also
caps the minus end of actin filaments and can sever
F-actin filaments (331). Standard preparations of erythrocyte ghosts have minus ends of actin filaments that are
capped (330), but it is difficult to be certain of the physiological significance in erythrocytes of the spectrin-4.1
capping activity, since these cells also contain the specific
minus end capping protein tropomodulin (reviewed in
Ref. 125).
A proportion of protein 4.1 remains membranebound after erythrocyte ghosts are extracted with low
ionic strength buffers. Several possible membrane associations have been identified including membrane lipids,
the anion exchanger band 3, and glycophorin C (e.g., Refs.
3, 137, 138, 169, 195, 266, 267, 320, 341). Protein 4.1 and
glycophorin C form a high-affinity ternary complex in
vitro with the membrane-associated guanylate kinase
(MAGUK) p55 (266). Genetic interactions between human
glycophorin C and p55 are indicated by loss of these
proteins from membranes of red blood cells from a patient with a 4.1 deficiency (3).
Protein 4.1 interactions in red blood cells are regulated by phosphorylation and by interaction with calmodulin. Protein 4.1 is a substrate for protein kinases A and C,
casein kinase II, and an erythrocyte tyrosine kinase. In-
terestingly, phosphorylation by all these kinases results in
downregulation of 4.1 membrane- and/or spectrin-actin
binding activities (e.g., Refs. 50, 51, 74, 180, 245, 332, 376).
Calmodulin alone (independent of Ca2⫹) can inhibit spectrin-actin and membrane binding (251, 306, 379).
B) MAMMALIAN GENES FOR PROTEIN 4.1 AND DOMAIN STRUCTURES. The prototypical 4.1 from erythrocytes is now
known as 4.1R (R for red blood cell) and is encoded by
the EPB41 gene. Three other genes encode close human
and mouse homologs of 4.1R; these are EPB41L2,
EPB41L3, and EPB41L1 (328). The corresponding proteins are 4.1G, 4.1B, and 4.1N. mRNA encoding 4.1R is
most abundant in hematopoietic tissue such as bone marrow and fetal liver, but the mRNA is expressed at lower
levels elsewhere (328). 4.1G is so named because of its
very general expression as determined by multiple tissue
Northern blots (317, 328). 4.1B is expressed most prominently in brain (316), and 4.1N also in brain, most particularly in subpopulations of neurons (412). In Drosophila
and C. elegans, single 4.1 genes are present. The D. melanogaster 4.1 protein Coracle has been extensively analyzed, as will be described below, and gives clues to the
functions of 4.1 proteins in a genetic background not
complicated by multiple potentially redundant 4.1 genes.
Products of all four mammalian genes are modular
proteins, with three functional domains linked by regions
relatively unconserved between them (see Refs. 65, 316).
This appears to be a general vertebrate pattern for 4.1
proteins. The two most conserved modules are a domain
that binds erythrocyte membranes and MAGUKs; the
other is a domain at the COOH terminus. The membrane
binding domain is a FERM domain, a “genetically mobile”
module found in ezrin, radixin, merlin, and moesin as well
as certain tyrosine phosphatases, talin, and a variety of
other metazoan and plant proteins (57). The structure of
the human 4.1R FERM domain has recently been solved
(Fig. 8) and is revealed to have three lobes (160). Each
lobe has a fold recognizable in other proteins. Most NH2terminal is a ubiquitin-like fold; the central region of
sequence is a four helix bundle similar to acyl-CoA binding protein; the COOH-terminal lobe is similar to the
PH/phosphotyrosine binding protein superfamily fold. Although the three lobes in 4.1R FERM domain appear to be
independent folding units, they have extensive contacts
and are suggested not to function independently. Interestingly, this structure reveals that individual lobes are regulated by differential mRNA splicing. Conboy et al. (69)
and Huang et al. (188) found that a common differential
mRNA splicing event is the excision of exons 4 and 5,
especially in brain and endothelial cell mRNAs. This
splice is predicted to remove most of the first lobe of the
FERM domain. Although the functional consequence of
loss of the ubiquitin-like domain has not yet been identified, mutational analysis of the D. melanogaster 4.1 pro-
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
Association of spectrins with F-actin is relatively
weak with measured KD values ranging from 6 ␮M for
recombinant ␤2-spectrin (238) to 250 ␮M for native erythrocyte dimer (309). In fact, spectrin most likely requires
accessory proteins in vivo to initiate assembly and to
stabilize actin interactions. Advantages of a low-affinity
spectrin-actin interaction with the potential for enhancement by accessory proteins include the ability to selectively stabilize spectrin-actin networks in specific locations, as well as providing an additional level of
regulation. Protein 4.1 (123, 400, 401) and adducin (23,
134, 135) have been identified in erythrocyte membranes
based on their activities in stabilizing spectrin-actin complexes. These proteins have been localized at the spectrinactin junction in erythrocyte membranes (91), as anticipated from their association with spectrin and actin in
vitro.
Volume 81
July 2001
SPECTRIN AND ANKYRIN-BASED PATHWAYS
1369
tein Coracle indicates that this eliminates membrane
binding activity (418).
The FERM domain is associated with a wide variety
of ligand binding activities including membrane proteins
(234), membrane lipids (especially acidic lipids and their
head groups) (114, 139), calmodulin (306), and 2,3-diphosphoglycerate (295). In cells other than red blood cells,
FERM domains interact with numerous transmembrane
proteins, including adhesion molecules of the neurexin
family, the heparan sulfate proteoglycans CD44 (206) and
syndecan (61), and a swelling-induced chloride conductance regulatory protein plCln (380). Neurexins and syndecans are discussed in more detail in section IIIG. The
structure of the 4.1R FERM domain and the recent structures of moesin (322) and radixin (158) FERM domains
illuminate the nature of the interactions. Band 3 (the
anion exchanger AE1) binds at a sequence LEEDY (195)
within the most NH2-terminal lobe. A region that binds
glycophorin C (and probably NCP neurexins and syndecan 2) is present on the central lobe (307). The binding
site for the MAGUK p55 (266) is present in the most
COOH-terminal lobe. The major protein binding sites are
therefore in separate lobes, so possibly differential splicing gives proteins that can (for example) bind distinct
classes of membrane proteins. A mechanism underlying
4.1 binding to a number of small phosphorylated ligands
such as inositol phosphates (114) or 2,3-diphosphoglycerate (295) may be indicated by the radixin structure. This
contains a binding site for IP3 at the junction of the NH2and COOH-terminal lobes: this site is composed of several
basic residues at the cleft between the two lobes. In 4.1R,
a deep cleft between the two lobes is surrounded by basic
residues, indicating the possible existence of a ligandbinding site.
The COOH-terminal domain (CTD) is the most diagnostic domain of 4.1 proteins. Whereas the FERM domain
is widespread, and the spectrin-actin binding domain not
highly conserved, the CTD is preserved among different
4.1 proteins. It is recognizable in D. melanogaster and C.
elegans 4.1 homologs, always in association with a FERM
domain. The CTD has not yet been found outside metazoa, indicating that like spectrin and ankyrin, 4.1 is an
animal protein. The COOH-terminal domain has been reported to bind the nuclear mitotic apparatus protein
NuMA and the immunophilin FKBP13 (413). Although a
4.1N-NuMA interaction is indicated to mediate some effects of nerve growth factor on pheochromocytoma
(PC12) cells (437), there is no evidence from D. melanogaster genetics of an essential role for 4.1 proteins in
neuronal differentiation (119).
The spectrin-actin binding (SAB) domain is well
characterized in human 4.1R. A 10-kDa fragment pro-
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
FIG. 8. Mammalian 4.1 proteins. Left: the four mammalian proteins are known as 4.1R, 4.1G, 4.1N, and 4.1B. They are
modular proteins, assembled from common domains linked by unconserved sequences. The common domains are a
FERM domain (a protein and lipid binding module common to ezrin, radixin, moesin, and some other proteins, Ref. 57);
a domain that binds spectrin and actin (the SAB domain, found only in vertebrate 4.1s so far); and a COOH-terminal
domain (CTD) common to all 4.1 proteins. [Modified from Parra et al. (316).] Right: the structure of the FERM domain
of moesin (PDB entry 1EF1A; Ref. 322). The 4.1 proteins have FERM domains homologous to the equivalent domains in
the ERM proteins. The figure shows the FERM domain of human moesin. Although the sequences of the human 4.1R and
moesin proteins are only ⬃32% identical, they probably fold very similarly. The FERM domain has three lobes. Lobe 1
(the NH2-terminal lobe) is similar to the fold of ubiquitin; the fold of lobe 2 is similar to that of the acyl-CoA binding
protein; and the lobe 3 fold is similar to that of the pleckstrin homology domain (from the PDB file 1EF1; Ref. 322).
1370
VANN BENNETT AND ANTHONY J. BAINES
part of the SAB domain, nuclear functions, like SAB,
probably evolved with vertebrates and are not conserved
among 4.1 proteins.
Coracle is required for correct functioning of at least
two D. melanogaster transmembrane proteins, the epidermal growth factor (EGF) receptor (119) and the cell adhesion molecule neurexin IV (418). Coracle mutations
suppress a hypermorphic allele of the EGF receptor ellipse, indicating that full expression of EGF receptor
activity requires Coracle. Coracle deficiency results in
partial loss of adult cuticular structures. A possible mechanism for this phenotype is that EGF receptor function is
compromised in flies with Coracle deficiency, resulting in
failure of cell proliferation in imaginal epithelia (226).
Neurexin and Coracle depend on each other for correct localization at the septate junction, and physically
interact since they coimmunoprecipitate from fly extracts
(7, 418). The coracle FERM domain is both necessary and
sufficient for correct localization of neurexin (418). The
mechanism underlying this is unclear but may require the
MAGUK disks lost (DLT; Ref. 27). DLT is essential for cell
polarity and is required for correct localization of neurexin IV at septate junctions. By analogy with the erythrocyte 4.1/p55/glycophorin C complex, it seems possible
that a ternary coracle/DLT/neurexin IV complex may form
at septate junctions, stabilizing the incorporation the
CAM at the junction, but at the time of writing, no published account has tested the existence of such a complex. Correct incorporation of neurexin in these junctions
seems to be essential for creation of the transepithelial barrier at the septate junction. In this context a recently described interaction of mammalian 4.1R with the tight junction protein ZO-2 (270) may indicate a general role for 4.1
proteins in barrier functions in vertebrates as well as flies.
D) FUNCTIONS OF MAMMALIAN 4.1R REVEALED BY GENETICS.
Deficiency or functional inactivation of erythrocyte 4.1R
has been linked for some years with the disease hereditary elliptocytosis (HE). Surprisingly, in view of the widespread expression of 4.1R mRNA, no other tissue in these
patients has been reported to be affected, even in patients
suffering total loss of 4.1R from their red blood cells. For
a more detailed analysis of 4.1R mutations in HE, readers
are referred to comprehensive reviews in this speciality,
e.g., Reference 87. Characterization of numerous mutations in the 4.1R gene has revealed the importance of the
various domains in erythrocyte function. Several mutations have been reported within the SAB domain and
confirm that spectrin-4.1-actin interaction is essential for
normal erythrocyte function. Loss of one or more exons
encoding this domain, or mutation of a critical lysine
residue in this domain, gives markedly reduced mechanical stability and deformability (66, 70, 95, 253, 265). These
mutations are also typically associated with some spectrin loss from the membrane. These cases of HE are
associated with anemia, in contrast to cases where the
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
duced in limited chrymotryptic digests of 4.1 promoted
spectrin-actin interaction (71, 72). This is encoded by
exons 16 and 17 (98, 99, 179). Exon 17 encodes sequence
required for the interaction with spectrin and actin, but
high-affinity binding is only achieved if sequence encoded
by exon 16 is present too. Because exon 16 expression is
upregulated during erythroblast differentiation, high-affinity SAB is effectively a gain of function during erythrocyte differentiation. This indicates that SAB may not be a
“core” function of the 4.1 proteins, a suggestion supported
by the lack of known SAB activity in D. melanogaster
Coracle. 4.1G has close conservation of the 4.1R SAB and
in vitro promotes spectrin-actin interactions (144). 4.1B
also promotes SAB (316). 4.1N does not have an especially well-conserved SAB domain, which is identical to
4.1R in only 26 of 66 residues.
More than one start codon has been identified in 4.1R
mRNAs. Erythrocyte 4.1 is predominantly an 80-kDa form.
The corresponding mRNA has a 17 nucleotide exon in the
5⬘-region (exon 2⬘) excised and translation starts at a
codon in a 210 nucleotide exon (exon 4) (188). If the 17
nucleotide exon is expressed, translation starts within
this exon yielding 135-kDa isoforms typical of nonerythroid 4.1R proteins. The extra NH2-terminal sequence contains a calmodulin-binding site (209, 230). So far, only one
translation initiation codon has been identified in 4.1B,
4.1G, and 4.1N, equivalent to the one that yields the
135-kDa form of 4.1R. However, as with 4.1R, these other
proteins are encoded by mRNAs that are extensively
spliced.
C) CORACLE, THE 4.1 PROTEIN IN D. MELANOGASTER. Fehon et
al. (119) have found a FERM domain protein in D. melanogaster they named Coracle. Coracle is distinguished as
a 4.1 protein by the presence of a conserved 4.1 CTD,
although there is little sequence evidence of a functional
SAB. Mutation of the Coracle gene resulted in defects in
dorsal closure; homozygotes are embryonic lethal.
Coracle does not obviously colocalize with either spectrin
or ankyrin in fly epithelial cells. Together with the lack of
an obvious SAB sequence, this indicates that coracle is
not a SAB protein. However, the sequence relationship of
the FERM and CTD regions of Coracle with mammalian
4.1 proteins provides a useful guide to their core functions.
Coracle is selectively localized at pleated septate
junctions of ectodermally derived epithelial cells. Smooth
septate junctions of, for example, the midgut do not express coracle. This contrasts again with the very wide
distribution of spectrin and actin in the fly. It is also clear
that there is no Coracle in fly nuclei. Nonerythroid 4.1
proteins of mammals have been found in nuclei, in association with NuMA (271) and spliceosomes (84). Nuclear
import requires sequence encoded by exon 16 of mammalian 4.1 (138), which is not present in Coracle. Because
exon 16 encodes both a nuclear localization sequence and
Volume 81
July 2001
SPECTRIN AND ANKYRIN-BASED PATHWAYS
2. Adducin
Adducin (from the Latin adducere, to draw together)
forms ternary complexes between spectrin and actin and
promotes association of spectrin with actin filaments (21,
135). Although adducin associates directly with the sides
(285, 382) as well as fast-growing ends of actin filaments
(223), adducin complexes with the combination of spectrin and actin at 5- to 20-fold lower concentrations than
with actin alone (239). Adducin exhibits the highest affinity for complexes between spectrin and the fast-growing
ends of actin filaments, which form with an association
constant of 15 nM adducin (239). The relative activities of
adducin for actin filament ends and sides in the presence
and absence of spectrin suggest that the preferred role of
adducin in cells is to form a complex with the fastgrowing ends of actin filaments that recruits spectrin and
prevents addition or loss of actin subunits (Fig. 9). Adducin thus is an actin-capping protein that recruits other
proteins to actin filament ends and could represent a new
class of assembly factor with the function of integrating
actin into other cell structures.
Adducin interactions with spectrin and actin are mediated by a COOH-terminal basic stretch of residues with
homology to the MARCKS family (myristolated alanine
rich C kinase substrate; Refs. 29, 298) (Fig. 9). The adducin MARCKS-related domain also contains the site of
phosphorylation by protein kinase C at the RTPS-serine
(268, 269) as well as for binding of Ca2⫹/calmodulin (135,
268). Adducin activities involving spectrin and actin are
all inhibited by Ca2⫹/calmodulin (135, 223) and by phosphorylation by protein kinase C (269). Antibodies specific
for RTPS-serine phosphorylated adducin reveal that adducin is an in vivo substrate for phorbol ester-activated
protein kinases that presumably include members of the
protein kinase C family (269). Gelsolin competes for adducin recruitment of spectrin to the fast-growing ends of
actin filaments (298) and may provide yet another means
of regulating adducin activity.
Adducin has been identified by Kaibuchi and colleagues (213) as a substrate for the Rho-dependent protein kinase at a site distinct from protein kinase C. Experiments with mutated forms of adducin lacking the
Rho-phosphorylation site suggest that Rho-dependent
phosphorylation of adducin plays an essential role in
promoting cell motility (130).
A) ADDUCIN DOMAINS, SUBUNITS, AND QUATERNARY STRUCTURE.
Adducin in humans and rodents includes three closely
related subunits termed ␣, ␤, and ␥ (102, 134, 201). Adducin subunits all contain an NH2-terminal globular domain
that participates in subunit interactions, a neck domain
that also promotes formation of oligomers, an unstructured tail domain, and finally a COOH-terminal MARCKShomology stretch containing the spectrin/actin interaction site as well as targets for calmodulin and protein
kinase C (Fig. 9). Erythrocyte adducin is comprised of
␣/␤-subunits in a 1:1 ratio, and in other cells include ␣/␥ as
well as ␣/␤ combinations of subunits (102, 134). Hydrodynamic and cross-linking experiments with erythrocyte
and brain adducins (134, 189, 200) support a physical
model of adducin as a mixture of dimers and tetramers
comprised of ball and chain-shaped subunits associated
via their globular domains with tail domains extending
into solution (Fig. 9). Adducin is represented by single
genes in C. elegans and D. melanogaster (see below) and
presumably exists as either homodimer or hometetramer
in these organisms.
Sites of interactions between adducin subunits include an oligomerization region within the neck domain
(298), as well as undefined sites in the globular head
domains (189). Mechanism(s) operate in vertebrate adducins that somehow ensure assembly of adducin heteromers with 1:1 ratios of ␣- and ␤-subunits or ␣- and ␥-subunits. One possibility is that one subunit is synthesized in
limiting amounts and monomeric subunits in excess of
available partners are degraded. However, recombinant
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
mutations are in the CTD. A very mild phenotype has been
reported in a homozygous HE family in which there are
mutations in the CTD (294). In these patients, 4.1R assembles correctly in the membrane skeleton, indicating this
domain is not required for any of the classical interactions
of 4.1 at the membrane. The relationship of the mutations
to the phenotype remains elusive.
So far, the only mouse 4.1 gene to be knocked out is
4.1R (361). Unlike coracle deficiency, loss of 4.1R is not
lethal. Indeed, the phenotype is surprisingly mild. However, functional redundancy among the products of the
four 4.1 genes could mask potential defects. As would be
expected, 4.1R-deficient mice suffer hemolytic anemia.
The membranes of erythrocytes are misshapen and frequently fragmented. There is a loss of ⬃30% of spectrin
from the membranes, complete loss of p55, and ⬎80% loss
of glycophorin C, i.e., 4.1 is required for stable accumulation of the latter two proteins in erythrocyte membranes. However, platelet function is generally normal,
and (at a gross level) the mice are generally unaffected.
Nevertheless, subtler defects are manifest in the nervous
system. 4.1R null mice exhibit deficits in learning, coordination, balance, and movement (413). The mechanism
underlying these defects is unclear, but they fit with the
normal pattern of expression of 4.1R mRNA, especially in
the dentate gyrus and cerebellum.
Like Coracle and neurexin IV, glycophorin and 4.1
have an interdependent relationship for accumulation at
the membrane. Leach elliptocytosis results from mutations in the glycophorin C gene (384) and is associated
with loss of both 4.1R and p55 from the erythrocyte
membrane (3).
1371
1372
VANN BENNETT AND ANTHONY J. BAINES
Volume 81
adducin constructs can form homodimers, suggesting additional aspects of regulation.
All of the spectrin/actin association activities of the
adducin are reproduced with recombinant polypeptides
containing the neck, tail, and MARCKS domains (298).
Tail/MARCKS and neck/tail domains are inactive. These
results and oligomerization activity of the neck domain
(see above) suggest that the active form is a parallel dimer
with dimerization provided by the neck domain and actin
contacts provided by the MARCKS domain (298).
The role of adducin globular domains beyond promoting subunit interactions currently is a mystery. As
noted above, the neck and tail domains of adducin are
necessary and sufficient for all activities involving spectrin and actin (298). The issue of undiscovered roles for
the head domain is particularly relevant in view of the
high degree of conservation of this domain and the exis-
tence of alternatively spliced variants of adducin lacking
the COOH-terminal MARCKS-related domain (see below).
A possible clue to the head domain function is a similarity
in primary sequence to an E. coli enzyme, aldolphosphofructohydrolase, which requires zinc for catalytic activity.
Histidines involved in zinc coordination are conserved in
adducin, although an aspartic acid residue required for
catalytic activity is missing. These considerations suggest
that adducin lacks enzymatic activity but may require zinc
for function of the globular domain. A corollary of a
requirement for zinc is that adducin may have been at
least partially inactive in previous biochemical experiments using adducin isolated with the metal chelator
EDTA (134).
B) GENES AND ALTERNATIVELY SPLICED VARIANTS. Adducins,
like spectrins, are, so far at least, found only in metazoans, although one of the adducin domains is expressed in
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
FIG. 9. Schematic models for adducin domain
organization (A) and adducin association with
spectrin and actin (B). A: folded domains of adducin deduced from limited proteolytic digestion
(189, 200); sites are indicated for phosphorylation
by protein kinase C and rho-kinase and association
with calcium/calmodulin. Amino acid sequences of
conserved COOH-terminal MARCKS homology domains of ␣-, ␤-, and ␥-adducins are noted. B: conformation of adducin dimers and tetramers (189),
and summary of modes of association of adducin
with spectrin and actin. Adducin associates with
the sides and fast-growing ends of actin filaments
both alone and with enhanced affinity in the presence of spectrin. The preferred mode of association
of adducin, spectrin, and F-actin occurs in a ternary
complex at the fast-growing ends of actin filaments
(239).
July 2001
SPECTRIN AND ANKYRIN-BASED PATHWAYS
III. PHYSIOLOGICAL FUNCTIONS
A. Overview
Hypotheses for roles of spectrin and ankyrin-related
genes have been generated through the classic maneuvers
of determining cellular localization and interacting proteins. Manipulation of gene expression in animal models
recently has provided critical tests of these hypotheses
and provided new functions as well. The purpose of this
section is to briefly summarize proposals for function
based on localization and protein neighbors, and then to
critically evaluate these ideas in the physiological context
of animals.
Initial efforts to explore functions of spectrin in cultured fibroblasts by microinjection of spectrin antibodies
resulted in aggregation and collapse of intermediate filaments in cells that otherwise were normal (264). These
findings suggested that spectrin was not likely to be required for survival of fibroblasts in culture, and apparently was not essential for fundamental metabolic processes. Subsequently, spectrin and ankyrin were
discovered to be localized at basolateral domains of a
variety of epithelial tissues (103, 104, 302). These morphological observations were accompanied by Nelson’s dis-
covery (304) that ankyrin associated with the Na⫹-K⫹ATPase, which is well known to be localized at
basolateral domains of many epithelial tissues. Nelson
and colleagues (277) subsequently discovered that expression of E-cadherin in fibroblasts promoted cell-cell
contact and recruited spectrin to sites of cell-cell contact.
These investigators also characterized a complex of spectrin, cadherin, and ankyrin that formed during assembly
of cultured Madin-Darby canine kidney (MDCK) cells into
epithelial sheets (301). Moreover, overexpression of
ankyrin-binding and actin-binding domains of ␤-spectrin
resulted in highly abnormal cells lacking polarized distribution of the Na⫹-K⫹-ATPase (92, 186). Taken together,
these and other observations have led to a widely accepted view that spectrin and ankyrin have a role either in
the initial formation of epithelial cell polarity immediately
following cadherin-based cell adhesion, or in maintenance of this polarity (438).
Spectrin and ankyrin have also been implicated in
intracellular roles involving the Golgi (11, 88). The concept of a Golgi-based spectrin skeleton is based on the
observation of immunoreactive forms of ␤-spectrin (9,
372) and ankyrin (10, 93, 327) associated with Golgi membranes. The finding that lysosomal membranes contain a
120-kDa ankyrin-G spliced form suggests other organelles
also utilize ankyrin and/or spectrin (178). Moreover, spectrin is associated with dynactin, an organelle-based microtubule motor, in cells overexpressing ARP2 (176). Evidence for function of spectrin in post-Golgi targeting is
based on disruption of polarized delivery of the Na⫹-K⫹ATPase in cultured MDCK cells overexpressing of the
NH2-terminal portion of ␤-spectrin (92).
Spectrin has been proposed to participate in axonal
transport based on the observation by Willard and colleagues (237) that spectrin (named fodrin by this group)
was present in multiple classes of axonally transported
proteins and organelles. Spectrin also has been suggested
to participate in tethering synaptic vesicles within nerve
terminals (363). This hypothesis is based on association
of spectrin with synaptic vesicle proteins synapsin (365)
and MUNC 13 (354) and observations of filament-shaped
structures the size of spectrin associated with synapses
(156).
D. melanogaster and C. elegans with their simplified
and completely characterized genomes provide model
systems to test function in the context of living animals.
The spectrin repertoire of both organisms is comprised of
three genes encoding one ␣, one generally expressed ␤
(␤-G), and one ␤-H subunit (Table 1). The pattern of
expression of spectrins in these organisms closely parallels their behavior in vertebrates. ␤-G spectrin of C. elegans is localized at sites of cell-cell contact beginning at
the two-cell embryo and continuing in adult epithelial
tissues (292). ␤-G spectrin in adult C. elegans, as in vertebrates, is most highly expressed in the nervous system
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
E. coli (see above). The C. elegans and D. melanogaster
genomes each contains a single adducin gene. Adducin in
D. melanogaster was discovered as a transposon-induced
mutation in oocyte development termed huli-tai shao or
hts (too-little nursing) (423, 444, 445). Adducin of vertebrates is encoded by three closely related genes termed
␣-, ␤-, and ␥-adducins (102, 201). ␣-Adducin is expressed
in most tissues, while ␤-adducin has a more restricted
pattern of expression limited to brain and hematopoietic
cells (201). ␥-Adducin is a likely companion for ␣-adducin
in cells lacking the ␤-subunit (102).
Alternative splicing is a general feature of metazoan
adducins. The D. melanogaster adducin gene encodes
four alternatively spliced forms, including one with a
basic COOH-terminal domain, which is expressed in somatic cells (423). Other D. melanogaster adducin transcripts in germ line cells lack the MARCKS-related domain and have distinct 3⬘-UTR regions resulting in
selective targeting of mRNAs within oocytes and nurse
cells (423). The C. elegans adducin gene encodes two
transcripts, one containing a MARCKS-related domain
and the other with a distinct sequence (S. Moorthy and V.
Bennett, unpublished data). In vertebrates, ␣- (241), ␤(389, 377) and ␥-adducins (60) also include variant transcripts lacking the COOH-terminal MARCKS-related domains. Other alternatively spliced versions of adducin
subunits have predicted internal deletions within the tail
as well as the head domain.
1373
1374
VANN BENNETT AND ANTHONY J. BAINES
B. Stabilization of Cell Surface Membranes
at Sites of Cell-Cell Contacts
A unifying role for ␣/␤-G spectrin emerging from
results with D. melanogaster and C. elegans is the stabi-
lization of cell-cell and cell-matrix attachments after these
structures have formed. The requirement for ␣/␤-G spectrin becomes most apparent in the larval stage when the
animals leave the supportive environment of the egg case
and presumably generate mechanical stress on their tissues during movement. Evidence for this idea is that
defects in muscle and nervous system are reduced in
unc-54 worms that exhibit reduced movement due to a
myosin mutation (159). Moreover, D. melanogaster
␣-spectrin null larvae that had successfully completed
embryonic morphogenesis and established normal cell
polarity subsequently exhibited loss of cell-cell contact
and cell-substratum contacts (232).
Conditional mutations have allowed evaluation of
spectrin function in the developing Drosophila ovary,
which is surrounded by a follicular epithelium. This epithelial layer is subjected to stress during oogenesis, especially when yolk formation in the oocyte requires an
increase in egg volume. ␣-Spectrin has been eliminated
selectively in follicular cells through site-specific recombination by the FLP/FRT system (231) and in both follicular cells and germ cells through rescue of ␣-spectrin null
animals with a temperature-sensitive mutation (89). In
either case, loss of ␣-spectrin resulted in disruption of the
follicular cell monolayer and changes in cell shape (89,
231). The absence of ␣-spectrin would be expected to
disrupt both laterally associated ␣/␤-G as well as apical
␣/␤-H complexes. Observations of ␤-H-deficient eggs suggest that the loss of ␣/␤-G is responsible for disruption of
the monolayer while loss of ␣/␤-H causes changes in cell
shape (446) (see below). ␤-Spectrin mutations in D. melanogaster have recently been characterized as embryonic
lethal, precluding a direct evaluation of ␣/␤ contribution
to follicular monolayer integrity (109). However, it will be
of interest in future work to target ␤-spectrin deletion to
follicular cells using the FLP/FRT technique.
Activity of ␣/␤ and ␣/␤-H spectrin in stabilizing cellcell contacts and in achieving normal columnar epithelial
cell shape requires formation of tetramers. The best evidence for spectrin tetramers as a functional unit is that
either a deletion or a point mutation of ␣-spectrin that
abolish head-head ␣-␤ interactions also prevent rescue of
␣-spectrin null D. melanogaster (89). The requirement for
spectrin tetramers suggests that cross-linking of actin
filaments is an important aspect of spectrin function and,
moreover, suggests an essential role for a spectrin-actin
network as resolved in erythrocytes. Coupling of cell
adhesion molecules to a spectrin lattice could be anticipated to promote their interaction with molecules on
adjacent cells through restricted diffusion and through
formation of closely spaced complexes linked to dual
sites on spectrin tetramers, thereby increasing the effective local concentration. Also, mechanical coupling of a
large number of adhesion molecules on both cytoplasmic
surfaces at sites of cell-cell contact into a transcellular
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
in axons as well as cell bodies and is localized in muscle
cells at sites of contact with the extracellular matrix
(292). ␤-H spectrin of D. melanogaster is localized in
apical domains of epithelial cells and is in an equivalent
location to ␤TW of chickens (85, 108, 147, 388).
Several observations, based on consequences of disruption of expression of spectrin genes in C. elegans and
Drosophila, suggest that spectrin, although essential for
survival beyond the embryonic stage, actually is not required for initial development of epithelial polarity, Golgi
function, axonal transport, or accumulation of synaptic
vesicles at presynaptic structures. Normal development
of epithelial polarity and normal progression of morphogenesis as well as embryonic viability occurs in C. elegans
in the absence of ␤-G spectrin (159, 292). Moreover, ␤-G
spectrin was not required for neuronal migration or axon
guidance of dorsal axons, for assembly of synaptic vesicles at nerve endings, or for polarized secretion of collagen. In addition, the plasma membranes of epithelial cells,
muscle, and neurons were normal in appearance in electron micrographs, with no evidence for a generalized loss
of membrane integrity. Equivalent phenotypes were observed with deficiency of ␤-G spectrin as a consequence
of either injection of double-stranded RNA (292) or a null
allele of unc-70 (159). Normal epithelial polarity in
␣-spectrin null mutants also has been observed in D.
melanogaster larvae (89, 232). Interpretation of these
findings was complicated by the possible contributions of
maternal spectrin (232, 325). However, the principal period of spectrin expression in both organisms occurs
during gastrulation, and spectrin synthesized at this time
is most likely not of maternal origin. Moreover, doublestranded RNA injection in C. elegans reduces levels of ␤-G
spectrin to ⬍2% of normal in early embryos.
In summary, direct evaluation of spectrin function in
C. elegans and D. melanogaster has raised serious doubts
regarding the previously hypothesized roles in maintenance of plasma membrane integrity, establishing cell
polarity, Golgi-related roles, axonal transport, and a structural role in the synapse. Nevertheless, all is not well for
spectrin mutants, which are lethally affected or subviable
as larvae and exhibit multiple defects.
For example, unc-70 worms that do survive under
specialized culture conditions (159) are paralyzed and
uncoordinated and exhibit loss of sarcomere structure as
well as abnormal axon extension. The elucidation of functions for spectrin, ankyrin, as well as their associated
proteins in animal models is just beginning, and these
results are summarized below.
Volume 81
July 2001
SPECTRIN AND ANKYRIN-BASED PATHWAYS
1375
and laterally integrated unit would be anticipated to create resistance to deformation in addition to enhanced
adhesion.
The molecular composition of the proposed transcellular cell adhesion molecule(s)/spectrin-actin network remains to be established but may involve ankyrin as well as
ankyrin-independent interactions. Ankyrin associates
with cell adhesion molecules in the L1 CAM family, which
are localized at sites of cell-cell contact of all cells in C.
elegans (Chen and Bennett, unpublished data) (see
above). Another candidate for a spectrin “receptor” is
NCAM, a member of the Ig superfamily of cell adhesion
molecules which associates directly with spectrin (337).
suggests that ␤-H spectrin physically interacts with adherens junction components and couples these to the
actomyosin contractile system. Initial targeting of ␤-H
spectrin to apical domains requires ␣-spectrin, while targeting of ␣-spectrin requires ␤-H spectrin (231, 446). The
“receptor” for ␣/␤-H spectrin thus requires both subunits.
The identity of molecular partners for ␤-H spectrin remains to be determined. DE cadherins or associated proteins are candidates for such an activity based on colocalization (388).
C. Cell Sheet Morphogenesis
Clustering of voltage-gated Na⫹ channels (NaCh) at
axon initial segments, nodes of Ranvier, and postsynaptic
folds of the neuromuscular junction is vital for generating
sufficient local current to overwhelm membrane capacitance and resistance and to initiate sufficient depolarization for effective signaling. Several clues point to a role of
ankyrin in NaCh clustering. Ankyrin and the NaCh copurify and associate in vitro as well as in cell models (263,
369). Moreover, 480/270-kDa isoforms of ankyrin-G are
localized at axon initial segments and nodes of Ranvier
(219), sites where the existence of high density of NaCh
has been well documented (48, 404, 430). As yet undefined
ankyrin-G spliced forms also colocalize with NaCh in
postsynaptic folds of mammalian neuromuscular junctions (122, 220, 431). Ankyrin-G also is associated with
NaCh clusters in the dystrophic mouse in regions lacking
myelination (86), as well as clusters of NaCh induced in
cultured retinal ganglion neurons (205). ␤ II spectrin, in
contrast to ankyrin-G, occurs along the length of axons,
does not exhibit increased density at nodes of Ranvier
(390) or axon initial segments (449), and thus is not likely
to play a role in NaCh localization. However, ␤ IV spectrin
is colocalized with 480/270-kDa ankyrin-G at nodal and
initial axon segments in the central and peripheral nervous system (24). Ankyrin-G and ␤ IV spectrin thus define
specialized domains within a more general spectrin-actin
network.
Evidence that ankyrin is required for localization of
both Na⫹ channels and L1 CAM at axon initial segments is
based on targeted knock-out of ankyrin-G expression in
the postnatal cerebellum of mice (453). Mutant mice lacking cerebellar 480/270-kDa ankyrin-G developed characteristic cerebellar defects with symptoms of abnormal
gait and tremor, and reduced locomotion that became
more severe with increasing age. Electrophysiological
data from analysis of cerebellar slices demonstrate that
Purkinje cell neurons of mutant mice were unable to fire
normal action potentials (453). Na⫹ channels were not
concentrated at initial segments of granule cells from
mutant mice (453). Neurofascin and NrCAM also exhib-
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
Creation of tissues and early embryos from ensembles of cells requires mechanisms for coordinating forces
generated in individual cells to produce morphogenesis.
For example, the developmental processes of embryonic
elongation of C. elegans, dorsal closure in Drosophila,
formation of enclosed epithelial tissues in metazoans, and
neurulation in vertebrates all involve transcellular transmission of forces resulting from constriction of apically
associated actin filaments. ␤-H spectrin has been implicated in these processes based on the phenotypes of ␤-H
mutations in C. elegans (276) and in D. melanogaster
(388, 446).
␤-H spectrin mutants in C. elegans, termed sma-1 for
their small size, are defective in embryonic elongation
(276). Normally, the epidermal actin filaments reorganize
soon after ventral enclosure to form an array oriented
around the circumference of the embryo. Contraction of
these actin fibers is accompanied by elongation of the
embryo. ␤-H mutant worms elongate at only 20% of the
rate of wild type and attain only half the final length (276).
Moreover, these mutants also exhibit defective morphogenesis of the pharynx and excretory cell. ␤-H mutant
worms, although mishaped, are viable.
␤-H localization and the phenotype of ␤-H mutations
in D. melanogaster provide strong evidence for a role in
epithelial morphogenesis. ␤-H spectrin is expressed in
most epithelial tissues and is restricted to apical cell
domains where it is present associated with zonula adherens as well as the brush border (231, 386, 388, 446).
Mutations of ␤-H result in extensive larval lethality as well
as defective eye morphology, wing morphogenesis, and
tracheal defects resulting in loss of hemolymph (388). In
addition, follicular epithelial cells lining egg chambers do
not undergo apical constriction but instead remain cuboidal (446).
Adherens junctions are abnormal in ␤-H mutant follicular epithelial cells (446). This observation combined
with lack of shape change in ␤-H deficient epithelial cells
D. Assembly of Voltage-Gated Naⴙ Channel-Rich
Domains in Excitable Cells
1376
VANN BENNETT AND ANTHONY J. BAINES
precede ankyrin-G and are candidates to either recruit or
stabilize ankyrin-G at these sites (228). Further observations at later stages of sciatic nerve development suggest
pairs of ankyrin/neurofascin/NrCAM/Na⫹ channel clusters fuse to form the mature node of Ranvier (228). Interestingly, similar clusters of Na⫹ channels have been reported in remyelinating peripheral nerve (111), which
may represent a reversion to an earlier developmental
stage of the axon as a response to injury.
Ankyrin-based clusters of Na⫹ channels persist in
Trembler mutant mice with defective myelin (228). These
clusters could have partial function in saltatory conduction of the action potential and could explain the relatively mild phenotype of trembler and the related disease
of Marie-Charcot-Tooth type 1A in humans.
The molecular events leading to assembly of ankyrinG and other components at nodes of Ranvier and axon
initial segments remain to be deciphered. Neurofascin
and NrCAM have been proposed to direct assembly, first,
of ankyrin at nodes of Ranvier and other sites, followed
by the localization of Na⫹ channels (228). However, the
finding that neurofascin is not distributed normally in the
absence of ankyrin-G (453) suggests that ankyrin-G is
required for the concentration of neurofascin as well as
the Na⫹ channel, at least at axon initial segments. The
domains required for restriction of GFP-tagged 270-kDa
ankyrin-G to axon initial segments of cultured dorsal root
ganglion neurons have been examined with the conclusion that the membrane-binding domain is not sufficient
(449). Moreover, both the spectrin-binding and tail domains of ankyrin-G exhibit activity in targeting GFP constructs to the plasma membrane of neurons. These findings suggest that ankyrin-G functions as an integrated unit
requiring all of its domains and imply unidentified
ankyrin-binding protein(s) upstream in the pathway to
formation of axon initial segments.
E. Targeting of Ca2-Release Channels to the Ca2ⴙ
Compartment of the ER
The ER of metazoan cells is a continuous system of
intracellular membranes segregated into subdomains
with specialized functions (407). One ER compartment
with particular relevance for cell signaling is devoted to
Ca2⫹ homeostasis and is a major determinant of intracellular Ca2⫹ levels (278). An ER Ca2⫹ homeostasis compartment is present in many cell types but is morphologically
best defined in striated muscle and the nervous system
(26, 129, 171). The ER Ca2⫹ compartment of striated
muscle, termed the sarcoplasmic reticulum (SR), is intimately integrated with sarcomeres. In heart muscle, this
configuration allows generation of Ca2⫹ waves that generate periodic cycles of contraction and relaxation by the
contractile apparatus. Ryanodine receptors mediate Ca2⫹
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
ited loss of restriction to axon initial segments of Purkinje
neurons of ankyrin-G mutant mice (453). It will be of
interest to determine if ankyrin-G also is required for
targeting of ␤ IV spectrin to axon initial segments.
Similarities between nodes of Ranvier and other sites
of Na⫹ channel concentration support the prediction that
ankyrin-G also is required for restriction of Na⫹ channels
at these domains. Nodes of Ranvier, the neuromuscular
junction, and axon initial segments are each highly specialized, suggesting the likely possibility that Na⫹ channel/ankyrin-G assemblies will include additional proteins
that perform specific functions adapted to each cell domain. Syntrophins, for example, associate with Na⫹ channels and are concentrated at the neuromuscular junction
(140, 357). The ␤-subunits of Na⫹ channels with their
extracellular Ig domains may also be candidates to mediate important domain-specific interactions as well as provide a linkage with ankyrin (263).
Ankyrin-G could form heterocomplexes between
Na⫹ channels and neurofascin in vivo by directly binding
to both proteins, since the ankyrin membrane-binding
domain has distinct binding sites for the Na⫹ channel,
located on subdomains 3 and 4 (370), and for neurofascin,
located on subdomains 2 and 3 as well as 3 and 4 (282)
(Fig. 7). Possible physiological roles for such Na⫹ channel/adhesion molecule/ankyrin complexes include directing extracellular interactions with adjacent cells to sites
of channel clusters. Another role of coclustering of Na⫹
channels with neurofascin/NrCAM could be related to the
fact that L1 CAM family members are palmitoylated at a
conserved cysteine site in their membrane-spanning segments (342). A high density of neurofascin-linked palmitoyl groups (likely to be in the range of 1,000 molecules/
␮m2) could perturb the local organization of phospholipids.
Potential consequences could be recruitment of palmitoylated or lipid-modified signaling proteins and an increased
membrane viscosity. It is pertinent in this regard that
evidence of a diffusion barrier for glycophosphatidylinositol (GPI)-linked proteins and membrane lipids at axon
initial segments has been reported, suggesting some difference in the lipid domain at this site (425).
Ankyrin-G, neurofascin, and the voltage-dependent
Na⫹ channel cosegregate into axon microdomains early
in the morphogenesis of the node of Ranvier (228). The
480-kDa ankyrin-G and 440-kDa ankyrin-B are coexpressed in premyelinated dorsal root ganglion axons at
early stages of axonal growth during embryonic life. However, at the onset of myelination in the sciatic nerve,
440-kDa ankyrin-B disappears and 480/270-kDa ankyrin-G
redistributes from a continuous distribution along premyelinated axons to localized patches adjacent to the ends of
myelin-associated glycoprotein (MAG)-staining processes
of myelinating Schwann cells. All foci of ankyrin-G also
contain neurofascin, NrCAM, and the voltage-dependent
Na⫹ channel. However, neurofascin and NrCAM clusters
Volume 81
July 2001
SPECTRIN AND ANKYRIN-BASED PATHWAYS
specialized sites within the SR. In this case, the basic
defect in ankyrin-B (⫺/⫺) cells underlying missorting of
multiple Ca2⫹-release channels could be a failure in some
step required for segregation of these proteins and/or
their transport between the ER and SR.
Ankyrin-B is a multifunctional protein that could participate in ER protein sorting at several levels. The multivalent ankyrin membrane-binding domain could bind to
and laterally segregate selected proteins within the plane
of the ER membrane (281, 282). Ankyrin-B also could
participate in coupling vesicles to transport systems
through interactions of the spectrin-binding domain or
the microtubule-association site (75, 88, 173). Finally,
ankyrin has recently been reported to associate with the
terminal domain of clathrin (283) and could therefore
interact with the coat proteins of certain vesicles.
F. Orientation of Mitotic Spindles in Asymmetric
Germ Cell Division in Drosophila
Germ line cell division in Drosophila as well as most
insects results in a stem cell, which remains undifferentiated, and a daughter cystoblast, which will form the
oocyte as well as nurse cells. The cystoblast subsequently
experiences four synchronized rounds of mitosis, with the
unusual feature of incomplete cytokinesis, resulting in a
16-cell syncytium connected through incompletely closed
cleavage furrows. These cells, termed cystocytes, are interconnected by a cytoplasmic structure called the fusome, which was first visualized by light microscopy (85,
275).
Fusomes are comprised of two components: membrane skeletal proteins and membrane vesicles and cisternae proposed to represent a specialized form of the ER
(233). Membrane skeletal proteins identified so far in
fusomes include adducin (encoded by the huli-tao shao
gene), ␣-spectrin, and ␤-spectrin (85, 243). ␤-H spectrin as
well as protein 4.1 and other members of the ERM family
are not present in fusomes (85, 272). Fusomes originate
from a structure in the stem cell termed the spectrosome,
which is enriched in ankyrin as well as ␣/␤-spectrin and
adducin (243). The membrane compartment of fusomes
includes a fly homolog of the transition ER ATPase
(TER94) (233). TER94 is believed to have a role in vesicle
fusion in assembly of ER cisternae, and the presence of
this protein in the fusome implies that fusome membranes
are derived from the ER and undergo fusion events.
Mutations in the huli-tao shao (hts) gene, named
from the Chinese for too-little nursing (444), and in
␣-spectrin (85) establish the essential role of these proteins in assembly of fusomes and the spectrosome. Moreover, the phenotypes of these mutants establish that the
fusome/spectrosome is required for several steps in successful maturation of oocytes. Hts mutants lack both the
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
release from SR stores and are localized adjacent to voltage-sensitive Ca2⫹ channels in t tubules, which are extensions of the plasma membrane (129). The SR/ER Ca2⫹ATPase (SERCA) is responsible for subsequent Ca2⫹
uptake into the SR lumen and is localized over actomyosin in the A-band at sites distinct from ryanodine receptors (198). The specialized neuronal ER involved in Ca2⫹
homeostasis is located in dendritic spines and growth
cones of axons and resembles the SR found in muscle as
visualized by electron microscopy (171). The Ca2⫹ compartment of dendritic spines may have a role in cellular
memory (26), while the same compartment in axon
growth cones modulates axon pathfinding (378).
Evidence for an essential role of ankyrin-B in targeting Ca2⫹-release channels to the SR of striated muscle has
come from disruption of the ankyrin-B gene in mice (397).
Knock-out of the ankyrin-B gene was initially characterized in the nervous system, where the 440-kDa ankyrin-B
isoform is required for postnatal survival of premyelinated axons (258) (see below). Ankyrin-B-deficient mice
subsesquently were discovered to have musculoskeletal
defects, elevated serum creatine kinase levels, as well as
localized sites of disorganized sarcomeres in skeletal
muscle (397). Ankyrin-B (⫺/⫺) cardiomyocytes have
highly irregular cytosolic Ca2⫹ waves, with reduced frequency and rate of uptake of Ca2⫹. Moreover, ryanodine
receptors were no longer distributed in a striated pattern
associated with sarcomeres in ankyrin-B (⫺/⫺) cardiomyocytes and skeletal muscle. IP3 receptors also exhibited altered localization in cardiomyocytes. The SR and
SR/t-tubule junctions are apparently preserved in a normal distribution in ankyrin-B (⫺/⫺) skeletal muscle based
on electron microscopy and the presence of a normal
pattern of triadin and the voltage-gated Ca2⫹ channel
(dyhyropyridine receptor). The abnormal localization of
IP3 and ryanodine receptors therefore represents a defect
in intracellular sorting of these proteins in striated muscle. Ankyrin-B (⫺/⫺) mice also exhibit thymic atrophy as
well as reduced expression of IP3 receptors in thymic
lymphocytes (397). The requirement for ankyrin-B for
proper targeting of Ca2⫹-release channels therefore is
likely to be a feature of other cell types with a specialized
Ca2⫹ compartment.
Several considerations support the hypothesis that
ankyrin-B operates at the level of transport and/or segregation of Ca2⫹-release channels into vesicles. Ankyrin-B is
not localized with SERCA or ryanodine receptors in striated muscle (397) but is associated with a novel class of
vesicles in striated muscle as well as brain (J. Q. Davis
and V. Bennett, unpublished data). Exchange of proteins
between ER and Golgi is well known to involve a cellular
machinery for recruitment of specific cargo proteins into
vesicles and transfer of these vesicles between organelle
compartments. A comparable system may also mediate
transfer of functionally defined proteins from the ER to
1377
1378
VANN BENNETT AND ANTHONY J. BAINES
G. A Role for Protein 4.1 in Selective and Directed
Membrane Protein Accumulation
1. The 4.1 proteins and the neurexin superfamily
A striking common feature of D. melanogaster
Coracle and mammalian 4.1 proteins is their requirement
for the stable accumulation of proteins related to glycophorin D/C and neurexins in plasma membranes. Both
human glycophorin C and D. melanogaster neurexin IV
are trapped and essentially immobilized in complexes
with 4.1 proteins. The binding site for 4.1 on glycophorin
C has been mapped (266) and shows strong sequence
similarity to a juxtamembrane sequence in the cytoplasmic domain of neurexin IV (15).
The neurexins are a family of three classes of CAMs
(15, 288, 335, 443). One class is the “classical” neurexins
I-III, which have roles in neuron-neuron adhesion. The
second class is the “NCP” group, which have roles in
mammalian brain in axon-glia contact, and in fly epithelia
in septate junction formation. The third class is represented so far only by the fly protein axotactin, a secreted
form of neurexin with a glia-neuronal signaling function.
The 4.1-binding sequence identified in glycophorin C and
D. melanogaster neurexin IV is also represented in all the
transmembrane members of the neurexin superfamily
(335).
In mammals, the junctions of axons and paranodal
loops from Schwann cells at the paranodal regions of
myelinated axons have been likened to septate junctions
(15, 113, 406). During development of myelinated nerves,
electron-dense junctions progressively form between the
Schwann cell loops and axons; these are incomplete at
day 10 of the postnatal life of a rat and may require up to
day 31 to assume their final form, substantially after
ankyrin-linked Na⫹ channels are clustered (433). The
NCP-group protein CASPR/paranodin is found in these
junctions (113, 279) and is localized to the paranodes
coincident with their formation (339). CASPR/paranodin
associates with protein 4.1 both from red blood cells and
brain extracts (279). Given that the neurexin IV/coracle
complex seems to be required for the formation of a
transepithelial barrier in fly epithelium (417, 418), it seems
probable that an analogous function is performed by a
paranodin/4.1 complex at the junction of axons with myelinating cells. The paranodal junction makes a partial
barrier to the diffusion of small ions and a complete
barrier to large molecules, and it will be important to test
in the 4.1R null mice whether there is evidence of breakdown of this barrier that might underlie their observed
neurological defects. CASPR was identified as a ligand in
cis on the neuronal cell surface for the GPI-linked molecule contactin (324); contactin is required for cell surface
expression of CASPR (115). Contactin-null mice have a
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
membrane and protein components of fusomes (243,
444). Adducin (or more accurately, the alternatively
spliced adducin isoform expressed in germ cells; see below) therefore is required for assembly of both membrane
skeletal proteins as well as membrane components of
fusomes. ␣-Spectrin mutations targeted to germ cells result in loss of fusome-associated ␤-spectrin and adducin,
but to a lesser extent than the hts mutants (85). ␣-Spectrin
mutants were not examined by electron microscopy, and
the state of membrane vesicles has not been determined.
Detailed analysis of hts mutants has demonstrated
several roles of the fusome in oogenesis (90, 243). A
striking observation is that the spectrosome is required
for orientation of the mitotic spindle in the first asymmetric cell division that produces the cystoblast and a renewing stem cell. Spindle orientation is a key event in asymmetric cell division events in generation of neuroblasts as
well as other examples (reviewed in Ref. 242). It will be of
interest to determine if adducin and other fusome components have a broader role in asymmetric cell division
events in addition to insect germ cells. Other consequences of the hts mutation that are likely to be specific
for germ cells included loss of synchronized cell division
in cytocytes and loss of polarized microtubule networks
(90).
Repasky and colleagues (28, 344) have discovered an
intracellular spectrin-rich structure in lymphocytes that
has interesting parallels with the fusome/spectrosome of
D. melanogaster germ cells. The lymphoycyte “spectrosome” also contains membrane vesicles (28), as well as
ankyrin (153). Other features of the lymphocyte spectrosome include association with a protein kinase C isoform
and relocation in response to extracellular signals.
Ankyrin-B is expressed in thymocytes, and ankyrin-B
(⫺/⫺) mice exhibit thymic atrophy (397), although the
spectrosome of ankyrin-B-deficient lymphocytes has not
been examined. The role of the spectrosome in lymphocyte physiology thus remains to be evalulated.
The relationship between the isoform(s) of adducin
expressed in germ cells and spectrin is not clear. Four
adducin transcripts are expressed in D. melanogaster
ovaries, and only the isoform found in somatic follicular
cells contains the COOH-terminal MARCKS-related domain that associates with spectrin and actin, while the
isoforms present in spectrosomes lack this domain and
presumably cannot interact directly with spectrin (423).
All adducin spliced variants contain a conserved globular
head domain, which at this point has an unknown function. The observation that vesicle components are missing
in hts mutants suggests the possibility that the head domain of adducin participates in organization of intracellular membranes and that spectrin and ankyrin are
secondarily recruited via interactions with adducin-associated membranes.
Volume 81
July 2001
SPECTRIN AND ANKYRIN-BASED PATHWAYS
2. The 4.1 proteins and syndecans
Syndecans are a major class of cell surface heparan
sulfate proteoglycan (HSPG), of which syndecan-2 is one
of the major neuronal representatives in the forebrain.
CASK and syndecan-2 interact in two-hybrid assays, are
coexpressed and colocalize in brain development, and
can be coimmunoprecipitated from brain extracts (61,
185). 4.1R interacts with CASK in vitro, and so is suggested to generate a tripartite complex (61); however,
simultaneous binding of the three proteins to each other
has not yet been demonstrated, even though syndecan-2
contains a putative 4.1-binding motif.
Bearing in mind that in flies Coracle is essential for
full expression of EGF receptor activity, the interaction
with syndecans has added interest in terms of signaling.
HSPGs are often considered to be coreceptors for heparin-binding factors: by binding these molecules, with
lower affinity and specificity than their cognate high-affinity tyrosine kinase receptors, they are regarded as increasing their effective concentration in the vicinity of the
plasma membrane (45). In vertebrates, neuregulin/ARIA
is a heparin-binding secreted factor that acts on EGF
receptor-like tyrosine kinases ErbB3 and ErbB4 (455).
Neuregulin/ARIA is concentrated in synaptic clefts of neu-
ronal-neuronal synapses and in the basal lamina of neuromuscular junctions (150, 355) and has a role in inducing
transcription of receptors for acetylcholine, N-methyl-Daspartate, and GABA (117, 314, 347). Syndecan-2 is concentrated in synapses and has been suggested to be the
coreceptor for ARIA/neuregulin (185). Both syndecans
and ErbB3/4 bind MAGUK proteins (61, 133, 185) and
seem likely to be coclustered in postsynaptic regions by
MAGUK interactions. Whether 4.1 has a role in linking this
receptor/HSPG complex to the spectrin cytoskeleton and
thereby stabilizing the mechanics of synaptic junctions, or
whether 4.1 is required for the stable accumulation of
either receptor or HSPG at the synapse remains to be
seen. CASK has a specific signaling role in brain development (184), thus adding an extra dimension to the potential significance of the 4.1-interactive proteins in brain.
IV. CLINICAL IMPLICATIONS
A general lesson from results of gene knock-outs in
model organisms of spectrin and ankyrin-related proteins
is that these genes are not essential for fundamental
cellular function, but act at the level of integration of cells
into tissues. A corollary is that mutations in these proteins
may be compatible with survival but with impaired physiological function, and therefore are candidates to cause
disease in humans. Gene knock-outs of ankyrins, protein
4.1 family members, and adducin in mice are now providing clues regarding general classes of phenotypes to be
expected as well as specific lesions. Moreover, these studies suggest the presence of pathways that involve additional proteins and potential disease candidates. A summary of currently known disorders due to abnormalities
of spectin, ankyrin, and associated proteins are discussed
below (see Table 2).
A. Functional Channelopathies Due to Defects
in Ankyrin-Dependent Targeting
Ankyrin-G- and ankyrin-B-deficient mice described
above both suffer from missorting of ion channels that are
the equivalent in phenotype to mutations in the channels
themselves. For example, mice missing cerebellar
ankyrin-G exhibited severe ataxia associated with loss of
ability of Purkinje neurons to fire action potentials (453).
Moreover, ankyrin-G (⫺/⫺) mice and mice with a mutation in the SNC6 Na⫹ channel subunit both exhibited
progressive degeneration of Purkinje neurons (32, 215).
Ankyrin-G is encoded by a complex set of tissue and
developmentally specific transcripts (93, 178, 219, 327).
Mutations could occur that would target specific tissues
as in the case of the cerebellar-restricted gene knock-out.
The potential channels targeted by ankyrin-G may include
the Na⫹-K⫹-ATPase (297, 304, 385), the Na⫹/Ca2⫹ ex-
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
severe ataxic phenotype (25), reminiscent of (but more
extreme than) part of the 4.1R-null phenotype (411).
Neighboring to the paranodal regions are the juxtaparanodes, in which another NCP protein CASPR2 (335)
is found. Here the Shaker-like K⫹ channels Kv1.1, Kv1.2,
and their ␤-subunit Kv␤2 are clustered and precisely colocalized with CASPR2. The COOH termini of CASPR2
and these K⫹ channels contain potential binding sites for
the PDZ domains of MAGUK proteins; CASPR2 contains a
putative 4.1 binding sequence. It seems likely that these
channels and CASPR2 are cross-linked in a complex with
MAGUKs and 4.1. These K⫹ channels are thought to modulate conduction failures at branch points of complex
axonal trees and to stabilize transition zones in myelinated axons (58), so it will be important to establish
whether these functions are compromised in 4.1R null
mice.
The classical neurexins are presynaptic adhesion
proteins (38, 288) that retain the conserved 4.1 binding
sequence (15). A cytoplasmic interaction of neurexin I
with a MAGUK protein, CASK, has been identified in yeast
two-hybrid assays (167). CASK is the human homolog of a
C. elegans MAGUK, LIN-2, a protein required for vulval
differentiation (183). CASK interacts with 4.1R (61), raising the possibility of a three-way neurexin/MAGUK/4.1
complex in presynaptic regions. Although such an idea is
not supported by fly genetics (since Coracle has not been
reported in presynaptic regions), rat 4.1N has been reported to be abundant in presynaptic terminals (311).
1379
1380
VANN BENNETT AND ANTHONY J. BAINES
2. Metazoan disorders due to abnormalities
of spectrin, ankyrin, and associated proteins
TABLE
Disorder
Hereditary
hemolytic
anemias
Ataxia
Muscular dystrophy/
myopathy
Nervous system
developmental
defects
Abnormal cognitive
function
Female infertility
Protein(s)
Reference
Nos.
393
Mouse
Mouse
Mouse
Mouse
Spectrins: ␣1, ␤1; AE1;
ankyrin-R;
glycophorin C;
protein 4.1R;
␤-adducin
Ankyrin-R;
ankyrin-G
Ankyrin-B
C. elegans
C. elegans
␤-G spectrin
␤-H spectrin
157, 292
276
Drosophila
Human,
mouse
␤-H spectrin
L1
446
212
Mouse
C. elegans
C. elegans
Mouse
Ankyrin-B
␤-G spectrin
Ankyrin/unc44
Protein 4.1R
358
157, 292
312
413
Drosophila
Drosophila
Drosophila
Adducin
␤-H spectrin
␣-Spectrin
444
388
231
Human,
mouse
143
326
453
397
changer (240), as well as others. The potential phenotypes
of ankyrin-G mutations therefore could range from abnormal Na⫹ absorption, defective Ca2⫹ dynamics, and abnormal heart rhythms to name a few. Moreover, the cellular
pathway utilized by ankyrin-G for Na⫹ channel targeting
is not known, but presumably involves additional proteins
that are candidates to contribute to a functional channelopathy.
Disruption of ankyrin-B in mice results in abnormal
targeting of Ca2⫹ signaling proteins including ryanodine
receptors and IP3 receptors (397). Ankyrin-B-deficient
mice exhibit multisystem dysfunction including severe
disorders in nervous system development, myopathy,
megacolon, atrophy of the thymus, and abnormal kidney
development and do not survive the neonatal period (358,
397). Complete ankyrin-B deficiency in humans therefore
would be anticipated to involve defective development of
the nervous system as well as severe dysfunction of striated muscle and immune system that would not be compatible with prolonged postnatal life. However, individuals with weak alleles or partial deficiency of ankyrin-B
may be viable and exhibit clinical disease.
Ankyrin-B (⫹/⫺) mice have reduced expression of
ankyrin-B but survive to adulthood (P. Mohler, A. Gramolini, and V. Bennett, unpublished data). These animals
may provide models for certain autosomal dominant human “channelopathies” involving Ca2⫹ release channels,
with loss of function due to missorting rather than direct
mutations in these proteins. A candidate disease involving
ankyrin-B is long Q-T syndrome type 4, an autosomal
dominant cardiac arrhythmia associated with sudden
death, that maps to the same chromosomal region of
4q25–27 as the ANK2 gene encoding ankyrin-B (356). In
further support of a connection between ankyrin-B and
heart disease, ankyrin-B (⫹/⫺) mice exhibit abnormalities in heart conduction and bradycardia, as well as an
increased Q-T interval (Mohler et al., unpublished data).
Ankyrin-B mutations also are likely to result in abnormalities in the immune and nervous systems. Coordinated assembly and spatial organization of Ca2⫹ release
channels within the ER is essential in cells of the immune
system as well as in striated muscle. Lymphocytes encode
information in the amplitude and frequency of intracellular waves of Ca2⫹ (101). Unlike the highly organized SR of
striated muscle, lymphocytes are likely to have variable
states of organization of IP3 receptors that depend on
their differentiation state. As a consequence of such plasticity, signals that elevate Ca2⫹ can have opposite effects
in naı̈ve and differentiated cells (101). Ankyrin-B deficiency has profound consequences for survival of neonatal thymic lymphocytes as expected if signals promoting
differentiation are interrupted (397).
The specialized neuronal ER involved in Ca2⫹ homeostasis is located in dendritic spines and growth cones
of axons and resembles the SR found in muscle as visualized by electron microscopy (171). The 220-kDa
ankyrin-B is expressed in neuron cell bodies and dendrites in the postnatal brain in a time frame that approximates development of the dendritic spine apparatus of
hippocampal and Purkinje neurons (225), whereas 440kDa ankyrin-B is targeted to axons early in development
(49, 224). These results suggest that 220-kDa ankyrin-B is
a good candidate to participate in targeting Ca2⫹-release
channels to their physiological sites in dendrites of neurons, whereas 440-kDa ankyrin-B may have a similar function in axons.
B. Use-Dependent Dystrophies of Muscle and
Nerves Due to Defects in the Spectrin-Based
Transcellular Mechanical Coupling Pathway
Consequences of spectrin deficiency in C. elegans
(159, 292) include degeneration of muscles and abnormalities in the nervous system that could be attributed to
mechanical stresses. Prediction of the phenotype in vertebrates resulting from defects in spectrin are complicated by the multiple closely related genes that may overlap in function. However, truncations of ␤-spectrin can
act in a dominant-negative fashion by poisoning ␣/␤-spectrin dimers and preventing formation of tetramers (186).
Such mutations conceivably could exhibit penetrance
even in the presence of other normal ␤-spectrin genes.
Examples of acquired disorders due to loss of mechanical support include unexplained myopathies and pe-
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
Morphogenetic
defects
Organism
Volume 81
July 2001
SPECTRIN AND ANKYRIN-BASED PATHWAYS
ripheral neuropathies. Demyelinated peripheral axons
would be particularly vulnerable to trauma if axo-glia
contacts were weakened. It is of interest in this regard
that a major defect in multiple sclerosis is severing of
axons at sites where myelin is interrupted (391). A potential therapeutic strategy in this case would be to restore
expression of 440-kDa ankyrin-B and of L1, which are
normally lost with myelination, and may play a role in
stabilizing premyelinated axons.
C. Abnormal Nervous System Development Due to
L1 CAM Defects
ceivable that mutations in L1 CAM family members may
have subtle consequences at the level of complex neural
function resulting in problems such as learning disabilities, thinking disorders, and overall mental retardation.
These activities are difficult to assess in mice, but could
be significant in a human context.
V. SUMMARY AND PERSPECTIVES
Molecular dissection of the spectrin-based membrane skeleton of the humble mammalian erythrocyte has
provided biologists with a set of proteins with diverse
roles in organization and survival of cells in metazoan
organisms. The lack of an essential function for membrane skeletal proteins in generic cells grown in culture
and the absence of their genes in the yeast genome have,
until recently, limited advances in understanding roles of
these proteins outside of erythrocytes. However, characterization of genes and alternatively spliced variants combined with completion of the genomes of simple metazoans and application of homologous recombination in mice
now are providing the first glimpses of the full scope of
physiological roles for spectrin, ankyrin, and their associated proteins. These functions now promise to include
organization of specialized compartments within the
plasma membrane and endoplasmic reticulum, mechanical stabilization at the tissue level based on transcellular
protein assemblies, participation in epithelial morphogenesis, and orientation of mitotic spindles in asymmetric
cell divisions.
These studies, in addition to stretching the erythrocyte paradigm beyond recognition, also are revealing new
pathways essential for metazoan life that may not be
present in fungi or plants. Examples include ankyrindependent targeting of proteins to the specialized compartments within the plasma membrane and ER, and components and function of the phospho-FIGQY tyrosine
pathway of L1 CAMs. A broader issue is the evolution of
these pathways, beginning with spectrin and ankyrin
themselves, and continuing with their legion of interacting partners and the cellular structures dependent on
these proteins. More immediate questions relate to the
roles of these pathways in a clinical context, either as the
basis for disease, or more positively as therapeutic targets. The newly recognized role of ankyrin-B in targeting
proteins to the Ca2⫹ compartment, for example, suggests
the potential for modulating functions of immune system,
the heart, and nervous system. These pathways could also
be influenced in a negative fashion by teratogens and
other toxic chemicals and have not yet been evaluated
due to lack of appropriate assays. These considerations
suggest that we have arrived at the end of the beginning in
terms of understanding basic roles of spectrin and
ankyrin and in applying this information.
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
The L1 CAM family comprises a major class of cell
adhesion molecules in the adult mammalian nervous system and has been implicated in diverse functions ranging
from axon fasciculation to synaptogenesis (39). Disorders
related to defects in L1 have provided initial insight into
the clinical implications of these functions. Mutations in
the human L1 gene, located on the X chromosome, are
responsible for a syndrome termed CRASH for corpus
callosum agenesis, mental retardation, adducted thumbs,
spastic paraplegia, and hydrocephalus (128, 212). Over 35
mutations in L1 have currently been characterized, and
these are distributed in all domains including several in
the cytoplasmic domain. Disruption of the L1 gene in mice
results in abnormal axon guidance in the corticospinal
tract, brain malformations including dilated ventricles,
and behavioral deficits (61, 73).
Several considerations implicate ankyrin and/or
phospho-FIGQY signaling in the pathophysiology of L1
defects. L1 and 440-kDa ankyrin-B are colocalized in premyelinated axon tracts in the developing nervous system
and are both downregulated after myelination (358).
Ankyrin-B (⫺/⫺) mice exhibit a phenotype similar to, but
more severe than, L1 (⫺/⫺) mice and share features of
human patients with L1 mutations (358). For example,
ankyrin-B (⫺/⫺) mice exhibit hypoplasia of the corpus
callosum and pyramidal tracts, dilated ventricles, all features of the CRASH syndrome associated with L1 mutations. One missense mutation in the L1 cytoplasmic domain results in conversion of FIGQY 1229 to H and
clinical disease (405). The Y1229H mutation of the FIGQY
tyrosine would abolish both ankyrin-binding activity (450)
as well as phospho-FIGQY tyrosine signaling. One or both
of these activities thus is essential for L1 function in
nervous system development.
Diseases have not yet been attributed to other L1
CAM family members. These genes are autosomal, in
contrast to L1, and presumably would exhibit a recessive
pattern of inheritance. It also is possible that mutations
could result in expression of dominant/negative forms of
polypeptides, as has been oberved with LAD-1 in C. elegans (Chen and Bennett, unpublished data). It is con-
1381
1382
VANN BENNETT AND ANTHONY J. BAINES
V. Bennett was supported in part by grants from the National Institutes of Health and the Muscular Dystrophy Association and by the Howard Hughes Medical Institute. A. J. Baines
was suported in part by the Biotechnology and Biological Science Research Council, the Wellcome Trust, and the British
Council Alliance. A travel grant from North American Treaty
Organization to the authors is acknowledged.
Address for reprint requests and other correspondence: V.
Bennett, Howard Hughes Medical Institute and Depts. of Cell
Biology and Biochemistry, Duke University Medical Center,
Durham, NC 27710 (E-mail: [email protected]).
18.
19.
20.
21.
22.
REFERENCES
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
membrane attachment site for human erythrocyte spectrin. J Biol
Chem 253: 2292–2299, 1978.
BENNETT V. Immunoreactive forms of human erythrocyte ankyrin
are present in diverse cells and tissues. Nature 281: 597–599, 1979.
BENNETT V AND DAVIS J. Erythrocyte ankyrin: immunoreactive analogs are associated with mitotic structures in cultured cells and
with microtubules in brain. Proc Natl Acad Sci USA 78: 7550 –7554,
1981.
BENNETT V, DAVIS J, AND FOWLER WE. Brain spectrin, a membraneassociated protein related in structure and function to erythrocyte
spectrin. Nature 299: 126 –131, 1982.
BENNETT V, GARDNER K, AND STEINER JP. Brain adducin: a protein
kinase C substrate that may mediate site-directed assembly at the
spectrin-actin junction. J Biol Chem 263: 5860 –5869, 1988.
BENNETT V AND STENBUCK PJ. The membrane attachment protein for
spectrin is associated with band 3 in human erythrocyte membranes. Nature 280: 468 – 473, 1979.
BENNETT V AND STENBUCK PJ. Identification and partial purification
of ankyrin, the high affinity membrane attachment site for human
erythrocyte spectrin. J Biol Chem 254: 2533–2541, 1979.
BERGHS S, AGGUAJARO D, DIRKX R JR, MAKSIMOVA E, STABACH P,
HERMEL JM, ZHANG JP, PHILBRICK W, SLEPNEV V, ORT T, AND SOLIMENA
M. BetaIV spectrin, a new spectrin localized at axon initial segments and nodes of Ranvier in the central and peripheral nervous
system. J Cell Biol 151: 985–1002, 2000.
BERGLUND EO, MURAI KK, FREDETTE B, SEKERKOVA G, MARTURANO B,
WEBER L, MUGNAINI E, AND RANSCHT B. Ataxia and abnormal cerebellar microorganization in mice with ablated contactin gene expression. Neuron 24: 739 –750, 1999.
BERRIDGE M. Neuronal calcium signaling. Neuron 21: 13–26, 1998.
BHAT MA, IZADDOOST S, LU Y, CHO KO, CHOI KW, AND BELLEN HJ.
Discs lost, a novel multi-PDZ domain protein, establishes and maintains epithelial polarity. Cell 96: 833– 845, 1999.
BLACK JD, KOURY ST, BANKERT RB, AND REPASKY EA. Heterogeneity in
lymphocyte spectrin distribution: ultrastructural identification of a
new spectrin-rich cytoplasmic structure. J Cell Biol 106: 97–109,
1988.
BLACKSHEAR PJ. The MARCKS family of cellular protein kinase C
substrates. J Biol Chem 268: 1501–1504, 1993.
BLANCO FJ, ORTIZ AR, AND SERRANO L. 1H and 15N NMR assignment
and solution structure of the SH3 domain of spectrin: comparison
of unrefined and refined structure sets with the crystal structure.
J Biomol NMR 9: 347–357, 1997.
BLOCH RJ AND MORROW JS. An unusual beta-spectrin associated with
clustered acetylcholine receptors. J Cell Biol 108: 481– 493, 1989.
BOAKES RJ, CANDY JM, DICK DJ, HARRIS JB, POLLARD S, AND THOMPSON
J. Abnormal electrophysiological and anatomical characteristics of
cerebellar Purkinje cells in the mutant mouse jolting (Abstract).
J Physiol (Lond) 346: 27P, 1984.
BODINE DM IV, BIRKENMEIER CS, AND BARKER JE. Spectrin deficient
inherited hemolytic anemias in the mouse: characterization by
spectrin synthesis and mRNA activity in reticulocytes. Cell 37:
721–729, 1984.
BORK P. Hundreds of ankyrin-like repeats in functionally diverse
proteins: mobile modules that cross phyla horizontally? Proteins
17: 363–374, 1993.
BOULEY M, TIAN MZ, PAISLEY K, SHEN YC, MALHOTRA JD, AND HORTSCH
MJ. The L1-type cell adhesion molecule neuroglian influences the
stability of neural ankyrin in the Drosophila embryo but not it
axonal localization. J Neurosci 20: 4515– 4523, 2000.
BOURGUIGNON LY, CHU A, JIN H, AND BRANDT NR. Ryanodine receptor-ankyrin interaction regulates internal Ca2⫹ release in mouse
T-lymphoma cells. J Biol Chem 270: 17917–17922, 1995.
BOURGUIGNON LY, JIN H, IIDA N, BRANDT NR, AND ZHANG SH. The
involvement of ankyrin in the regulation of inositol 1,4,5-trisphosphate receptor-mediated internal Ca2⫹ release from Ca2⫹ storage
vesicles in mouse T-lymphoma cells. J Biol Chem 268: 7290 –7297,
1993.
BROSE N. Synaptic cell adhesion proteins and synaptogenesis in the
mammalian central nervous system. Naturwissenschaften 86: 516 –
524, 1999.
BRUMMENDORF T AND RATHJEN FG. Molecular interactions involving
immunoglobulin superfamily adhesion molecules. In: Ig Superfam-
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
1. AGRE P, CASELLA JF, ZINKHAM WH, MCMILLAN C, AND BENNETT V.
Partial deficiency of erythrocyte spectrin in hereditary spherocytosis. Nature 314: 380 –383, 1985.
2. AGRE P, ORRINGER EP, AND BENNETT V. Deficient red-cell spectrin in
severe, recessively inherited spherocytosis. N Engl J Med 306:
1155–1161, 1982.
3. ALLOISIO N, DALLA VENEZIA N, RANA A, ANDRABI K, TEXIER P, GILSANZ
F, CARTRON JP, DELAUNAY J, AND CHISHTI AH. Evidence that red blood
cell protein p55 may participate in the skeleton-membrane linkage
that involves protein 4.1 and glycophorin C. Blood 82: 1323–1327,
1993.
4. ANSTEE DJ, HEMMING NJ, AND TANNER MJ. Functional factors in the
red cell membrane: interactions between the membrane and its
underlying skeleton. Immunol Invest 24: 187–198, 1995.
5. APPELL KC AND LOW PS. Partial structural characterization of the
cytoplasmic domain of the erythrocyte membrane protein, band 3.
J Biol Chem 256: 11104 –11111, 1981.
6. BANUELOS S, SARASTE M, AND CARUGO KD. Structural comparisons of
calponin homology domains: implications for actin binding. Structure 6: 1419 –1431, 1998.
7. BAUMGARTNER S, LITTLETON JT, BROADIE K, BHAT MA, HARBECKE R,
LENGYEL JA, CHIQUETEHRISMANN R, PROKOP A, AND BELLEN HJ. A
Drosophila neurexin is required for septate junction and bloodnerve barrier formation and function. Cell 87: 1059 –1068, 1996.
8. BEAVEN GH AND GRATZER WB. Interaction of divalent cations with
human red cell cytoskeletons. Biochim Biophys Acta 600: 140 –149,
1980.
9. BECK KA, BUCHANAN JA, MALHOTRA V, AND NELSON WJ. Golgi spectrin:
identification of an erythroid ␤-spectrin homolog associated with
the Golgi complex. J Cell Biol 127: 707–723, 1994.
10. BECK KA, BUCHANAN JA, AND NELSON WJ. Golgi membrane skeleton:
identification, localization and oligomerization of a 195 kDa
ankyrin isoform associated with the Golgi complex. J Cell Sci 110:
1239 –1249, 1997.
11. BECK KA AND NELSON WJ. A spectrin membrane skeleton of the
Golgi complex. Biochim Biophys Acta 1404: 153–160, 1998.
12. BECKER PS, SCHWARTZ MA, MORROW JS, AND LUX SE. Radiolabeltransfer cross-linking demonstrates that protein 4.1 binds to the
NH2-terminal region of ␤ spectrin and to actin in binary interactions. Eur J Biochem 193: 827– 836, 1990.
13. BECKER PS, TSE WT, LUX SE, AND FORGET BG. Beta spectrin kissimmee: a spectrin variant associated with autosomal dominant hereditary spherocytosis and defective binding to protein 4.1 J Clin
Invest 92: 612– 616, 1993.
14. BEGG GE, HARPER SL, MORRIS MB, AND SPEICHER DW. Initiation of
spectrin dimerization involves complementary electrostatic interactions between paired triple-helical bundles. J Biol Chem 275:
3279 –3287, 2000.
15. BELLEN HJ, LU Y, BECKSTEAD R, AND BHAT MA. Neurexin IV, caspr
and paranodin—novel members of the neurexin family: encounters
of axons and glia. Trends Neurosci 21: 444 – 449, 1998.
16. BENNETT H AND CONDEELIS J. Isolation of an immunoreactive analog
of brain fodrin that is associated with the cell cortex of Dictyostelium amoebae. Cell Motil Cytoskeleton 11: 303–317, 1988.
17. BENNETT V. Purification of an active proteolytic fragment of the
Volume 81
July 2001
40.
41.
42.
43.
44.
46.
47.
48.
49.
50.
51.
52.
53.
55.
56.
57.
58.
59.
60.
61.
62.
63.
ily Molecules in the Nervous System, edited by Sonderegger P.
Amsterdam: Harwood Academic, 1998, p. 23–56.
BURRIDGE K, KELLY T, AND MANGEAT P. Nonerythrocyte spectrins:
actin-membrane attachment proteins occurring in many cell types.
J Cell Biol 95: 478 – 486, 1982.
BYERS TJ, BRANDIN E, LUE RA, WINOGRAD E, AND BRANTON D. The
complete sequence of Drosophila beta-spectrin reveals supra-motifs comprising eight 106-residue segments. Proc Natl Acad Sci USA
89: 6187– 6191, 1992.
BYERS TJ AND BRANTON D. Visualization of the protein associations
in the erythrocyte membrane skeleton. Proc Natl Acad Sci USA 82:
6153– 6157, 1985.
BYERS TJ, HUSAIN-CHISHTI A, DUBREUIL RR, BRANTON D, AND GOLDSTEIN LS. Sequence similarity of the amino-terminal domain of
Drosophila beta spectrin to ␣ actinin and dystrophin. J Cell Biol
109: 1633–1641, 1989.
CALVERT R, BENNETT P, AND GRATZER W. Properties and structural
role of the subunits of human spectrin. Eur J Biochem 107: 355–
361, 1980.
CAREY DJ. Syndecans: multifunctional cell-surface co-receptors.
Biochem J 327: 1–16, 1997.
CARUGO KD, BANUELOS S, AND SARASTE M. Crystal structure of a
calponin homology domain. Nat Struct Biol 4: 175–179, 1997.
CASTRESANA J AND SARASTE M. Does Vav bind to F-actin through a CH
domain? FEBS Lett 374: 149 –151, 1995.
CATTERALL WA. Localization of sodium channels in cultured neural
cells. J Neurosci 1: 777–783, 1981.
CHAN W, KORDELI E, BENNETT V. 440-kD ankyrinB: structure of the
major developmentally regulated domain and selective localization
in unmyelinated axons. J Cell Biol 123: 1463–1473, 1993.
CHAO TS AND TAO M. Effect of 2,3-diphosphoglycerate on the phosphorylation of protein 4.1 by protein kinase C. Arch Biochem
Biophys 285: 221–226, 1991.
CHAO TS AND TAO M. Modulation of protein 4.1 binding to inside-out
membrane vesicles by phosphorylation. Biochemistry 30: 10529 –
10535, 1991.
CHASIS JA, AGRE P, AND MOHANDAS N. Decreased membrane mechanical stability and in vivo loss of surface area reflect spectrin deficiencies in hereditary spherocytosis. J Clin Invest 82: 617– 623,
1988.
CHE A, MORRISON IE, PAN R, AND CHERRY RJ. Restriction by ankyrin
of band 3 rotational mobility in human erythrocyte membranes and
reconstituted lipid vesicles. Biochemistry 36: 9588 –9595, 1997.
CHERRY L, MENHART N, AND FUNG LW. Interactions of the ␣-spectrin
N-terminal region with beta-spectrin. Implications for the spectrin
tetramerization reaction. J Biol Chem 274: 2077–2084, 1999.
CHISHTI AH. Function of p55 and its nonerythroid homologues.
Curr Opin Hematol 5: 116 –121, 1998.
CHISHTI AH, KIM AC, MARFATIA SM, LUTCHMAN M, HANSPAL M, JINDAL
H, LIU SC, LOW PS, ROULEAU GA, MOHANDAS N, CHASIS JA, CONBOY JG,
GASCARD P, TAKAKUWA Y, HUANG SC, BENZ EJ, BRETSCHER A, FEHON
RG, GUSELLA AF, RAMESH V, SOLOMON F, MARCHESI VT, TSUKITA S,
ARPIN M, LOUVARD D, TONKS NK, ANDERSON JM, FANNING AS, BRYANT
PJ, WOODS DF, AND HOOVER KB. The FERM domain: a unique
module involved in the linkage of cytoplasmic proteins to the
membrane. Trends Biochem Sci 23: 281–282, 1998.
CHIU SY, ZHOU L, ZHANG CL, AND MESSING A. Analysis of potassium
channel functions in mammalian axons by gene knockouts. J Neurocytol 28: 349 –364, 1999.
CHOW CW. Regulation and intracellular localization of the epithelial
isoforms of the Na⫹/H⫹ exchangers NHE2 and NHE3. Clin Invest
Med 22: 195–206, 1999.
CITTERIO L, AZZANI T, DUGA S, AND BIANCHI G. Genomic organization
of the human gamma adducin gene. Biochem Biophys Res Commun 266: 110 –114, 1999.
COHEN AR, WOODS DF, MARFATIA SM, WALTHER Z, CHISHTI AH, ANDERSON JM, AND WOOD DF. Human CASK/LIN-2 binds syndecan-2 and
protein 4.1 and localizes to the basolateral membrane of epithelial
cells. J Cell Biol 142: 129 –138, 1998.
COHEN CM, DOTIMAS E, AND KORSGREN C. Human erythrocyte membrane protein band 4.2 (pallidin). Semin Hematol 30: 119 –137,
1993.
COHEN CM AND LANGLEY RC JR. Functional characterization of hu-
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
1383
man erythrocyte spectrin alpha and beta chains: association with
actin and erythrocyte protein 4.1. Biochemistry 23: 4488 – 4495,
1984.
COHEN NR, TAYLOR JS, SCOTT LB, GUILLERY RW, SORIANO P, AND
FURLEY AJ. Errors in corticospinal axon guidance in mice lacking
the neural cell adhesion molecule L1. Curr Biol 8: 26 –33, 1997.
CONBOY J. The role of alternative premRNA splicing in regulating
the structure and function of skeletal protein 4.1. Proc Soc Exp Biol
Med 220: 73–78, 1999.
CONBOY J, MARCHESI S, KIM R, AGRE P, KAN YW, AND MOHANDAS N.
Molecular analysis of insertion deletion mutations in protein-4.1 in
elliptocytosis. 2. Determination of molecular genetic origins of
rearrangements. J Clin Invest 86: 524 –530, 1990.
CONBOY J, MOHANDAS N, TCHERNIA GY, AND KAN W. Molecular basis of
hereditary elliptocytosis due to protein 4.1 deficiency. N Engl
J Med 315: 680 – 685, 1986.
CONBOY JG. Structure, function, and molecular genetics of erythroid membrane skeletal protein 4.1 in normal and abnormal red
blood cells. Semin Hematol 30: 58 –73, 1993.
CONBOY JG, CHAN JY, CHASIS JA, KAN YW, AND MOHANDAS N. Tissuespecific and development-specific alternative RNA splicing regulates expression of multiple isoforms of erythroid membrane protein 4.1. J Biol Chem 266: 8273– 8280, 1991.
CONBOY JG, SHITAMOTO R, PARRA M, WINARDI R, KABRA A, SMITH J, AND
MOHANDAS N. Hereditary elliptocytosis due to both qualitative and
quantitative defects in membrane skeletal protein 4.1. Blood 78:
2438 –2443, 1991.
CORREAS I, LETO TL, SPEICHER DW, AND MARCHESI VT. Identification
of the functional site of erythrocyte protein 4.1 involved in spectrinactin associations. J Biol Chem 261: 3310 –3315, 1986.
CORREAS I, SPEICHER DW, AND MARCHESI VT. Structure of the spectrin-actin binding-site of erythrocyte protein 4.1. J Biol Chem 261:
3362–3366, 1986.
DAHME M, BARTSCH U, MARTINI R, ANLIKER B, SCHACHNER M, AND
MANTEI N. Disruption of the mouse L1 gene leads to malformations
of the nervous system. Nat Genet 17: 346 –349, 1997.
DANILOV YN, FENNELL R, LING E, AND COHEN CM. Selective modulation of band 4.1 binding to erythrocyte membranes by protein
kinase C. J Biol Chem 265: 2556 –2562, 1990.
DAVIS JQ AND BENNETT V. Brain ankyrin. A membrane-associated
protein with binding sites for spectrin, tubulin, and the cytoplasmic
domain of the erythrocyte anion channel. J Biol Chem 259: 13550 –
13559, 1984.
DAVIS JQ AND BENNETT V. Ankyrin binding activity shared by the
neurofascin/L1/NrCAM family of nervous system cell adhesion molecules. J Biol Chem 269: 27163–27166, 1994.
DAVIS JQ, LAMBERT S, AND BENNETT V. Molecular composition of the
node of Ranvier: identification of ankyrin-binding cell adhesion
molecules neurofascin (mucin⫹/third FNIII domain-) and NrCAM
at nodal axon segments. J Cell Biol 135: 1355–1367, 1996.
DAVIS L, LUX SE, AND BENNETT V. Mapping the ankyrin-binding site
of the human erythrocyte anion exchanger. J Biol Chem 264:
9665–9672, 1989.
DAVIS JQ, MCLAUGHLIN T, AND BENNETT V. Ankyrin-binding proteins
related to nervous system cell adhesion molecules: candidates to
provide transmembrane and intercellular connections in adult
brain. J Cell Biol 121: 121–133, 1993.
DAVIS LH AND BENNETT V. Identification of two regions of beta G
spectrin that bind to distinct sites in brain membranes. J Biol Chem
269: 4409 – 4416, 1994.
DAVIS LH, DAVIS JQ, AND BENNETT V. Ankyrin regulation: an alternatively spliced segment of the regulatory domain functions as an
intramolecular modulator. J Biol Chem 267: 18966 –18972, 1992.
DAVIS LH, OTTO E, AND BENNETT V. Specific 33-residue repeat(s) of
erythrocyte ankyrin associate with the anion exchanger. J Biol
Chem 266: 11163–11169, 1991.
DEBANT A, SERRA-PAGES C, SEIPEL K, O’BRIEN S, TANG M, PARK SH,
AND STREULI M. The multidomain protein Trio binds the LAR transmembrane tyrosine phosphatase, contains a protein kinase domain, and has separate rac-specific and rho-specific guanine nucleotide exchange factor. Proc Natl Acad Sci USA 93: 5466 –5471,
1996.
DECARCER G, LALLENA WJ, AND CORREAS I. Protein 4.1 is a component
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
45.
SPECTRIN AND ANKYRIN-BASED PATHWAYS
1384
85.
86.
87.
88.
89.
91.
92.
93.
94.
95.
96.
97.
98.
99.
100.
101.
102.
103.
104.
of the nuclear matrix of mammalian cells. Biochem J 312: 871– 877,
1995.
DE CUEVAS M, LEE JK, AND SPRADLING AC. Alpha-spectrin is required
for germline cell division and differentiation in the Drosophila
ovary. Development 122: 3959 –3968, 1996.
DEERINCK TJ, LEVINSON SR, BENNETT GV, AND ELLISMAN EH. Clustering of voltage-sensitive sodium channels on axons is independent
of direct Schwann cell contact in the dystrophic mouse. J Neurosci
17: 5080 –5088, 1997.
DELAUNAY J, ALLOISIO N, MORLE L, BAKLOUTI F, DALLAVENEZIA N,
MAILLET P, AND WILMOTTE R. Molecular genetics of hereditary elliptocytosis and hereditary spherocytosis. Ann Genet 39: 209 –221,
1996.
DE MATTEIS MA AND MORROW JS. The role of ankyrin and spectrin in
membrane transport and domain formation. Curr Opin Cell Biol
10: 542–549, 1998.
DENG H, LEE JK, GOLDSTEIN LS, AND BRANTON D. Drosophila development requires spectrin network formation. J Cell Biol 128: 71–79,
1995.
DENG W AND LIN H. Spectrosomes and fusomes anchor mitotic
spindles during asymmetric germ cell divisions and facilitate the
formation of apolarized microtubule array for oocyte specification
in Drosophila. Dev Biol 189: 79 –94, 1997.
DERICK LH, LIU SC, CHISHTI AH, AND PALEK J. Protein immunolocalization in the spread erythrocyte membrane skeleton. Eur J Cell
Biol 57: 317–320, 1992.
DEVARAJAN P, STABACH PR, DE MATTEIS MA, AND MORROW JS.
DevNa,K-ATPase transport from endoplasmic reticulum to Golgi
requires the Golgi spectrin-ankyrin G119 skeleton in Madin-Darby
canine kidney cells. Proc Natl Acad Sci USA 94: 10711–10716, 1997.
DEVARAJAN P, STABACH PR, MANN AS, ARDITO T, KASHGARIAN M, AND
MORROW JS. Identification of a small cytoplasmic ankyrin
(AnkG119) in the kidney and muscle that binds beta I sigma spectrin and associates with the Golgi apparatus. J Cell Biol 133:
819 – 830, 1996.
DHERMY D, GALAND C, BOURNIER O, CYNOBER T, MECHINAUD F,
TCHEMIA G, AND GARBARZ M. Hereditary spherocytosis with spectrin
deficiency related to null mutations of the beta-spectrin gene. Blood
Cells Mol Dis 24: 251–261, 1998.
DHERMY D, GARBARZ M, LECOMTE MC, FEO C, BOURNIER O, CHAVEROCHE I, GAUTERO H, GALAND C, AND BOIVIN P. Hereditary elliptocytosis: clinical, morphological and biochemical studies of 38 cases.
Nouv Rev Fr Hematol 28: 129 –140, 1986.
DING Y, KOBAYASHI S, AND KOPITO R. Mapping of ankyrin binding
determinants on the erythroid anion exchanger, AE1. J Biol Chem
271: 22494 –22498, 1996.
DISCHER DE. New insights into erythrocyte membrane organization
and microelasticity. Curr Opin Hematol 7: 117–122, 2000.
DISCHER DE, PARRA M, CONBOY JG, AND MOHANDAS N. Mechanochemistry of the alternatively spliced spectrin-actin binding domain in
membrane skeletal protein-4.1. J Biol Chem 268: 7186 –7195, 1993.
DISCHER DE, WINARDI R, SCHISCHMANOFF PO, PARRA M, CONBOY JG,
AND MOHANDAS N. Mechanochemistry of protein 4.1s spectrin-actinbinding domain: ternary complex interactions, membrane-binding,
network integration, structural strengthening. J Cell Biol 130: 897–
907, 1995.
DJINOVIC-CARUGO K, YOUNG P, GAUTEL M, AND SARASTE M. Structure of
the alpha-actinin rod: molecular basis for cross-linking of actin
filaments. Cell 98: 537–546, 1999.
DOLMETSCH R, LEWIS RS, GOODNOW CG, AND HEALY JI. Differential
activation of transcription factors induced by calcium response
amplitude and duration. Nature 386: 855– 858, 1997.
DONG L, CHAPLINE C, MOUSSEAU B, FOWLER L, RAMSAY K, STEVENS JL,
AND JAKEN S. 35h, a sequence isolated as a protein kinase C binding
protein, is a novel member of the adducin family. J Biol Chem 270:
25534 –25540, 1995.
DRENCKHAHN D AND BENNETT V. Polarized distribution of Mr 210,000
and 190,000 analogs of erythrocyte ankyrin along the plasma membrane of transporting epithelia, neurons and photoreceptors. Eur
J Cell Biol 43: 479 – 486, 1987.
DRENCKHAHN D, SCHLUTER K, ALLEN DP, AND BENNET V. Colocalization of band 3 with ankyrin and spectrin at the basal membrane of
intercalated cells in the rat kidney. Science 230: 1287–1289, 1985.
Volume 81
105. DUBREUIL RR, BYERS TJ, SILLMAN AL, BAR-ZVI D, GOLDSTEIN LS, AND
BRANTON D. The complete sequence of Drosophila alpha-spectrin:
conservation of structural domains between alpha-spectrins and
alpha-actinin. J Cell Biol 109: 2197–2205, 1989.
106. DUBREUIL RR, BYERS TJ, STEWART CT, AND KIEHART DP. A betaspectrin isoform from Drosophila (beta H) is similar in size to
vertebrate dystrophin. J Cell Biol 111: 1849 –1858, 1990.
107. DUBREUIL RR, MACVICAR G, DISSANAYAKE S, LIU C, HOMER D, AND
HORTSCH M. Neuroglian-mediated cell adhesion induces assembly
of the membrane skeleton at cell contact sites. J Cell Biol 133:
647– 655, 1996.
108. DUBREUIL RR, MADDUX PB, GRUSHKO TA, AND MACVICAR GR. Segregation of two spectrin isoforms: polarized membrane-binding sites
direct polarized membrane skeleton assembly. Mol Biol Cell 8:
1933–1942, 1997.
109. DUBREUIL RR, WANG P, DAHL S, LEE J, AND GOLDSTEIN LS. Drosophila
beta spectrin functions independently of alpha spectrin to polarize
the Na,K ATPase in epithelial cells. J Cell Biol 149: 647– 656, 2000.
110. DUBREUIL RR AND YU J. Ankyrin and beta-spectrin accumulate independently of alpha-spectrin in Drosophila. Proc Natl Acad Sci
USA 91: 10285–10289, 1994.
111. DUGANDZIJA-NOVAKOVIC S, KOSZOWSKI AG, LEVINSON SR, AND SHRAGER
P. Clustering of Na⫹ channels and node of Ranvier formation in
remyelinating axons. J Neurosci 15: 492–503, 1995.
112. EBER SW, GONZALEZ JM, LUX ML, SCARPA AL, TSE WT, DORNWELL M,
HERBERS J, KUGLER W, OZCAN R, PEKRUN A, GALLAGHER PG, SCHROTER
W, FORGET BG, AND LUX SE. Ankyrin-1 mutations are a major cause
of dominant and recessive hereditary spherocytosis. Nat Genet 13:
214 –218, 1996.
113. EINHEBER S, ZANAZZI G, CHING W, SCHERER S, MILNER TA, PELES E, AND
SALZER JL. The axonal membrane protein Caspr, a homologue of
neurexin IV, is a component of the septate-like paranodal junctions
that assemble during myelination. J Cell Biol 139: 1495–1506, 1997.
114. EL OUGGOUTI S, BOURNIER O, BOIVIN P, BERTRAND O, AND DHERMY D.
Purification of erythrocyte protein 4.1 by selective interaction with
inositol hexaphosphate. Protein Exp Purif 3: 488 – 496, 1992.
115. FAIVRE-SARRAILH C, GAUTHIER F, DENISENKO-NEHRBASS N, LE BIVIC A,
ROUGON G, AND GIRAULT JA. The glycosylphosphatidyl inositol-anchored adhesion molecule F3/contactin is required for surface
transport of paranodin/contactin-associated protein (caspr). J Cell
Biol 149: 491–502, 2000.
116. FAIX J, STEINMETZ M, BOVES H, KAMMERER RA, LOTTSPEICH F, MINTERT
U, MURPHY J, STOCK A, AEBI U, AND GERISCH G. Cortexillins, major
determinants of cell shape and size, are actin-bundling proteins
with a parallel coiled-coil tail. Cell 86: 631– 642, 1996.
117. FALLS DL, ROSEN KM, CORFAS G, LANE WS, AND FISCHBACH GD. Aria,
a protein that stimulates acetylcholine receptor synthesis, is a
member of the neu ligand family. Cell 72: 801– 815, 1993.
118. FARADAY CD AND SPANSWICK RM. Evidence for a membrane skeleton
in higher plants. A spectrin-like polypeptide co-isolates with rice
root plasma membranes. FEBS Lett 318: 313–316, 1993.
119. FEHON RG, DAWSON IA, AND ARTAVANISTSAKONAS S. A Drosophila
homolog of membrane-skeleton protein-4.1 is associated with septate junctions and is encoded by the coracle gene. Development
120: 545–557, 1994.
120. FEO CJ, FISCHER S, PIAU JP, GRANGE MJ, AND TCHERNIA G. First
instance of the absence of an erythrocyte membrane protein [band
4(1)] in a case of familial elliptocytic anemia. Nouv Rev Fr Hematol
22: 315–325, 1980.
121. FITZLI D, STOECKLI ET, KUNZ S, SIRIBOUR K, RADER C, KUNZ B, KOZLOV
SV, BUCHSTALLER A, LANE RP, SUTER DM, DREYER WJ, AND SONDEREGGER P. A direct interaction of axonin-1 with NgCAM-related cell
adhesion molecule (NrCAM) results in guidance, but not growth of
commissural axons. J Cell Biol 149: 951–968, 2000.
122. FLUCHER BE AND DANIELS MP. Distribution of Na⫹ channels and
ankyrin in neuromuscular junctions is complementary to that of
acetylcholine receptors and the 43 kD protein. Neuron 3: 163–175,
1989.
123. FOWLER V AND TAYLOR DL. Spectrin plus band 4.1 cross-link actin.
Regulation by micromolar calcium. J Cell Biol 85: 361–376, 1980.
124. FOWLER VM. Tropomodulin: a cytoskeletal protein that binds to the
end of erythrocyte tropomyosin and inhibits tropomyosin binding
to actin. J Cell Biol 111: 471– 481, 1990.
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
90.
VANN BENNETT AND ANTHONY J. BAINES
July 2001
SPECTRIN AND ANKYRIN-BASED PATHWAYS
148. GLOOR S, ANTONICEK H, SWEADNER KJ, PAGLIUSI S, FRANK R, MOOS M,
AND SCHACHNER M. The adhesion molecule on glia (AMOG) is a
homologue of the beta subunit of the Na,K-ATPase. J Cell Biol 110:
165–174, 1990.
149. GOLDSMITH SC, POKALA N, SHEN W, FEDOROV AA, MATSUDAIRA P, AND
ALMO SC. The structure of an actin-crosslinking domain from human fimbrin. Nat Struct Biol 4: 708 –712, 1997.
150. GOODEARL AD, YEE AG, SANDROCK AW JR, CORFAS G, AND FISCHBACH
GD. Aria is concentrated in the synaptic basal lamina of the developing chick neuromuscular junction. J Cell Biol 130: 1423–1434,
1995.
151. GOODMAN SR, ZAGON IS, AND KULIKOWSKI RR. Identification of a
spectrin-like protein in nonerythroid cells. Proc Natl Acad Sci USA
78: 7570 –7574, 1981.
152. GREENQUIST AC, SHOHET SB, AND BERNSTEIN SE. Marked reduction of
spectrinin hereditary spherocytosis in the common house mouse.
Blood 51: 1149 –1155, 1978.
153. GREGORIO CC, REPASKY EA, FOWLER VM, AND BLACK JD. Dynamic
properties of ankyrin in T lymphocytes: colocalization with spectrin and protein kinase C beta. J Cell Biol 125: 345–358, 1994.
154. GREGORY SL AND BROWN NH. Kakapo, a gene required for adhesion
between and within cell layers in Drosophila, encodes a large
cytoskeletal linker protein related to plectin and dystrophin. J Cell
Biol 143: 1271–1282, 1998.
155. GRUM VL, LI D, MACDONALD RI, AND MONDRAGON A. Structures of two
repeats of spectrin suggest models of flexibility. Cell 98: 523–535,
1999.
156. GULLEY RL AND REESE TS. Cytoskeletal organization at the postsynaptic complex. J Cell Biol 91: 298 –302, 1981.
157. HALL TG AND BENNETT V. Regulatory domains of erythrocyte
ankyrin. J Biol Chem 262: 10537–10545, 1987.
158. HAMADA K, SHIMIZU T, MATSUI T, TSUKITA S, AND HAKOSHIMA T. Structural basis of the membrane-targeting and unmasking mechanisms
of the radixin FERM domain. EMBO J 19: 4449 – 4462, 2000.
159. HAMMARLUND M, DAVIS WS, AND JORGENSEN EM. Mutations in betaspectrin disrupt axon outgrowth and sarcomere structure. J Cell
Biol 149: 931–942, 2000.
160. HAN BG, NUNOMURA W, TAKAKUWA Y, MOHANDAS N, AND JAP BK.
Protein 4.1R core domain structure and insights into regulation of
cytoskeletal organization. Nat Struct Biol 7: 871– 875, 2000.
161. HANEIN D, VOLKMANN N, GOLDSMITH S, MICHON AM, LEHMAN W, CRAIG
R, DEROSIER D, ALMO S, AND MATSUDAIRA P. An atomic model of
fimbrin binding to F-actin and its implications for filament
crosslinking and regulation. Nat Struct Biol 5: 787–792, 1998.
162. HARRIS AS, CROALL DE, AND MORROW JS. The calmodulin-binding site
in alpha-fodrin is near the calcium-dependent protease-I cleavage
site. J Biol Chem 263: 15754 –15761, 1988.
163. HARRIS HW JR AND LUX SE. Structural characterization of the phosphorylation sites of human erythrocyte spectrin. J Biol Chem 255:
11512–11520, 1980.
164. HARTWIG JH. Actin-binding proteins. 1. Spectrin super family. Protein Profile 2: 703– 800, 1995.
165. HASSEL B, RATHJEN FG, AND VOLKMER H. Organization of the neurofascin gene and analysis of developmentally regulated alternative
splicing. J Biol Chem 272: 28742–28749, 1997.
166. HASSOUN H AND PALEK J. Hereditary spherocytosis: a review of the
clinical and molecular aspects of the disease. Blood Rev 10: 129 –
147, 1996.
167. HATA Y, BUTZ S, AND SUDHOF TC. Cask, a novel DLG/PSD95 homolog
with an N-terminal calmodulin-dependent protein-kinase domain
identified by interaction with neurexins. J Neurosci 16: 2488 –2494,
1996.
168. HAYES NV, SCOTT C, HEERKENS E, OHANIAN V, MAGGS AM, PINDER JC,
KORDELI E, AND BAINES AJ. Identification of a novel C-terminal
variant of betaII spectrin: two isoforms of betaII spectrin have
distinct intracellular locations and activities. J Cell Sci 113: 2023–
2034, 2000.
169. HEMMING NJ, ANSTEE DJ, MAWBY WJ, REID ME, AND TANNER MJ.
Localization of the protein 4.1-binding site on human erythrocyte
glycophorins C and D. Biochem J 299: 191–196, 1994.
170. HEMMING NJ, ANSTEE DJ, STARICOFF MA, TANNER MJ, AND MOHANDAS
N. Identification of the membrane attachment sites for protein 4.1
in the human erythrocyte. J Biol Chem 270: 5360 –5366, 1995.
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
125. FOWLER VM. Regulation of actin filament length in erythrocytes and
striated muscle. Curr Opin Cell Biol 8: 86 –96, 1996.
126. FOWLER VM. Capping actin filament growth: tropomodulin in muscle and nonmuscle cells. Soc Gen Physiol Ser 52: 79 – 89, 1997.
127. FOWLER VM AND BENNETT V. Erythrocyte membrane tropomyosin.
Purification and properties. J Biol Chem 259: 5978 –5989, 1984.
128. FRANSEN E, LEMMON V, VAN CAMP G, VITS L, COUCKE P, AND WILLEMS
PJ. Crash syndrome: clinical spectrum of corpus callosum hypoplasia, retardation, adducted thumbs, spastic paraparesis and hydrocephalus due to mutations in one single gene, L1. Eur J Hum Genet
3: 273–284, 1995.
129. FRANZINI-ARMSTRONG C AND PROTASI F. Ryanodine receptors of striated muscles: a complex channel capable of multiple interactions.
Physiol Rev 77: 699 –729, 1997.
130. FUKATA Y, OSHIRO N, KINOSHITA N, KAWANO Y, MATSUOKA Y, BENNETT
V, MATSUURA Y, AND KAIBUCHI K. Phosphorylation of adducin by
Rho-kinase plays a crucial role in cell motility. J Cell Biol 145:
347–361, 1999.
131. GALLAGHER PG AND FERRIERA JD. Molecular basis of erythrocyte
membrane disorders. Curr Opin Hematol 4: 128 –135, 1997.
132. GALLAGHER PG, TSE WT, SCARPA AL, LUX SE, AND FORGET BG. Structure and organization of the human ankyrin-1 gene. Basis for complexity of premRNA processing. J Biol Chem 272: 19220 –19228,
1997.
133. GARCIA RA, VASUDEVAN K, AND BUONANNO A. The neuregulin receptor
ErbB-4 interacts with PDZ-containing proteins at neuronal synapses. Proc Natl Acad Sci USA 97: 3596 –3601, 2000.
134. GARDNER K AND BENNETT V. A new erythrocyte membrane-associated protein with calmodulin binding activity. Identification and
purification. J Biol Chem 261: 1339 –1348, 1986.
135. GARDNER K AND BENNETT V. Modulation of spectrin-actin assembly
by erythrocyte adducin. Nature 328: 359 –362, 1987.
136. GARVER TD, REN Q, TUVIA S, AND BENNETT V. Tyrosine phosphorylation at a site highly conserved in the L1 family of cell adhesion
molecules abolishes ankyrin binding and increases lateral mobility
of neurofascin. J Cell Biol 137: 703–714, 1997.
137. GASCARD P AND COHEN CM. Absence of high-affinity band 4.1 binding
sites from membranes of glycophorin C- and D-deficient (Leach
phenotype) erythrocytes. Blood 83: 1102–1108, 1994.
138. GASCARD P, NUNOMURA W, LEE G, WALENSKY LD, KRAUSS SW,
TAKAKUWA Y, CHASIS JA, MOHANDAS N, AND CONBOY JG. Deciphering
the nuclear import pathway for the cytoskeletal red cell protein
4.1R. Mol Biol Cell 10: 1783–1798, 1999.
139. GASCARD P, PAWELCZYK T, LOWENSTEIN JM, AND COHEN CM. The role
of inositol phospholipids in the association of band 4.1 with the
human erythrocyte-membrane. Eur J Biochem 211: 671– 681, 1993.
140. GEE SH, MADHAVAN R, LEVINSON SR, CALDWELL JH, SEALOCK R, AND
FROEHNER SC. Interaction of muscle and brain sodium channels
with multiple members of the syntrophin family of dystrophinassociated proteins. J Neurosci 18: 128 –137, 1998.
141. GEORGATOS SD AND MARCHESI VT. The binding of vimentin to human
erythrocyte membranes: a model system for the study of intermediate filament-membrane interactions. J Cell Biol 100: 1955–1961,
1985.
142. GILLIGAN DM, LIEMAN J, AND BENNETT V. Assignment of the human
beta-adducin gene (ADD2) to 2p13–p14 by in situ hybridization.
Genomics 28: 610 – 612, 1995.
143. GILLIGAN DM, LOZOVATSKY L, GWYNN B, BRUGNARA C, MOHANDAS N,
AND PETERS LL. Targeted disruption of the beta adducin gene
(Add2) causes red blood cell spherocytosis in mice. Proc Natl Acad
Sci USA 96: 10717–10722, 1999.
144. GIMM JA AND MOHANDAS N. Actin-binding motif of protien 4.1, a
novel sequence. Mol Biol Cell 10: 776, 1999.
145. GIMONA M AND MITAL R. The single CH domain of calponin is neither
sufficient nor necessary for F-actin binding. J Cell Sci 111: 1813–
1821, 1998.
146. GLENNEY JR JR, GLENNEY P, OSBORN M, AND WEBER K. An F-actin- and
calmodulin-binding protein from isolated intestinal brush borders
has a morphology related to spectrin. Cell 28: 843– 854, 1982.
147. GLENNEY JR JR, GLENNEY P, AND WEBER K. The spectrin-related
molecule, TW-260/240, cross-links the actin bundles of the microvillus rootlets in the brush borders of intestinal epithelial cells. J Cell
Biol 96: 1491–1496, 1983.
1385
1386
VANN BENNETT AND ANTHONY J. BAINES
193. JACOBS MD AND HARRISON SC. Structure of an IkappaBalpha/NFkappaB complex. Cell 95: 749 –758, 1998.
195. JONS T AND DRENCKHAHN D. Identification of the binding interface
involved in linkage of cytoskeletal protein 4.1 to the erythrocyte
anion exchanger. EMBO J 11: 2863–2867, 1992.
196. JONS T AND DRENCKHAHN D. Anion exchanger 2 (AE2) binds to
erythrocyte ankyrin and is colocalized with ankyrin along the basolateral plasma membrane of human gastric parietal cells. Eur
J Cell Biol 75: 232–236 1998.
197. JORDAN C, PUSCHEL B, KOOB R, AND DRENCKHAHN D. Identification of
a binding motif for ankyrin on the alpha-subunit of Na⫹,K⫹ATPase. J Biol Chem 270: 29971–29975, 1995.
198. JORGENSEN AO, SHEN AC, ARNOLD W, MCPHERSON PS, AND CAMPBELL
KP. The Ca2⫹-release channel/ryanodine receptor is localized in
junctional and corbular sarcoplasmic reticulum in cardiac muscle.
J Cell Biol 120: 969 –980, 1993.
199. JOSEPH SK AND SAMANTA S. Detergent solubility of the inositol
trisphosphate receptor in rat brain membranes. Evidence for association of the receptor with ankyrin. J Biol Chem 268: 6477– 6486,
1993.
200. JOSHI R AND BENNETT V. Mapping the domain structure of human
erythrocyte adducin. J Biol Chem 265: 13130 –13136, 1990.
201. JOSHI R, GILLIGAN DM, OTTO E, MCLAUGHLIN T, AND BENNETT V.
Primary structure and domain organization of human alpha and
beta adducin. J Cell Biol 115: 665– 675, 1991.
202. KAISER HW, O’KEEFE E, AND BENNETT V. Adducin: Ca2⫹-dependent
association with sites of cell-cell contact. J Cell Biol 109: 557–569,
1989.
203. KALOMIRIS EL AND BOURGUIGNON LY. Mouse T lymphoma cells contain a transmembrane glycoprotein (GP85) that binds ankyrin.
J Cell Biol 106: 319 –327, 1988.
204. KAMAL A, YING Y, AND ANDERSON RG. Annexin VI-mediated loss of
spectrin during coated pit budding is coupled to delivery of LDL to
lysosomes. J Cell Biol 142: 937–947, 1998.
205. KAPLAN MR, MEYER-FRANKE A, LAMBERT S, BENNETT V, DUNCAN ID,
LEVINSON SR, AND BARRES BA. Induction of sodium channel clustering by oligodendrocytes. Nature 386: 724 –728, 1997.
206. KARAKESISOGLOU I, YANG Y, AND FUCHS E. An epidermal plakin that
integrates actin and microtubule networks at cellular junctions.
J Cell Biol 149: 195–208, 2000.
207. KARINCH AM, ZIMMER WE, AND GOODMAN SR. The identification and
sequence of the actin-binding domain of human red blood cell
beta-spectrin. J Biol Chem 265: 11833–11840, 1990.
208. KEEP NH, WINDER SJ, MOORES CA, WALKE S, NORWOOD FL, AND
KENDRICK-JONES JJ. Crystal structure of the actin-binding region of
utrophin reveals a head-to-tail dimer. Struct Fold Des 7: 1539 –1546,
1999.
209. KELLY GM, ZELUS BD, AND MOON RT. Identification of a calciumdependent calmodulin-binding domain in Xenopus membrane skeleton protein 4.1. J Biol Chem 266: 12469 –12473, 1991.
210. KENNEDY SP, WARREN SL, FORGET BG, AND MORROW JS. Ankyrin binds
to the 15th repetitive unit of erythroid and nonerythroid betaspectrin. J Cell Biol 115: 267–277, 1991.
211. KENNEDY SP, WEED SA, FORGET BG, AND MORROW JS. A partial
structural repeat forms the heterodimer self-association site of all
beta-spectrins. J Biol Chem 269: 11400 –11408, 1994.
212. KENWRICK S, WATKINS A, AND ANGELIS ED. Neural cell recognition
molecule L1: relating biological complexity to human disease mutations. Hum Mol Genet 9: 879 – 886, 2000.
213. KIMURA K, FUKATA Y, MATSUOKA Y, BENNETT V, MATSUURA Y, OKAWA K,
IWAMATSU A, AND KAIBUCHI K. Regulation of the association of adducin with actin filaments by Rho-associated kinase (Rho-kinase)
and myosin phosphatase. J Biol Chem 273: 5542–5548, 1998.
214. KOENIG E AND REPASKY E. A regional analysis of alpha-spectrin in the
isolated Mauthner neuron and in isolated axons of the goldfish and
rabbit. J Neurosci 5: 705–714, 1985.
215. KOHRMAN DC, SMITH AL, GOLDIN J, HARRIS MR, AND MEISLER MH. A
missense mutation in the sodium channel Scn8a is responsible for
cerebellar ataxia in the mouse mutant jolting. J Neurosci 16: 5993–
5999, 1996.
216. KOOB R, ZIMMERMANN M, SCHONER W, AND DRENCKHAHN D. Colocalization and coprecipitation of ankyrin and Na⫹,K⫹-ATPase in kidney epithelial cells. Eur J Cell Biol 45: 230 –237, 1988.
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
171. HENKART M, LANDIS DM, AND REESE TS. Similarity of junctions between plasma membranes and endoplasmic reticulum in muscle
and neurons. J Cell Biol 70: 338 –347, 1976.
172. HERRMANN JM, MALKUS P, AND SCHEKMAN R. Out of the ER-outfitters,
escorts and guides. Trends Cell Biol 9: 5–7, 1999.
173. HOLLERAN EA AND HOLZBAUR EL. Speculating about spectrin: new
insights into the Golgi-associated cytoskeleton. Trends Cell Biol 8:
26 –29, 1998.
174. HOLLERAN EA, TOKITO MK, KARKI S, AND HOLZBAUR EL. Centractin
(ARP1) associates with spectrin revealing a potential mechanism
to link dynactin to intracellular organelles. J Cell Biol 135: 1815–
1829, 1996.
175. HOLTZMAN DA, WERTMAN KF, AND DRUBIN DG. Mapping actin surfaces required for functional interactions in vivo. J Cell Biol 126:
423– 432, 1994.
176. HOLZINGER A, DE RUIJTER N, EMONS AM, AND LUTZ-MEINDL U. Spectrin-like proteins in green algae (Desmidiaceae). Cell Biol Int 23:
335–344, 1999.
177. HONTS JE, SANDROCK TS, BROWER SM, O’DELL JL, AND ADAMS AE.
Actin mutations that show suppression with fimbrin mutations
identify a likely fimbrin-binding site on actin. J Cell Biol 126:
413– 422, 1994.
178. HOOCK TC, PETERS LL, AND LUX SE. Isoforms of ankyrin-3 that lack
the NH2-terminal repeats associate with mouse macrophage lysosomes. J Cell Biol 136: 1059 –1070, 1997.
179. HORNE WC, HUANG SC, BECKER PS, TANG TK, AND BENZ E JR. Tissuespecific alternative splicing of protein 4.1 inserts an exon necessary
for formation of the ternary complex with erythrocyte spectrin and
F-actin. Blood 82: 2558 –2563, 1993.
180. HORNE WC, LETO TL, AND MARCHESI VT. Differential phosphorylation
of multiple sites in protein 4.1 and protein 4 9 by phorbol esteractivated and cyclic AMP-dependent protein-kinases. J Biol Chem
260: 9073–9076, 1985.
181. HORTSCH M. Structural and functional evolution of the L1 family: are
four adhesion molecules better than one? Mol Cell Neurosci 15:
1–10, 2000.
182. HORTSCH M, HOMER D, MALHOTRA JD, CHANG S, FRANKEL J, JEFFORD G,
AND DUBREUIL RR. Structural requirements for outside-in and insideout signaling by Drosophila neuroglian, a member of the L1 family
of cell adhesion molecules. J Cell Biol 142: 251–261, 1998.
183. HOSKINS R, HAJNAL AF, HARP SA, AND KIM SK. The C. elegans vulvar
induction gene lin-2 encodes a member of the MAGUK family of
cell junction proteins. Development 122: 97–111, 1996.
184. HSUEH YP, WANG TF, YANG FC, AND SHENG M. Nuclear translocation
and transcription regulation by the membrane-associated guanylate kinase CASK/LIN-2. Nature 404: 298 –302, 2000.
185. HSUEH YP, YANG FC, KHARAZIA V, NAISBITT S, COHEN AR, WEINBERG
RJ, AND SHENG M. Direct interaction of CASK/LIN-2 and syndecan
heparan sulfate proteoglycan and their overlapping distribution in
neuronal synapses. J Cell Biol 142: 139 –151, 1998.
186. HU J, MOORTHY S, AND BENNETT V. Expression of functional domains
of beta G-spectrin disrupts epithelial morphology in cultured cells.
J Cell Biol 128: 1069 –1080, 1995.
187. HU J, WATANABE M, AND BENNETT V. Characterization of human brain
cDNA encoding the general isoform of beta-spectrin. J Biol Chem
267: 18715–18722, 1992.
188. HUANG JP, TANG CJ, KOU GH, MARCHESI VT, BENZ E JR, AND TANG TK.
Genomic structure of the locus encoding protein 4.1. Structural
basis for complex combinational patterns of tissue-specific alternative RNA splicing. J Biol Chem 268: 3758 –3766, 1993.
189. HUGHES CA AND BENNETT V. Adducin: a physical model with implications for function in assembly of spectrin-actin complexes. J Biol
Chem 270: 18990 –18996, 1995.
190. HYVONEN M, MACIAS MJ, NILGES M, OSCHKINAT H, SARASTE M, AND
WILMANNS M. Structure of the binding-site for inositol phosphates in
a PH domain. EMBO J 14: 4676 – 4685, 1995.
191. ICHIMURA T AND ELLISMAN MH. Three-dimensional fine structure of
cytoskeletal-membrane interactions at nodes of Ranvier. J Neurocytol 20: 667– 681, 1991.
192. IIDA N, LOKESHWAR VB, AND BOURGUIGNON LY. Mapping the fodrin
binding domain in CD45, a leukocyte membrane-associated tyrosine phosphatase. J Biol Chem 269: 28576 –28583, 1994.
Volume 81
July 2001
SPECTRIN AND ANKYRIN-BASED PATHWAYS
239.
240.
241.
242.
243.
244.
245.
246.
247.
248.
249.
250.
251.
252.
253.
254.
255.
256.
257.
258.
259.
260.
261.
domains required for formation of spectrin/adducin/actin complexes. J Biol Chem 271: 15695–15702, 1996.
LI X, MATSUOKA Y, AND BENNETT V. Adducin preferentially recruits
spectrin to the fast growing ends of actin filaments in a complex
requiring the MARCKS-related domain and a newly defined oligomerization domain. J Biol Chem 273: 19329 –19338, 1998.
LI ZP, BURKE EP, FRANK JS, BENNETT V, AND PHILIPSON KD. The
cardiac Na⫹-Ca2⫹ exchanger binds to the cytoskeletal protein
ankyrin. J Biol Chem 268: 11489 –11491, 1993.
LIN B, NASIR J, MCDONALD H, GRAHAM R, ROMMENS JM, GOLDBERG YP,
AND HAYDEN MR. Genomic organization of the human alpha-adducin
gene and its alternately spliced isoforms. Genomics 25: 93–99, 1995.
LIN H AND SCHAGAT T. Neuroblasts: a model for the asymmetric
division of stem cells. Trends Genet 13: 33–39, 1997.
LIN H AND SPRADLING AC. The Drosophila fusome, a germlinespecific organelle, contains membrane skeletal proteins and functions in cyst formation. Development 120: 947–956, 1994.
LIN H AND SPRADLING AC. Fusome asymmetry and oocyte determination in Drosophila. Dev Genet 16: 6 –12, 1995.
LING E, DANILOV YN, AND COHEN CM. Modulation of red cell band 4.1
function by cAMP-dependent kinase and protein kinase C phosphorylation. J Biol Chem 263: 2209 –2216, 1988.
LING E, GARDNER K, AND BENNETT V. Protein kinase C phosphorylates a recently identified membrane skeleton-associated calmodulin-binding protein in human erythrocytes. J Biol Chem 261:
13875–13878, 1986.
LIU C, DERICK LH, AND PALEK J. Visualization of the hexagonal lattice
in the erythrocyte membrane skeleton. J Cell Biol 104: 527–536,
1987.
LOKESHWAR VB AND BOURGUIGNON LY. Tyrosine phosphatase activity
of lymphoma CD45 (GP180) is regulated by a direct interaction
with the cytoskeleton. J Biol Chem 267: 21551–21557, 1992.
LOKESHWAR VB, FREGIEN N, AND BOURGUIGNON LY. Ankyrin-binding
domain of CD44(GP85) is required for the expression of hyaluronic
acid-mediated adhesion function. J Cell Biol 126: 1099 –1109, 1994.
LOMBARDO CR AND LOW PS. Calmodulin modulates protein-4.1 binding to human erythrocyte membranes. Arch Biochem Biophys
1196: 139 –144, 1994.
LOMBARDO CR, WEED SA, KENNEDY SP, FORGET BG, AND MORROW JS.
Beta II-spectrin (fodrin) and beta I epsilon 2-spectrin (muscle)
contain NH2- and COOH-terminal membrane association domains
(MAD1 and MAD2). J Biol Chem 269: 29212–29219, 1994.
LORENZ M, BISIKIRSKA B, HANUS-LORENZ B, STRZALKA K, AND SIKORSKI
AF. Proteins reacting with anti-spectrin antibodies are present in
Chlamydomonas cells. Cell Biol Int 19: 83–90, 1995.
LORENZO F, DALLAVENEZIA N, MORLE L, BAKLOUTI F, ALLOISIO N,
DUCLUZEAU MT, RODA L, LEFRANCOIS P, AND DELAUNAY J. Protein 4.1
deficiency associated with an altered binding to the spectrin-actin
complex of the red-cell membrane skeleton. J Clin Invest 94:
1651–1656, 1994.
LUKOWSKI S, LECOMTE MC, MIRA JP, MARIN P, GAUTERO H, RUSSOMARIE F, AND GENY B. Inhibition of phospholipase D activity by
fodrin. An active role for the cytoskeleton. J Biol Chem 271:
24164 –24171, 1996.
LUKOWSKI S, MIRA JP, ZACHOWSKI A, AND GENY B. Fodrin inhibits
phospholipases A2, C, and D by decreasing polyphosphoinositide
cell content. Biochem Biophys Res Commun 248: 278 –284, 1998.
LUNA EJ, KIDD GH, AND BRANTON D. Identification by peptide analysis of the spectrin-binding protein in human erythrocytes. J Biol
Chem 254: 2526 –2532, 1979.
LUNDBERG S, BUEVICH AV, SETHSON I, EDLUND U, AND BACKMAN L.
Calcium-binding mechanism of human nonerythroid alpha-spectrin
EF-structures. Biochemistry 36: 7199 –7208, 1997.
LUNDBERG S, LEHTO VP, AND BACKMAN L. Characterization of calciumbinding to spectrins. Biochemistry 31: 5665–5671, 1992.
LUO L. Trio quartet in D. Neuron 26: 1–2, 2000.
LUX SE, JOHN KM, AND BENNETT V. Analysis of cDNA for human
erythrocyte ankyrin indicates a repeated structure with homology
to tissue-differentiation and cell-cycle control proteins. Nature 344:
36 – 42, 1990.
MA Y, ZIMMER WE, RIEDERER BM, BLOOM ML, BARKER JE, GOODMAN
SR, AND GOODMAN SM. The complete amino acid sequence for brain
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
217. KORDELI E AND BENNETT V. Distinct ankyrin isoforms at neuron cell
bodies and nodes of Ranvier resolved using erythrocyte ankyrindeficient mice. J Cell Biol 114: 1243–1259, 1991.
218. KORDELI E, DAVIS J, TRAPP B, AND BENNETT V. An isoform of ankyrin
is localized at nodes of Ranvier in myelinated axons of central and
peripheral nerves. J Cell Biol 110: 1341–1352, 1990.
219. KORDELI E, LAMBERT S, AND BENNETT V. AnkyrinG. A new ankyrin
gene with neural-specific isoforms localized at the axonal initial
segment and node of Ranvier. J Biol Chem 270: 2352–2359, 1995.
220. KORDELI E, LUDOSKY MA, DEPRETTE C, FRAPPIER T, AND CARTAUD J.
AnkyrinG is associated with the postsynaptic membrane and the
sarcoplasmic reticulum in the skeletal muscle fiber. J Cell Sci 111:
2197–2207, 1998.
221. KOTULA L, DESILVA TM, SPEICHER DW, AND CURTIS PJ. Functional
characterization of recombinant human red cell alpha-spectrin
polypeptides containing the tetramer binding site. J Biol Chem 268:
14788 –14793, 1993.
222. KUHLMAN PA AND FOWLER VM. Purification and characterization of
an alpha 1 beta 2 isoform of CapZ from human erythrocytes:
cytosolic location and inability to bind to Mg2⫹ ghosts suggest that
erythrocyte actin filaments are capped by adducin. Biochemistry
36: 13461–13472, 1997.
223. KUHLMAN PA, HUGHES CA, BENNETT V, AND FOWLER VM. A new
function for adducin. Calcium/calmodulin-regulated capping of the
barbed ends of actin filaments. J Biol Chem 271: 7986 –7991, 1996.
224. KUNIMOTO M. A neuron-specific isoform of brain ankyrin, 440-kD
ankyrinB, is targeted to the axons of rat cerebellar neurons. J Cell
Biol 131: 1821–1829, 1995.
225. KUNIMOTO M, OTTO E, AND BENNETT V. A new 440-kD isoform is the
major ankyrin in neonatal rat brain. J Cell Biol 115: 1319 –1331,
1991.
226. LAMB RS, WARD RE, SCHWEIZER L, AND FEHON RG. Drosophila
coracle, a member of the protein 4.1 superfamily, has essential
structural functions in the septate junctions and developmental
functions in embryonic and adult epithelial cells. Mol Biol Cell 9:
3505–3519, 1998.
227. LAMBERT S AND BENNETT V. Postmitotic expression of ankyrinR and
beta R-spectrin in discrete neuronal populations of the rat brain.
J Neurosci 13: 3725–3735, 1993.
228. LAMBERT S, DAVIS JQ, AND BENNETT V. Morphogenesis of the node of
Ranvier: co-clusters of ankyrin and ankyrin-binding integral proteins define early developmental intermediates. J Neurosci 17:
7025–7036, 1997.
229. LAMBERT S, YU H, PRCHAL JT, LAWLER J, RUFF P, SPEICHER D, CHEUNG
MC, KAN YW, AND PALEK J. cDNA sequence for human erythrocyte
ankyrin. Proc Natl Acad Sci USA 87: 1730 –1734, 1990.
230. LECLERC E AND VETTER S. Characterization of a calcium-dependent
calmodulin-binding domain in the 135-kD human protein 4.1 isoform. Eur J Biochem 258: 567–571, 1998.
231. LEE JK, BRANDIN E, BRANTON D, AND GOLDSTEIN LS. alpha-Spectrin is
required for ovarian follicle monolayer integrity in Drosophila
melanogaster. Development 124: 353–362, 1997.
232. LEE JK, COYNE RS, DUBREUIL RR, GOLDSTEIN LS, AND BRANTON D. Cell
shape and interaction defects in alpha-spectrin mutants of Drosophila melanogaster. J Cell Biol 123: 1797–1809, 1993.
233. LEON A AND MCKEARIN D. Identification of TER94, an AAA ATPase
protein, as a Bam-dependent component of the Drosophila fusome.
Mol Biol Cell 10: 3825–3834, 1999.
234. LETO TL AND MARCHESI VT. A structural model of human-erythrocyte
protein-4.1. J Biol Chem 259: 4603– 4608, 1984.
235. LETO TL, PLEASIC S, FORGET BG, BENZ EJ JR, AND MARCHESI VT.
Characterization of the calmodulin-binding site of nonerythroid
alpha-spectrin. Recombinant protein and model peptide studies.
J Biol Chem 264: 5826 –5830, 1989.
236. LEUNG CL, SUN D, ZHENG M, KNOWLES DR, AND LIEM RK. Microtubule
actin cross-linking factor (MACF): a hybrid of dystonin and dystrophin that can interact with the actin and microtubule cytoskeletons. J Cell Biol 147: 1275–1286, 2001.
237. LEVINE J AND WILLARD M. Fodrin: axonally transported polypeptides
associated with the internal periphery of many cells. J Cell Biol 90:
631– 642, 1981.
238. LI X AND BENNETT V. Identification of the spectrin subunit and
1387
1388
262.
263.
264.
265.
267.
268.
269.
270.
271.
272.
273.
274.
275.
276.
277.
278.
279.
280.
281.
beta spectrin (beta fodrin): relationship to globin sequences. Brain
Res Mol Brain Res 18: 87–99, 1993.
MACIAS MJ, MUSACCHIO A, PONSTINGL H, NILGES M, SARASTE M, AND
OSCHKINAT H. Structure of the pleckstrin homology domain from
beta-spectrin. Nature 369: 675– 677, 1994.
MALHOTRA JD, KAZEN-GILLESPIE K, HORTSCH M, AND ISOM LL. Sodium
channel beta subunits mediate homophilic cell adhesion and recruit ankyrin to points of cell-cell contact. J Biol Chem 275: 11383–
11388, 2000.
MANGEAT PH AND BURRIDGE K. Immunoprecipitation of nonerythrocyte spectrin within live cells following microinjection of specific
antibodies: relation to cytoskeletal structures. J Cell Biol 98: 1363–
1377, 1984.
MARCHESI SL, CONBOY J, AGRE P, LETSINGER JT, MARCHESI VT,
SPEICHER DW, AND MOHANDAS N. Molecular analysis of insertion
deletion mutations in protein-4.1 in elliptocytosis. I. Biochemical
identification of rearrangements in the spectrin actin binding domain and functional characterizations. J Clin Invest 86: 516 –523,
1990.
MARFATIA SM, LEU RA, BRANTON D, AND CHISHTI AH. Identification of
the protein 4.1 binding interface on glycophorin C and p55, a
homologue of the Drosophila discs-large tumor suppressor protein. J Biol Chem 270: 715–719, 1995.
MARFATIA SM, LUE RA, BRANTON D, AND CHISHTI AH. In vitro bindingstudies suggest a membrane-associated complex between erythroid P55, protein-4.1, and glycophorin-C. J Biol Chem 269: 8631–
8634, 1994.
MATSUOKA Y, HUGHES CA, AND BENNETT V. Adducin regulation. Definition of the calmodulin-binding domain and sites of phosphorylation by protein kinases A and C. J Biol Chem 271: 25157–25166,
1996.
MATSUOKA Y, LI X, AND BENNETT V. Adducin is an in vivo substrate
for protein kinase C: phosphorylation in the MARCKS-related domain inhibits activity in promoting spectrin-actin complexes and
occurs in many cells, including dendritic spines of neurons. J Cell
Biol 142: 485– 497, 1998.
MATTAGAJASINGH SN, HUANG SC, HARTENSTEIN JS, AND BENZ EJ JR.
Characterization of the interaction between protein 4.1R and ZO-2:
a possible link between the tight junction and the actin cytoskeleton. J Biol Chem. In press.
MATTAGAJASINGH SN, HUANG SC, HARTENSTEIN JS, SNYDER M,
MARCHESI VT, AND BENZ EJ. A nonerythroid isoform of protein 4.1R
interacts with the nuclear mitotic apparatus (NuMA) protein. J Cell
Biol 145: 29 – 43, 1999.
MCCARTNEY BM AND FEHON RG. Distinct cellular and subcellular
patterns of expression imply distinct functions for the Drosophila
homologues of moesin and the neurofibromatosis 2 tumor suppressor, merlin. J Cell Biol 133: 843– 852, 1996.
MCGOUGH A, WAY M, AND DEROSIER D. Determination of the alphaactinin-binding site on actin filaments by cryoelectron microscopy
and image analysis. J Cell Biol 126: 433– 443, 1994.
MCGOUGH AM AND JOSEPHS R. On the structure of erythrocyte spectrin in partially expanded membrane skeletons. Proc Natl Acad Sci
USA 7: 5208 –5212, 1990.
MCKEARIN D. The Drosophila fusome, organelle biogenesis and
germ cell differentiation: if you build it. Bioessays 19: 147–152,
1997.
MCKEOWN C, PRAITIS V, AND AUSTIN J. sma-1 encodes a betaHspectrin homolog required for Caenorhabditis elegans morphogenesis. Development 125: 2087–2098, 1998.
MCNEILL H, OZAWA M, KEMLER R, AND NELSON WJ. Novel function of
the cell adhesion molecule uvomorulin as an inducer of cell surface
polarity. Cell 62: 309 –316, 1990.
MELDOLESI J AND POZZAN T. The heterogeneity of ER calcium stores
has a key role in nonmuscle cell signaling and function. J Cell Biol
142: 1395–1398, 1998.
MENEGOZ M, GASPAR P, LEBERT M, GALVEZ T, BURGAYA F, PALFREY C,
EZAN P, AMOS F, AND GIRAULT JA. Paranodin, a glycoprotein of
neuronal paranodal membranes. Neuron 19: 319 –331, 1997.
MICHAELY P AND BENNETT V. The membrane-binding domain of
ankyrin contains four independently folded subdomains, each comprised of six ankyrin repeats. J Biol Chem 268: 22703–22709, 1993.
MICHAELY P AND BENNETT V. The ANK repeats of erythrocyte ankyrin
282.
283.
284.
285.
286.
287.
288.
289.
290.
291.
292.
293.
294.
295.
296.
297.
298.
299.
300.
301.
302.
Volume 81
form two distinct but cooperative binding sites for the erythrocyte
anion exchanger. J Biol Chem 270: 22050 –22057, 1995.
MICHAELY P AND BENNETT V. Mechanism for binding site diversity on
ankyrin. Comparison of binding sites on ankyrin for neurofascin
and the Cl⫺/HCO⫺
3 anion exchanger. J Biol Chem 270: 31298 –
31302, 1995.
MICHAELY P, KAMAL A, ANDERSON RG, AND BENNETT V. A requirement
for ankyrin binding to clathrin during coated pit budding. J Biol
Chem 274: 35908 –35913, 1999.
MICHAUD D, GUILLET G, ROGERS PA, AND CHAREST PM. Identification
of a 220 kDa membrane-associated plant cell protein immunologically related to human beta-spectrin. FEBS Lett 294: 77– 80, 1991.
MISCHE SM, MOOSEKER MS, AND MORROW JS. Erythrocyte adducin: a
calmodulin-regulated actin-bundling protein that stimulates spectrin-actin binding. J Cell Biol 105: 2837–2845, 1987.
MISHRA L, CAI T, LEVINE A, WENG D, MEZEY E, MISHRA B, AND GEARHART JJ. Identification of elf1, a beta-spectrin, in early mouse liver
development. Int J Dev Biol 42: 221–224, 1998.
MISHRA L, CAI T, YU P, MONGA SP, AND MISHRA B. Elf3 encodes a
novel 200-kD beta-spectrin: role in liver development. Oncogene 18:
353–364, 1999.
MISSLER M AND SUDHOF TC. Neurexins: three genes and 1001 products. Trends Genet 14: 20 –26, 1998.
MOHANDAS N AND EVANS E. Mechanical properties of the red cell
membrane in relation to molecular structure and genetic defects.
Annu Rev Biophys Biomol Struct 23: 787– 818, 1994.
MOLDAY LL, COOK NJ, KAUPP UB, AND MOLDAY RS. The cGMP-gated
cation channel of bovine rod photoreceptor cells is associated with
a 240-kDa protein exhibiting immunochemical cross-reactivity with
spectrin. J Biol Chem 265: 18690 –18695, 1990.
MOORES NH, KEEP CA, AND KENDRICK-JONES J. Structure of the Utrophin actin-binding domain bound to F-actin reveals binding by an
induced fit mechanism. J Mol Biol 297: 465– 480, 2000.
MOORTHY S, CHEN L, AND BENNETT V. Caenorhabditis elegans beta-G
spectrin is dispensable for establishment of epithelial polarity, but
essential for muscular and neuronal function. J Cell Biol 149:
915–930, 2000.
MORGANS CW AND KOPITO RR. Association of the brain anion exchanger, AE3, with the repeat domain of ankyrin. J Cell Sci 105:
1137–1142, 1993.
MORINIERE M, RIBEIRO L, DALLA VENEZIA N, DEGUILLIEN M, MAILLET P,
CYNOBER T, DELHOMMEAU F, ALMEIDA H, TAMAGNINI G, DELAUNAY J,
AND BAKLOUTI F. Elliptocytosis in patients with C-terminal domain
mutations of protein 4.1 correlates with encoded messenger RNA
levels rather than with alterations in primary protein structure.
Blood 95: 1834 –1841, 2000.
MORIYAMA R, LOMBARDO CR, WORKMAN RF, AND LOW PS. Regulation of
linkages between the erythrocyte membrane and its skeleton by
2,3-diphosphoglycerate. J Biol Chem 268: 10990 –10996, 1993.
MORRIS MB AND LUX SE. Characterization of the binary interaction
between human erythrocyte protein-4.1 and actin. Eur J Biochem
231: 644 – 650, 1995.
MORROW JS, CIANCI CD, ARDITO T, MANN AS, AND KASHGARIAN M.
Ankyrin links fodrin to the alpha subunit of Na,K-ATPase in MadinDarby canine kidney cells and in intact renal tubule cells. J Cell
Biol 108: 455– 465, 1989.
MUSACCHIO A, NOBLE M, PAUPTIT R, WIERENGA R, AND SARASTE M.
Crystal structure of a Src-homology 3 (SH3) domain. Nature 359:
851– 855, 1992.
NEHLS V, DRENCKHAHN D, JOSHI R, AND BENNETT V. Adducin in erythrocyte precursor cells of rats and humans: expression and compartmentalization. Blood 78: 1692–1696, 1991.
NELSON WJ AND LAZARIDES E. Switching of subunit composition of
muscle spectrin during myogenesis in vitro. Nature 304: 364 –368,
1983.
NELSON WJ, SHORE EM, WANG AZ, AND HAMMERTON RW. Identification of a membrane-cytoskeletal complex containing the cell adhesion molecule uvomorulin (E-cadherin), ankyrin, and fodrin in
Madin-Darby canine kidney epithelial cells. J Cell Biol 11: 349 –357,
1990.
NELSON WJ AND VESHNOCK PJ. Dynamics of membrane-skeleton
(fodrin) organization during development of polarity in Madin-
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
266.
VANN BENNETT AND ANTHONY J. BAINES
July 2001
303.
304.
305.
306.
307.
309.
310.
311.
312.
313.
314.
315.
316.
317.
318.
319.
320.
321.
322.
323.
324.
Darby canine kidney epithelial cells. J Cell Biol 103: 1751–1765,
1986.
NELSON WJ AND VESHNOCK PJ. Modulation of fodrin (membrane
skeleton) stability by cell-cell contact in Madin-Darby canine kidney epithelial cells. J Cell Biol 104: 1527–1537, 1987.
NELSON WJ AND VESHNOCK PJ. Ankyrin binding to (Na⫹
⫹ K⫹)ATPase and implications for the organization of membrane
domains in polarized cells. Nature 328: 533–536, 1987.
NIGG E AND CHERRY RJ. Dimeric association of band 3 in the
erythrocyte membrane demonstrated by protein diffusion measurements. Nature 277: 493– 494, 1979.
NUNOMURA W, TAKAKUWA Y, PARRA M, CONBOY JG, AND MOHANDAS N.
Ca(2⫹)-dependent and Ca(2⫹)-independent calmodulin binding
sites in erythrocyte protein 4.1. Implications for regulation of protein 4 1 interactions with transmembrane proteins. J Biol Chem
275: 6360 – 6367, 2000.
NUNOMURA TAKAKUWA WY, PARRA M, CONBOY J, AND MOHANDAS N.
Regulation of protein 4.1R, p55, and glycophorin C ternary complex
in human erythrocyte membrane. J Biol Chem 275: 24540 –24546,
2000.
NUNOMURA W, TAKAKUWA Y, TOKIMITSU R, KRAUSS SW, KAWASHIMA M,
AND MOHANDAS N. Regulation of CD44-protein 4.1 interaction by
Ca2⫹ and calmodulin: implications for modulation of CD44-ankyrin
interaction. J Biol Chem 272: 30322–30328, 1997.
OHANIAN V, WOLFE LC, JOHN KM, PINDER JC, LUX SE, AND GRATZER
WB. Analysis of the ternary interaction of the red cell membrane
skeletal proteins spectrin, actin, and 4.1. Biochemistry 23: 4416 –
4420, 1984.
OHARA O, OHARA R, YAMAKAWA H, NAKAJIMA D, AND NAKAYAMA M.
Characterization of a new beta-spectrin gene which is predominantly expressed in brain. Mol Brain Res 57: 181–192, 1998.
OHARA R, YAMAKAWA H, NAKAYAMA M, YUASA S, AND OHARA O. Cellular
and subcellular localization of a newly identified member of the
protein 4.1 family, brain 4 1, in the cerebellum of adult and postnatally developing rats. Brain Res 117: 127–138, 1999.
OTSUKA AJ, FRANCO R, YANG B, SHIM KH, TANG LZ, ZHANG YY,
BOONTRAKULPOONTAWEE P, JEYAPRAKASH A, HEDGECOCK E, WHEATON
VI, AND SOBERY A. An ankyrin-related gene (unc-44) is necessary for
proper axonal guidance in Caenorhabditis elegans. J Cell Biol 129:
1081–1092, 1995.
OTTO E, KUNIMOTO M, MCLAUGHLIN T, AND BENNETT V. Isolation and
characterization of cDNAs encoding human brain ankyrins reveal a
family of alternatively spliced genes. J Cell Biol 114: 241–253, 1991.
OZAKI M, SASNER M, YANO R, LU HS, AND BUONANNO A. Neuregulinbeta induces expression of an NMDA-receptor subunit. Nature 390:
691– 694, 1997.
PALEK J AND LAMBERT S. Genetics of the red cell membrane skeleton.
Semin Hematol 27: 290 –332, 1990.
PARRA M, GASCARD P, WALENSKY LD, GIMM JA, BLACKSHAW S, CHAN N,
TAKAKUWA Y, BERGER T, LEE G, CHASIS JA, SNYDER SH, MOHANDAS N,
AND CONBOY JG. Molecular and functional characterization of protein 4.1B, a novel member of the protein 4 1 family with high level,
focal expression in brain. J Biol Chem 275: 3247–3255, 2000.
PARRA M, GASCARD P, WALENSKY LD, SNYDER SH, MOHANDAS N, AND
CONBOY JG. Cloning and characterization of 4.1G (EPB41L2), a new
member of the skeletal protein 4 1 (EPB41) gene family. Genomics
49: 298 –306, 1998.
PASCUA J, CASTRESANA J, AND SARASTE M. Evolution of the spectrin
repeat. Bioessays 19: 811– 817, 1997.
PASCUAL J, PFUHL M, WALTHER D, SARASTE M, AND NILGES M. Solution
structure of the spectrin repeat: a left-handed antiparallel triplehelical coiled-coil. J Mol Biol 273: 740 –751, 1997.
PASTERNACK G, ANDERSON R, LETO TL, AND MARCHESI VT. Interactions
between protein 4.1 and Band 3. J Biol Chem 260: 3676 –3683, 1985.
PAWSON T AND SCOTT JD. Signaling through scaffold, anchoring, and
adaptor proteins. Science 278: 2075–2080, 1997.
PEARSON MA, RECZEK D, BRETSCHER A, AND KARPLUS PA. Structure of
the ERM protein moesin reveals the FERM domain fold masked by
an extended actin binding tail domain. Cell 101: 259 –270, 2000.
PEDRONI S, LECOMTE MC, GAUTERO H, AND SPEICHER DW. Characterization of the phosphorylation sites of human erythrocyte spectrin.
Mol Biol Cell 6: 1569 –1569, 1995.
PELES E, NATIV M, LUSTIG M, GRUMET M, SCHILLING J, MARTINEZ R,
325.
326.
327.
328.
329.
330.
331.
332.
333.
334.
335.
336.
337.
338.
339.
340.
341.
342.
343.
344.
345.
346.
1389
PLOWMAN GD, AND SCHLESSINGER J. Identification of a novel contactin-associated transmembrane receptor with multiple domains implicated in protein-protein interactions. EMBO J 16: 978 –988, 1997.
PESACRETA TC, BYERS TJ, DUBREUIL R, KIEHART DP, AND BRANTON D.
Drosophila spectrin: the membrane skeleton during embryogenesis. J Cell Biol 108: 1697–1709, 1989.
PETERS LL, BIRKENMEIER CS, BRONSON RT, WHITE RA, LUX SE, OTTO E,
BENNETT V, HIGGINS A, AND BARKER JE. Purkinje cell degeneration
associated with erythroid ankyrin deficiency in nb/nb mice. J Cell
Biol 114: 1233–1241, 1991.
PETERS LL, JOHN KM, LU FM, EICHER EM, HIGGINS A, YIALAMAS M,
TURTZO LC, OTSUKA AJ, AND LUX SE. Ank3 (epithelial ankyrin), a
widely distributed new member of the ankyrin gene family and the
major ankyrin in kidney, is expressed in alternatively spliced
forms, including forms that lack the repeat domain. J Cell Biol 130:
313–330, 1995.
PETERS LL, WEIER HU, WALENSKY LD, SNYDER SH, PARRA M, MOHANDAS N, AND CONBOY JG. Four paralogous protein 4.1 genes map to
distinct chromosomes in mouse and human. Genomics 54: 348 –
350, 1998.
PINDER JC, GARDNER B, AND GRATZER WB. Interaction of protein-4.1
with the red-cell membrane: effects of phosphorylation by proteinkinase-C. Biochem Biophys Res Commun 210: 478 – 482, 1995.
PINDER JC AND GRATZER WB. Structural and dynamic states of actin
in the erythrocyte. J Cell Biol 96: 768 –775, 1983.
PINDER JC, OHANIAN V, AND GRATZER WB. Spectrin and protein 4.1 as
an actin filament capping complex. FEBS Lett 169: 161–164, 1984.
PINDER JC, PEKRUN A, MAGGS AM, BRAIN AP, AND GRATZER WB.
Association state of human red blood cell band 3 and its interaction
with ankyrin. Blood 85: 2951–2961, 1995.
PINDER JC, UNGEWICKELL E, BRAY D, AND GRATZER WB. The spectrinactin complex and erythrocyte shape. J Supramol Struct 8: 439 –
445, 1978.
PINTO-CORREIA C, GOLDSTEIN EG, BENNETT V, AND SOBEL JS. Immunofluorescence localization of an adducin-like protein in the chromosomes of mouse oocytes. Dev Biol 146: 301–311, 1991.
POLIAK S, GOLLAN L, MARTINEZ R, CUSTER A, EINHEBER S, SALZER JL,
TRIMMER JS, SHRAGER P, AND PELES E. Caspr2, a new member of the
neurexin superfamily, is localized at the juxtaparanodes of myelinated axons and associates with K⫹ channels. Neuron 24: 1037–
1047, 1999.
POLLARD TD. Purification of a high molecular weight actin filament
gelation protein from Acanthamoeba that shares antigenic determinants with vertebrate spectrins. J Cell Biol 99: 1970 –1980, 1984.
POLLERBERG GE, BURRIDGE K, KREBS KE, GOODMAN SR, AND
SCHACHNER M. The 180-kD component of the neural cell adhesion
molecule N-CAM is involved in a cell-cell contacts and cytoskeleton-membrane interactions. Cell Tissue Res 250: 227–236, 1987.
PORTER GA, SCHER MG, RESNECK WG, PORTER NC, FOWLER VM, AND
BLOCH RJ. Two populations of beta-spectrin in rat skeletal muscle.
Cell Motil Cytoskeleton 37: 7–19, 1997.
RASBAND MN, TRIMMER JS, PELES E, LEVINSON SR, AND SHRAGER P. K⫹
channel distribution and clustering in developing and hypomyelinated axons of the optic nerve. J Neurocytol 28: 319 –331, 1999.
REBECCHI MJ AND SCARLATA S. Pleckstrin homology domains: a
common fold with diverse functions. Annu Rev Biophys Biomol
Struct 27: 503–528, 1998.
REID ME, TAKAKUWA Y, CONBOY J, TCHERNIA G, AND MOHANDAS N.
Glycophorin C content of human erythrocyte membrane is regulated by protein 4.1. Blood 75: 2229 –2234, 1990.
REN Q AND BENNETT V. Palmitoylation of neurofascin at a site in the
membrane-spanning domain highly conserved among the L1 family
of cell adhesion molecules. J Neurochem 70: 1839 –1849, 1998.
REPASKY EA, GRANGER BL, AND LAZARIDES E. Widespread occurrence
of avian spectrin in nonerythroid cells. Cell 29: 821– 833, 1982.
REPASKY EA, SYMER DE, AND BANKERT RB. Spectrin immunofluorescence distinguishes a population of naturally capped lymphocytes
in situ. J Cell Biol 99: 350 –355, 1984.
RIEDERER BM, ZAGON IS, AND GOODMAN SR. Brain spectrin(240/235)
and brain spectrin(240/235E): two distinct spectrin subtypes with
different locations within mammalian neural cells. J Cell Biol 102:
2088 –2097, 1986.
RIEF M, PASCUAL J, SARASTE M, AND GAUB HE. Single molecule force
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
308.
SPECTRIN AND ANKYRIN-BASED PATHWAYS
1390
347.
348.
349.
350.
351.
353.
354.
355.
356.
357.
358.
359.
360.
361.
362.
363.
364.
365.
366.
367.
spectroscopy of spectrin repeats: low unfolding forces in helix
bundles. J Mol Biol 286: 553–561, 1999.
RIEFF HI, RAETZMAN LT, SAPP DW, YEH HH, SIEGEL RE, AND CORFAS G.
Neuregulin induces GABA(A) receptor subunit expression and neurite outgrowth in cerebellar granule cells. J Neurosci 19: 10757–
10766, 1999.
RODRIGUEZ MM, RON D, TOUHARA K, CHEN CH, AND MOCHLY-ROSEN D.
Rack1, a protein kinase C anchoring protein, coordinates the binding of activated protein kinase C and select pleckstrin homology
domains in vitro. Biochemistry 38: 13787–13794, 1999.
ROTIN D, BAR-SAGI D, O’BRODOVICH H, MERILAINEN J, LEHTO VP,
CANESSA CM, ROSSIER BC, AND DOWNEY GP. An SH3 binding region in
the epithelial Na⫹ channel (alpha rENaC) mediates its localization
at the apical membrane. EMBO J 13: 4440 – 4450, 1994.
RUTISHAUSER U. Defining a role and mechanism for IgCAM function
in vertebrate axon guidance. J Cell Biol 149: 757–760, 2000.
RYBAKOVA IN, AMANN KJ, AND ERVASTI JM. A new model for the
interaction of dystrophin with F-actin. J Cell Biol 135: 661– 672,
1996.
SADQI M, CASARES S, ABRIL MA, LOPEZ-MAYORGA O, CONEJERO-LARA F,
AND FREIRE E. The native state conformational ensemble of the SH3
domain from alpha-spectrin. Biochemistry 38: 8899 – 8906, 1999.
SAHR KE, LAURILA P, KOTULA L, SCARPA AL, COUPAL E, LETO TL,
LINNENBACH AJ, WINKELMANN JC, SPEICHER DW, AND MARCHESI VT.
The complete cDNA and polypeptide sequences of human erythroid alpha-spectrin. J Biol Chem 265: 4434 – 4443, 1990.
SAKAGUCHI G, ORITA S, NAITO A, MAEDA M, IGARASHI H, SASAKI T, AND
TAKAI Y. A novel brain-specific isoform of beta spectrin: isolation
and its interaction with Munc13. Biochem Biophys Res Commun
248: 846 – 851, 1998.
SANDROCK AW JR, GOODEARL AD, YIN QW, CHANG D, AND FISCHBACH
GD. Aria is concentrated in nerve terminals at neuromuscular
junctions and at other synapses. J Neurosci 15: 6124 – 6136, 1995.
SCHOTT JJ, CHARPENTIER F, PELTIER S, FOLEY P, DROUIN E, BOUHOUR
JB, MAREC HL, PASCAL O, DONNELLY P, VERGNAUD G, BACHNER L, AND
MOISAN JP. Mapping of a gene for long QT syndrome to chromosome 4q25–27. Am J Hum Genet 57: 1114 –1122, 1995.
SCHULTZ J, HOFFMULLER U, KRAUSE G, ASHURST J, MACIAS PMJ,
SCHMIEDER P, SCHNEIDER-MERGENER J, AND OSCHKINAT H. Specific
interactions between the syntrophin PDZ domain and voltage-gated
sodium channels. Nat Struct Biol 5: 19 –24, 1998.
SCOTLAND P, ZHOU D, BENVENISTE H, AND BENNETT V. Nervous system
defects of AnkyrinB (⫺/⫺) mice suggest functional overlap between the cell adhesion molecule L1 and 440-kDAnkyrinB in premyelinated axons. J Cell Biol 143: 1305–1315, 1998.
SEDGWICK SG AND SMERDON SJ. The ankyrin repeat: a diversity of
interactions on a common structural framework. Trends Biochem
Sci 24: 311–316, 1999.
SHEN BW, JOSEPHS R, AND STECK TL. Ultrastructure of the intact
skeleton of the human erythrocyte membrane. J Cell Biol 102:
997–1006, 1986.
SHI ZT, AFZAL V, COLLER B, PATEL D, CHASIS JA, PARRA M, LEE G,
PASZTY C, STEVENS M, WALENSKY L, PETERS LL, MOHANDAS N, RUBIN E,
AND CONBOY JG. Protein 4.1R-deficient mice are viable but have
erythroid membrane skeleton abnormalities. J Clin Invest 103:
331–340, 1999.
SHOTTON DM, BURKE BE, AND BRANTON D. The molecular structure of
human erythrocyte spectrin. Biophysical and electron microscopic
studies. J Mol Biol 131: 303–329, 1979.
SIKORSKI AF, SANGERMAN J, GOODMAN SR, AND CRITZ SD. Spectrin
(betaSpIIsigma1) is an essential component of synaptic transmission. Brain Res 852: 161–166, 2000.
SIKORSKI AF, SWAT W, BRZEZINSKA M, WROBLEWSKI Z, AND BISIKIRSKA B.
A protein cross-reacting with anti-spectrin antibodies is present in
higher plant cells. Z Naturforsch 48: 580 –583, 1993.
SIKORSKI AF, TERLECKI G, ZAGON IS, AND GOODMAN SR. Synapsin
I-mediated interaction of brain spectrin with synaptic vesicles.
J Cell Biol 114: 313–318, 1991.
SPEICHER DW, DESILVA TM, SPEICHER KD, URSITTI JA, HEMBACH P, AND
WEGLARZ L. Location of the human red cell spectrin tetramer binding site and detection of a related “closed” hairpin loop dimer using
proteolytic footprinting. J Biol Chem 268: 4227– 4235, 1993.
SPEICHER DW AND MARCHESI VT. Erythrocyte spectrin is comprised
368.
369.
370.
371.
372.
373.
374.
375.
376.
377.
378.
379.
380.
381.
382.
383.
384.
385.
386.
387.
388.
389.
Volume 81
of many homologous triple helical segments. Nature 311: 177–180,
1984.
SPEICHER DW, WEGLARZ L, AND DESILVA TM. Properties of human red
cell spectrin heterodimer (side-to-side) assembly and identification
of an essential nucleation site. J Biol Chem 267: 14775–14782, 1992.
SRINIVASAN Y, ELMER L, DAVIS J, BENNETT V, AND ANGELIDES K. Ankyrin
and spectrin associate with voltage-dependent sodium channels in
brain. Nature 333: 177–180, 1988.
SRINIVASAN Y, LEWALLEN M, AND ANGELIDES K. Mapping the binding
site on ankyrin for the voltage-dependent sodium channel from
brain. J Biol Chem 267: 7483–7489, 1992.
STABACH PR AND MORROW JS. Identification and characterization of
beta V spectrin, a mammalian ortholog of Drosophila beta H spectrin. J Biol Chem 275: 21385–21395, 2000.
STANKEWICH MC, TSE WT, PETERS LL, CH’NG Y, JOHN KM, STABACH PR,
DEVARAJAN P, MORROW JS, AND LUX SE. A widely expressed betaIII
spectrin associated with Golgi and cytoplasmic vesicles. Proc Natl
Acad Sci USA 95: 14158 –14163, 1998.
STEINER JP AND BENNETT V. Ankyrin-independent membrane protein-binding sites for brain and erythrocyte spectrin. J Biol Chem
263: 14417–14425, 1988.
STEINER JP, WALKE HT JR, AND BENNETT V. Calcium/calmodulin
inhibits direct binding of spectrin to synaptosomal membranes.
J Biol Chem 264: 2783–2791, 1989.
STEVEN R, KUBISESKI TJ, ZHENG H, KULKARNI S, MANCILLAS J, RUIZ
MORALES A, HOGUE CW, PAWSON T, AND CULOTTI J. Unc-73 activates
the Rac GTPase and is required for cell and growth cone migrations
in C. elegans. Cell 92: 785–795, 1998.
SUBRAHMANYAM G, BERTICSAND PJ, AND ANDERSON RA. Phosphorylation of protein 4.1 on tyrosine-418 modulates its function in vitro.
Proc Natl Acad Sci USA 88: 5222–5226, 1991.
SURIYAPPERUMA SP, LOZOVATSKY L, CICIOTTE SL, PETERS LL, AND GILLIGAN DM. The mouse adducin gene family: alternative splicing and
chromosomal localization. Mamm Genome 11: 16 –23, 2000.
TAKEI K, SHIN RM, INOUE T, KATO K, AND MIKOSHIBA K. Regulation of
nerve growth mediated by inositol 1,4,5-trisphosphate receptors in
growth cones. Science 282: 1705–1708, 1998.
TANAKA T, KADOWAKI K, LAZARIDES E, AND SOBUE K. Ca2⫹-dependent
regulation of the spectrin actin interaction by calmodulin and
protein 4.1. J Biol Chem 266: 1134 –1140, 1991.
TANG CJC AND TANG TK. The 30-kD domain of protein 4.1 mediates
its binding to the carboxyl terminus of pICln, a protein involved in
cellular volume regulation. Blood 92: 1442–1447, 1998.
TANNER MJ. The structure and function of band 3 (AE1): recent
developments (review). Mol Membr Biol 14: 155–165, 1997.
TAYLOR KA AND TAYLOR DW. Formation of two-dimensional complexes of F-actin and crosslinking proteins on lipid monolayers:
demonstration of unipolar alpha-actinin-F-actin crosslinking. Biophys J 67: 1976 –1983, 1994.
TCHERNIA G, MOHANDAS N, AND SHOHET SB. Deficiency of skeletal
membrane protein band 4.1 in homozygous hereditary elliptocytosis. Implications for erythrocyte membrane stability. J Clin Invest
68: 454 – 460, 1981.
TELEN MJ, LE VAN KIM C, CHUNG A, CARTRON JP, AND COLIN Y.
Molecular basis for elliptocytosis associated with glycophorin C
and D deficiency in the Leach phenotype. Blood 78: 1603–1606,
1991.
THEVANANTHER S, KOLLI AH, AND DEVARAJAN P. Identification of a
novel ankyrin isoform (AnkG190) in kidney and lung that associates with the plasma membrane and binds alpha-Na,K-ATPase.
J Biol Chem 273: 23952–23058, 1998.
THOMAS GH AND KIEHART DP. Beta heavy-spectrin has a restricted
tissue and subcellular distribution during Drosophila embryogenesis. Development 120: 2039 –2050, 1994.
THOMAS GH, NEWBERN EC, KORTE CC, BALES MA, MUSE SV, CLARK
AG, AND KIEHART DP. Intragenic duplication and divergence in the
spectrin superfamily of proteins. Mol Biol Evol 14: 1285–1295, 1997.
THOMAS GH, ZARNESCU DC, JUEDES AE, BALES MA, LONDERGAN A,
KORTE CC, AND KIEHART DP. Drosophila beta heavy-spectrin is
essential for development and contributes to specific cell fates in
the eye. Development 125: 2125–2134, 1998.
TISMINETZKY S, DEVESCOVI G, TRIPODI G, MURO A, BIANCHI G, COLOMBI
M, MORO L, BARLATI S, TUTEJA R, AND BARALLE FE. Genomic organi-
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
352.
VANN BENNETT AND ANTHONY J. BAINES
July 2001
390.
391.
392.
393.
394.
395.
397.
398.
399.
400.
401.
402.
403.
404.
405.
406.
407.
408.
409.
410.
411.
412.
sation and chromosomal localisation of the gene encoding human
beta adducin. Gene 167: 313–316, 1995.
TRAPP BD, ANDREWS SB, WONG A, O’CONNELL M, AND GRIFFIN JW.
Co-localization of the myelin-associated glycoprotein and the microfilament components, F-actin and spectrin, in Schwann cells of
myelinated nerve fibres. J Neurocytol 18: 47– 60, 1989.
TRAPP BD, PETERSON J, RANSOHOFF RM, RUDICK R, MORK S, AND BO L.
Axonal transection in the lesions of multiple sclerosis. N Engl
J Med 338: 278 –285, 1998.
TRAVE G, LACOMBE PJ, PFUHL M, SARASTE M, AND PASTORE A. Molecular mechanism of the calcium-induced conformational change in
the spectrin EF-hands. EMBO J 14: 4922– 4931, 1995.
TRAVE G, PASTORE A, HYVONEN M, AND SARASTE M. The C-terminal
domain of alpha-spectrin is structurally related to calmodulin. Eur
J Biochem 227: 35– 42, 1995.
TSCHOPP J, MARTINON F, AND HOFMANN K. Apoptosis: silencing the
death receptors. Curr Biol 9: R381–R384, 1999.
TSE WT, LECOMTE MC, COSTA FF, GARBARZ M, FEO C, BOIVIN P,
DHERMY D, AND FORGET BG. Point mutation in the beta-spectrin gene
associated with alpha I/74 hereditary elliptocytosis. Implications
for the mechanism of spectrin dimer self-association. J Clin Invest
86: 909 –916, 1990.
TSE WT AND LUX SE. Red blood cell membrane disorders. Br J
Hematol 104: 2–13, 1999.
TUVIA S, BUHUSI M, DAVIS L, REEDY M, AND BENNETT V. Ankyrin-B is
required for intracellular sorting of structurally diverse Ca2⫹ homeostasis proteins. J Cell Biol 147: 995–1008, 1999.
TUVIA S, GARVER TD, AND BENNETT V. The phosphorylation state of
the FIGQY tyrosine of neurofascin determines ankyrin-binding activity and patterns of cellsegregation. Proc Natl Acad Sci USA 94:
12957–12962, 1997.
TYLER JM, HARGREAVES DR, AND BRANTON D. Purification of two
spectrin-binding proteins: biochemical and electron microscopic
evidence for site-specific reassociation between spectrin and bands
2.1 and 4.1. Proc Natl Acad Sci USA 76: 5192–5196, 1979.
TYLER JM, REINHARDT BN, AND BRANTON D. Associations of erythrocyte membrane proteins. Binding of purified bands 2.1 and 4.1 to
spectrin. J Biol Chem 255: 7034 –7039, 1980.
UNGEWICKELL E, BENNETT PM, CALVERT R, OHANIAN V, AND GRATZER
WB. In vitro formation of a complex between cytoskeleton proteins
of the human erythrocyte. Nature 280: 811– 814, 1979.
URSITTI JA AND FOWLER VM. Immunolocalization of tropomodulin,
tropomyosin and actin in spread human erythrocyte skeletons.
J Cell Sci 107: 1633–1639, 1994.
URSITTI JA, KOTULA L, DESILVA TM, CURTIS PJ, AND SPEICHER DW.
Mapping the human erythrocyte beta-spectrin dimer initiation site
using recombinant peptides and correlation of its phasing with the
alpha-actinin dimer site. J Biol Chem 271: 6636 – 6644, 1996.
VABNICK I, NOVAKOVIC SD, LEVINSON SR, SCHACHNER M, AND SHRAGER
P. The clustering of axonal sodium channels during development of
the peripheral nervous system. J Neurosci 16: 4914 – 4922, 1996.
VAN CAMP G, FRANSEN E, VITS L, RAES G, AND WILLEMS PJ. A locusspecific mutation database for the neural cell adhesion molecule
L1CAM (Xq28). Hum Mutat 8: 391, 1996.
VASILESCU V AND FILIP DA. The Ranvier node as a chemo-electric
pulsatory unit: a study of its structure-functions relations. Physiologie 16: 83–91, 1979.
VERTEL BM, WALTERS LM, AND MILLS D. Subcompartments of the
endoplasmic reticulum. Semin Cell Biol 3: 325–341, 1992.
VIEL A. Alpha-actinin and spectrin structures: an unfolding family
story. FEBS Lett 460: 391–394, 1999.
VIEL A AND BRANTON D. Interchain binding at the tail end of the
Drosophila spectrin molecule. Proc Natl Acad Sci USA 91: 10839 –
10843, 1994.
VIEL A AND BRANTON D. Spectrin: on the path from structure to
function. Curr Opin Cell Biol 8: 49 –55, 1996.
VIEL A, GEE MS, TOMOOKA L, AND BRANTON D. Motifs involved in
interchain binding at the tail-end of spectrin. Biochim Biophys
Acta 1384: 396 – 404, 1998.
WALENSKY LD, BLACKSHAW S, LIAO D, WATKINS CC, WEIER HU, PARRA
M, HUGANIR RL, CONBOY JG, MOHANDAS N, AND SNYDER SH. A novel
neuron-enriched homolog of the erythrocyte membrane cytoskeletal protein 4.1. J Neurosci 19: 6457– 6467, 1999.
1391
413. WALENSKY LD, SHI ZT, BLACKSHAW S, DEVRIES AC, DEMAS GE, GASCARD
P, NELSON RJ, CONBOY JG, RUBIN EM, SNYDER SH, AND MOHANDAS N.
Neurobehavioral deficits in mice lacking the erythrocyte membrane cytoskeletal protein 4.1. Curr Biol 8: 1269 –1272, 1998.
414. WALLIS C, WENEGIEME E, AND BABITCH J. Characterization of calcium
binding to brain spectrin. J Biol Chem 267: 4333– 4337, 1992.
415. WANG DS, MILLER R, SHAW R, AND SHAW G. The pleckstrin homology
domain of human beta I sigma II spectrin is targeted to the plasma
membrane in vivo. Biochem Biophys Res Commun 225: 420 – 426,
1996.
416. WANG DS AND SHAW G. The association of the C-terminal region of
beta I sigma II spectrin to brain membranes is mediated by a PH
domain, does not require membrane proteins, and coincides with a
inositol-1,4,5 triphosphate binding site. Biochem Biophys Res Commun 217: 608 – 615, 1995.
417. WARD RE AND FEHON RG. Structure/function analysis of Drosophila
coracle, a protein 4.1 homologue required for morphogenesis and
cell proliferation. Mol Biol Cell 7: 3521–3521, 1996.
418. WARD RE, LAMB RS, AND FEHON RG. A conserved functional domain
of Drosophila coracle is required for localization at the septate
junction and has membrane-organizing activity. J Cell Biol 140:
1463–1473, 1998.
419. WASENIUS VM, SARASTE M, SALVEN P, ERAMAA M, HOLM L, AND LEHTO
VP. Primary structure of the brain alpha-spectrin. J Cell Biol 108:
79 –93, 1989.
420. WATANABE T, INUI M, CHEN BY, IGA M, AND SOBUE K. Annexin
VI-binding proteins in brain. Interaction of annexin VI with a membrane skeletal protein, calspectin (brain spectrin or fodrin). J Biol
Chem 269: 17656 –17662, 1994.
421. WEBER A, PENNISE CR, BABCOCK GG, AND FOWLER VM. Tropomodulin
caps the pointed ends of actin filaments. J Cell Biol 127: 1627–1635,
1994.
422. WECHSLER A AND TEICHBERG VI. Brain spectrin binding to the NMDA
receptor is regulated by phosphorylation, calcium and calmodulin.
EMBO J 17: 3931–3939, 1998.
423. WHITTAKER KL, DING D, FISHER WW, AND LIPSHITZ HD. Different
3⬘-untranslated regions target alternatively processed hu-li tai shao
(hts) transcripts to distinct cytoplasmic locations during Drosophila oogenesis. J Cell Sci 112: 3385–3398, 1999.
424. WILLARDSON BM, THEVENIN BJ, HARRISON ML, BENSON WB, LOW MD,
AND LOW PS. Localization of the ankyrin-binding site on erythrocyte
membrane protein, band 3. J Biol Chem 264: 15893–15899, 1989.
425. WINCKLER B, FORSCHER P, AND MELLMAN I. A diffusion barrier maintains distribution of membrane proteins in polarized neurons. Nature 397: 698 –701, 1999.
426. WINDER SJ, HEMMINGS L, MACIVER SK, BOLTON SJ, TINSLEY JM, DAVIES
KE, CRITCHLEY DR, AND KENDRICK-JONES J. Utrophin actin binding
domain: analysis of actin binding and cellular targeting. J Cell Sci
108: 63–71, 1995.
427. WINKELMANN JC, CHANG JG, TSE WT, SCARPA AL, MARCHESI VT, AND
FORGET BG. Full-length sequence of the cDNA for human erythroid
beta-spectrin. J Biol Chem 265: 11827–11832, 1990.
428. WINKELMANN JC, COSTA FF, LINZIE BL, AND FORGET BG. Beta spectrin
in human skeletal muscle. Tissue-specific differential processing of
3⬘ beta spectrin premRNA generates a beta spectrin isoform with a
unique carboxyl terminus. J Biol Chem 265: 20449 –20454, 1990.
429. WINKELMANN JC AND FORGET BG. Erythroid and nonerythroid. Blood
81: 3173–3185, 1993.
430. WOLLNER DA AND CATTERALL WA. Localization of sodium channels in
axon hillocks and initial segments of retinal ganglion cells. Proc
Natl Acad Sci USA 83: 8424 – 8428, 1986.
431. WOOD SJ AND SLATER CR. Beta-spectrin is colocalized with voltagegated sodium channels and ankyrinG at the adult rat neuromuscular
junction. J Cell Biol 140: 675– 684, 1998.
432. XU J, ZIEMNICKA D, MERZ GS, AND KOTULA L. Human spectrin src
homology 3 domain binding protein 1 regulates macropinocytosis
in NIH 3T3 cells. J Cell Sci 113:3805–3814, 2000.
433. YAMAMOTO K, MERRY AC, AND SIMA AA. An orderly development of
paranodal axoglial junctions and bracelets of Nageotte in the rat
sural nerve. Brain Res 96: 36 – 45, 1996.
434. YAN Y, WINOGRAD E, VIEL A, CRONIN T, HARRISON SC, AND BRANTON D.
Crystal structure of the repetitive segments of spectrin. Science
262: 2027–2030, 1993.
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
396.
SPECTRIN AND ANKYRIN-BASED PATHWAYS
1392
VANN BENNETT AND ANTHONY J. BAINES
447. ZHANG P, TALLURI S, DENG H, BRANTON D, AND WAGNER G. Solution
structure of the pleckstrin homology domain of Drosophila betaspectrin. Structure 3: 1185–1195, 1995.
448. ZHANG X AND BENNETT V. Identification of O-linked N-acetylglucosamine modification of ankyrinG isoforms targeted to nodes of
Ranvier. J Biol Chem 271: 31391–31398, 1996.
449. ZHANG X AND BENNETT V. Restriction of 480/270 kD ankyrin-G to
axon proximal segments requires multiple ankyrin-G-specific domains. J Cell Biol 142: 1571–1581, 1998.
450. ZHANG X, DAVIS JQ, CARPENTER S, AND BENNETT V. Structural requirements for association of neurofascin with ankyrin. J Biol Chem
273: 30785–30794, 1998.
451. ZHANG Z, DEVARAJAN P, DORFMAN AL, AND MORROW JS. Structure of
the ankyrin-binding domain of alpha-Na,K-ATPase. J Biol Chem
273: 18681–18684, 1998.
452. ZHOU D, BIRKENMEIER CS, WILLIAMS MW, SHARP JJ, BARKER JE, AND
BLOCH RJ. Small, membrane-bound, alternatively spliced forms of
ankyrin 1 associated with the sarcoplasmic reticulum of mammalian skeletal muscle. J Cell Biol 136: 621– 631, 1997.
453. ZHOU D, LAMBERT S, MALEN PL, CARPENTER S, BOLAND LM, AND
BENNETT V. AnkyrinG is required for clustering of voltage-gated Na
channels at axon initial segments and for normal action potential.
J Cell Biol 143: 1295–1304, 1998.
454. ZHOU D, URSITTI JA, AND BLOCH RJ. Developmental expression of
spectrins in rat skeletal muscle. Mol Biol Cell 9: 47– 61, 1998.
455. ZHU X, LAI C, THOMAS S, AND BURDEN SJ. Neuregulin receptors, erbB3
and erbB4, are localized at neuromuscular synapses. EMBO J 14:
5842–5848, 1995.
456. ZIEMNICKA-KOTULA D, XU J, GU H, POTEMPSKA A, KIM KS, JENKINS EC,
TRENKNER E, AND KOTULA L. Identification of a candidate human
spectrin Src homology 3 domain-binding protein suggests a general
mechanism of association of tyrosine kinases with the spectrinbased membrane skeleton. J Biol Chem 273: 13681–13692, 1998.
457. ZUCKERMAN JB, CHEN X, JACOBS JD, HU B, KLEYMAN TR, AND SMITH PR.
Association of the epithelial sodium channel with Apx and alphaspectrin in A6 renal epithelial cells. J Biol Chem 274: 23286 –23295,
1999.
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on July 4, 2017
435. YANG Y, BAUER C, STRASSER G, WOLLMAN R, JULIEN JP, AND FUCHS E.
Integrators of the cytoskeleton that stabilize microtubules. Cell 98:
229 –238, 1999.
436. YANG Y, DOWLING J, YU QC, KOUKLIS P, CLEVELAND DW, AND FUCHS E.
An essential cytoskeletal linker protein connecting actin microfilaments to intermediate filaments. Cell 86: 655– 665, 1996.
437. YE K, COMPTON DA, LAI MM, WALENSKY LD, AND SNYDER SH. Protein
4.1N binding to nuclear mitotic apparatus protein in PC12 cells
mediates the antiproliferative actions of nerve growth factor.
J Neurosci 19: 10747–10756, 1999.
438. YEAMAN C, GRINDSTAFF KK, AND NELSON WJ. New perspectives on
mechanisms involved in generating epithelial cell polarity. Physiol
Rev 79: 73–98, 1999.
439. YI SJ, LIU SC, DERICK LH, MURRAY J, BARKER JE, CHO MR, PALEK J,
AND GOLAN DE. Red cell membranes of ankyrin-deficient nb/nb mice
lack band 3 tetramers but contain normal membrane skeletons.
Biochemistry 36: 9596 –9604, 1997.
440. YU J, FISCHMAN DA, AND STECK TL. Selection solubilization of proteins and phospholipids from red blood cell membranes by nonionic detergents. J Supramol Struct 1: 233–248, 1973.
441. YU J AND GOODMAN SR. Syndeins: the spectrin-binding protein(s) of
the human erythrocyte membrane. Proc Natl Acad Sci USA 76:
2340 –2344, 1979.
442. YU J AND STECK TL. Isolation and characterization of band 3, the
predominant polypeptide of the human erythrocyte membrane.
J Biol Chem 250: 9170 –9175, 1975.
443. YUAN LL AND GANETZKY B. A glial-neuronal signaling pathway revealed by mutations in a neurexin-related protein. Science 283:
1343–1345, 1999.
444. YUE L AND SPRADLING AC. Hu-li tai shao, a gene required for ring
canal formation during Drosophila oogenesis, encodes a homolog
of adducin. Genes Dev 6: 2443–2454, 1992.
445. ZACCAI M AND LIPSHITZ HD. Role of Adducin-like (hu-li tai shao)
mRNA and protein localization in regulating cytoskeletal structure
and function during Drosophila oogenesis and early embryogenesis. Dev Genet 19: 249 –257, 1996.
446. ZARNESCU DC AND THOMAS GH. Apical spectrin is essential for epithelial morphogenesis but not apicobasal polarity in Drosophila.
J Cell Biol 146: 1075–1086, 1999.
Volume 81