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Review Article
Anatomy of the red cell membrane skeleton: unanswered questions
Samuel E. Lux IV
Dana-Farber/Boston Children’s Cancer and Blood Disorders Center, and Department of Medicine, Boston Children’s Hospital, Boston, MA
The red cell membrane skeleton is a
pseudohexagonal meshwork of spectrin,
actin, protein 4.1R, ankyrin, and actinassociated proteins that laminates the
inner membrane surface and attaches to
the overlying lipid bilayer via band 3–
containing multiprotein complexes at the
ankyrin- and actin-binding ends of spectrin. The membrane skeleton strengthens
the lipid bilayer and endows the membrane with the durability and flexibility to
survive in the circulation. In the 36 years
since the first primitive model of the red
cell skeleton was proposed, many additional proteins have been discovered, and
their structures and interactions have
been defined. However, almost nothing
is known of the skeleton’s physiology,
and myriad questions about its structure
remain, including questions concerning the
structure of spectrin in situ, the way spectrin
and other proteins bind to actin, how the
membrane is assembled, the dynamics of
the skeleton when the membrane is deformed or perturbed by parasites, the role
lipids play, and variations in membrane
structure in unique regions like lipid rafts.
This knowledge is important because the red
cell membrane skeleton is the model for
spectrin-based membrane skeletons in all
cells, and because defects in the red cell
membrane skeleton underlie multiple hemolytic anemias. (Blood. 2016;127(2):187-199)
History
Modern knowledge of the red cell plasma membrane and its membrane
skeleton began with Marchesi and Steers’s identification of spectrin in
1968.1 Prior to that year, almost the only thing known about membranes
was that they contained a lipid bilayer.2 Indeed, there was a period in the
1960s when it was believed that red cell membranes contained only a
single 22.5-kDa protein called “structural protein.”3 The fertile decade
following the discovery of spectrin and the more or less coincident
introduction of sodium dodecyl sulfate polyacrylamide gel electrophoresis as an analytic tool led to the isolation and characterization of several
major red cell membrane proteins. The first simple model of the
membrane skeleton appeared 36 years ago (Figure 1),4 and although it
contained the core elements of the modern model, a great deal has been
learned in the intervening decades (Figure 25). Hundreds of individuals
have contributed to our knowledge of the red cell membrane skeleton,
but a few deserve special recognition, including Peter Agre, David
Anstee, G. Vann Bennett, Daniel Branton, Lesley Bruce, Jean Delaunay,
David Discher, Velia Fowler, Patrick Gallagher, Walter Gratzer, Philip
Low, Vincent Marchesi, Narla Mohandas, Jon Morrow, Jeri Palek,
Luanne Peters, David Speicher, Ted Steck, and Michael Tanner.
This review focuses on our current understanding of the anatomy of
the membrane skeleton, particularly its spectrin-actin core, and on some
of the key unanswered questions. Because of space limitations, many
important aspects of the red cell membrane are not discussed. These
include integral membrane proteins not attached to the skeleton, the lipid
bilayer, membrane biogenesis, posttranslational modifications of the
membrane, red cell aging, and mouse and human membrane diseases.
For interested readers, a few other recent reviews are recommended.6-10
The red cell membrane skeleton
The red cell membrane contains ;20 major proteins and at least 850
minor ones.11 Some of the important ones are described in Table 1. The
Submitted December 2, 2014; accepted March 30, 2015. Prepublished online
as Blood First Edition paper, November 4, 2015; DOI 10.1182/blood-2014-12512772.
BLOOD, 14 JANUARY 2016 x VOLUME 127, NUMBER 2
integral membrane proteins are organized into macromolecular complexes centered on band 3, the anion-exchange channel. Most of the
peripheral membrane proteins form the membrane skeleton, a protein
meshwork 40- to 90-nm thick that laminates the inner membrane
surface. The skeleton is composed principally of spectrin, actin and its
associated proteins (tropomyosin, tropomodulin, adducin, and dematin),
protein 4.1R, and ankyrin.
Spectrin
Erythrocyte spectrin is a long, flexible, wormlike protein composed of 2
parallel chains (a- and b-spectrin) oriented in opposite directions. Each
chain contains multiple spectrin-type repeats, with specialized functional domains at the “head” end for spectrin dimer-tetramer association
and for erythrocyte ankyrin (also known as ankyrin-1 or ankyrin-R)
binding, and domains at the “tail” end for binding to protein 4.1R,
protein 4.2, short filaments of actin, and other proteins. The details of
spectrin structure are shown in Figure 3.12-26 On average, 6 spectrins
bind per actin filament, leading to a pseudohexagonal arrangement.27
Isolated a- and b-spectrin chains bind to each other electrostatically
at a pair of nucleation sites formed by repeats near the spectrin tail
(Figure 3A). After this initial connection, the rest of the a and b chains
zip together, coiling around each other approximately every 428 repeats.
The side-to-side interactions beyond the nucleation site are relatively
weak,29 allowing the 2 chains to slide past each other when the spectrin
molecule flexes and extends during membrane deformation.
Spectrin a and b chains associate with each other at the head end.
Basically, the complementary partial repeats (a0 and b17) at the end of
each chain join to form a complete 3-helix repeat: a0/b17 (Figure 3A).
In spectrin ab dimers, the longer a chain folds back on itself, so the
a0/b17 repeat interacts with repeat a4 and the a1 and a3 repeats contact each other (Figure 3A).13 In the a2b2-heterotetramers, there are 3
such side-to-side interactions between the longer a chains (Figure 3C).
These lateral interactions, though weak, contribute to self-association.
© 2016 by The American Society of Hematology
187
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BLOOD, 14 JANUARY 2016 x VOLUME 127, NUMBER 2
a continuous helix across the junction between repeats (Figure 3B).
There is evidence that the helical linker may bend in certain directions at
the junction35 and that helices within some repeats may “melt” and
rearrange, shortening the repeat.35 In addition, some of the repeats are
unstable at physiological temperatures (starred repeats in Figure 3A)23
and may partially unfold when red cells deform, which would
contribute to flexibility.36
The actin protofilament and its associated proteins
Figure 1. The first model of the red cell membrane skeleton, published 36
years ago. Reprinted from Lux4 with permission.
Higher oligomers (hexamers, octamers, and so forth) form by the same
mechanism and can also be seen on the membrane.27,30,31 Even though
nearly 95% of the spectrin is in the tetramer or oligomer forms,32 spectrin
self-association is uniquely weak in the red cell.33 Tetramers dissociate
and reform under physiological conditions, and this is greatly accentuated when the membrane is distorted by shear forces.34 This mechanism may be an evolutionary accommodation to permit the enormous
distortions that the red cell undergoes in parts of the microvasculature.
Spectrin is a highly flexible molecule, but it is not certain how it
achieves its flexibility because crystal structures of tandem repeats show
Actin. Red cells contain short, double-helical filaments of nonmuscle
or b-actin, termed “protofilaments” (Figure 2). These lie roughly
parallel to the membrane plane (620°) and are randomly oriented.37
There are ;30 000 to 40 000 protofilaments per red cell, with 6 to 8
actin monomers in each of the 2 strands.
Not much is known about how these unique actin filaments form,
but some hints are emerging from defects in actin assembly. Mice that
lack both Rac1 and Rac2 GTPases,38 or lack Hem-1, a member of the
Wiskott-Aldrich syndrome verprolin-homologous (WAVE) protein
complex that regulates F-actin polymerization,39 develop hemolytic
anemias with misshaped red cells and clumped or irregular skeletons.
This finding suggests that Rac GTPases and Hem-1 help organize or
regulate the red cell membrane skeleton, acting through pathways that
stimulate actin polymerization in many cell types. It is unclear whether
such regulation occurs dynamically in the mature red cell or just during
erythropoiesis.
Tropomyosin. Erythrocyte tropomyosin (TM) is a long rodlike
dimer of a- and g-TM isoforms (hTM5b and hTM5, respectively).10
One TM dimer binds to each of the 2 strands in the actin protofilament
Figure 2. Current model of the red cell membrane. Most of the known protein contacts are shown, but the relative positions of the proteins to each other within the various
complexes are mostly not known. The major proteins are drawn roughly to scale, but the shapes are mostly imaginary. Approximately 40% of the band 3 molecules are
tetramers in a complex with ankyrin and other integral proteins near the spectrin self-association site (Ankyrin complex). An approximately similar fraction of the band 3
molecules, probably dimers, are located near the spectrin-actin junction and bind to spectrin via protein 4.1R (4.1), protein 4.2 (4.2), and adducin (Actin junctional complex). As
described later in the text, it is likely that these 2 complexes, with their associated proteins, are large enough that they sometimes contact each other. The remaining band 3
dimers float untethered within the lipid bilayer (Unbound band 3). The actin protofilament lies parallel to the membrane. The complexes of proteins associated with band 3 are
not constant; that is, some proteins are present in much smaller numbers than others (eg, Kell, Kx, CD44, CD47, DARC/Duffy, LW, phosphofructokinase, and aldolase). The
amounts of p55, adducin, and dematin are also insufficient to interact with all the spectrin/protein 4.1/actin complexes. As a consequence, the ankyrin and actin junctional
complexes must vary in composition and mobility. For visual clarity, peroxiredoxin 2 (Prx2) is shown attached only to unbound band 3, but there is no evidence for this
selectivity. CH, calponin homology; CH1 and CH2, actin binding domains of b-spectrin; EF, calcium ion–binding EF hand domain of a-spectrin; F-actin, filamentous actin;
GEC, glycolytic enzyme complex (glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, lactic dehydrogenase, pyruvate kinase, aldolase, and enolase); Glut1,
glucose transporter 1; GPA, glycophorin A; GPB, glycophorin B; GPC/D, glycophorins C and D; LW, Landsteiner-Wiener; RhAG, Rh-associated glycoprotein. Professional
illustration by Somersault18:24. Adapted from Korsgren et al5 with permission.
11.5
GPD
82
143
170-250
1000
200-700
500
130
6-13
17
6-10
Unknown
100
heterodimer?
Monomer? Homo or
Homo or heterodimer
Dimer and tetramer
Tetramer
Monomer or trimer?
Unknown
Monomer
Unknown
Unknown
Unknown
Dimer or tetramer
Monomer
124 6 11
1200
Tetramer
Heterodimer/ tetramer
Oligomer (;14)
Oligomeric state
20
30
30
500
2
?
1
,1
2 (dimer)
1
,1
Complex
6
?
3
,1
6 (dimer)
3
,1
Unit cell‡
2
6
1-6
1-6 (dimer)
1-3
1 (if trimer)
3 (if monomer)
,1
,1
1-6 (dimer)
,1
1 (dimer)
1
Complex and unit cell‡
Oligomers in actin
junction complex
Functions in mature erythrocyte membranes
p55, and band 3. Carries Gerbich blood group antigens.
Links actin junctional complex to membrane via interactions with protein 4.1R,
antigens and much of the red cell’s negative surface charge.
facilitate band 3 transfer to the membrane. Carries MNSsU blood group
Binds band 3 in the ankyrin-linked band 3 complex, and maybe to RhAG. May
band 3.
Links actin to the membrane via interactions with dematin, adducin, and
Transports glucose. Transports L-dehydroascorbic acid when bound to stomatin.
LDH.
Forms glycolytic metabolon on band 3 with aldolase, PFK, enolase, PK, and
attach actin to the membrane via Glut1.
Binds and bundles actin filaments. Facilitates spectrin-actin binding. Helps
Plasmodium vivax. Function in normal red cell unknown.
Chemokine receptor. Carries the Duffy (Fy) blood group antigen. Receptor for
ankyrin-linked band 3 complex. Inhibits phagocytosis.
Binds to protein 4.2 and to the Rh complex (RhAG/RhD/RhCE) within the
nonerythroid cells. Function in the red cell is unknown.
Binds to protein 4.1R and maybe to ankyrin. Multifunctional protein in
transport and CO2 elimination.
channel. Catalyzes interconversion of CO2 and HCO32, which aids in HCO32
Attaches to the outside (CAIV) and inside (CAII) surfaces of band 3, the HCO32
deoxyhemoglobin. Carries Ii and many other blood group antigens.
metabolon with G3PD, PFK, PGK, aldolase, LDH, and PK. Binds
4.2, p55, adducin, and Glut1. Binds peroxiredoxin. Forms a glycolytic
protein 4.2, and linked to the actin junctional complex via protein 4.1R, protein
stomatin, and other proteins that are linked to spectrin via ankyrin-1 and
CAIV. Forms membrane complexes with GPA, GPB, the Rh complex, Glut1,
Anion-exchange channel (especially HCO32 and Cl2) with the aid of CAII and
RhAG in the ankyrin-linked band 3 complex.
Attaches b-spectrin and the membrane skeleton to band 3 and to protein 4.2 and
Forms glycolytic complex on band 3 with PFK, enolase, G3PD, PK, and LDH.
chorein.
on actin. Links actin junction to band 3, Glut1, and maybe stomatin. Binds
Caps the fast-growing (barbed) end of actin and recruits spectrin to nearby sites
tropomyosin, tropomodulin, adducin, dematin, and chorein.
membrane skeleton. Binds spectrin with the aid of protein 4.1R. Binds
Forms ;37-nm long filaments that serve as a molecular junction in the
Ankyrin-1, erythrocyte ankyrin; BCAM, basal cell adhesion molecule; CAII, carbonic anhydrase II; CAIV, carbonic anhydrase IV; Cl , chloride ion; CO2, carbon dioxide; G3PD, glyceraldehyde-3-phosphate dehydrogenase; HCO32,
bicarbonate; ICAM4, intercellular adhesion molecule 4; LDH, lactic dehydrogenase; Lu, Lutheran; NH3/NH41, ammonia/ammonium ion; PFK, phosphofructokinase; PGK, phosphoglycerate kinase; PK, pyruvate kinase; TM, tropomyosin.
*Size of the mature protein chain without any signal peptide, propeptides, or initiator methionine, but not including posttranslational modifications.
†References for the numbers of copies of each protein are listed in the footnotes of Table 2 found in Lux6. It must be emphasized that the numbers of many red cell membrane proteins are roughly estimated but not accurately measured,
which affects the estimates of the composition of the ankyrin complex and actin junctional complex.
‡The unit cell is defined in Figure 8.
§Data shown are for full-length ankyrin (band 2.1), the major isoform, except for the number of copies per cell, which includes the smaller isoforms: bands 2.2, 2.3, and 2.6.
13.8
35.6
DARC/Duffy
GPC
33.3
CD47
7.7
37.1
CD44
GPB
33.1
CAIV
14.3
29.1
CAII
GPA
101.8
Band 3 (SLC4A1)
54.1
206.0
Ankyrin-1§
Glut1
39.4
Aldolase A
35.9
80.7
b-Adducin
G3PD
81.0
a-Adducin
43.1/45.5
41.6
b-Actin
Monomers
per cell† 3 1023
Oligomers in ankyrin
complex
BLOOD, 14 JANUARY 2016 x VOLUME 127, NUMBER 2
Dematin
Molecular
mass, kDa*
Protein
Table 1. Some important erythrocyte membrane proteins
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ANATOMY OF THE RED CELL MEMBRANE SKELETON
189
64.3
27.0
Lu/BCAM
LW glycoprotein/
85.2
66.4
79.8/76.9
PFK (muscle)
Protein 4.1R
Protein 4.2
31.7
40.6
32.9
32.9
Stomatin
Tropomodulin 1
Tropomyosin 1 (a-TM)
Tropomyosin 3 (g-TM)
Oligomeric state
Monomer?
;280
100-200
140
30
200-400?
242
242
100-200
Homodimer
Monomer
Oligomer (9-12)?
tetramer/oligomer
Heterodimer/
RhD, and RhCE?
Heterotrimer of RhAG,
Monomer
;240
100-200
Heterotetramer
Dimer or decamer
Dimer?
Monomer
Unknown
Heterodimer with Kell
Heterodimer with Kx
6
200
80
3-5
1.5-8
Unknown
3-18
Monomers
per cell† 3 1023
?
2 (dimer)
1
2
,1
?
,1
Complex
?
6 (dimer)
3
6
,1
?
,1
Unit cell‡
Oligomers in ankyrin
complex
,1
2
2
1
1
6 (dimer)
1
6
,1
?
1
the Kell blood group antigen. Kx probably has an undiscovered transport
,1
May help specify actin filament lengths in the red cell.
Binds to and stabilizes actin filaments in a magnesium ion–dependent manner.
Caps slow-growing (pointed) end of actin. Binds TM, which strengthens capping.
3, adducin, and peroxiredoxin. Located in lipid rafts.
Binds to Glut1 and converts it into an ascorbic acid transporter. Also binds band
ankyrin-1 and Lu/BCAM at the head end.
hand domain. Self-associates into tetramers and oligomers and binds to
protein 4.1R, adducin, and dematin. Binds protein 4.2 via the a-spectrin EF
Cross-links actin filaments into a hexagonal lattice at the tail end with the help of
ankyrin-linked band 3 complex. RhAG transports NH3/NH41 and maybe CO2.
The RhAG/RhD/RhCE trimer binds to CD47, LW, ankyrin, CD47, and GPB in the
binds to the EF hand domain of a-spectrin.
Binds to band 3, ankyrin, and CD47 in the ankyrin-linked band 3 complex. Also
band 3. Competes with ankyrin for binding to band 3.
interaction. Links actin to the membrane via interactions with GPC/D, p55, and
Binds to both b-spectrin and actin and greatly strengthens the spectrin-actin
LDH.
Forms glycolytic metabolon on band 3 with aldolase, G3PD, enolase, PK, and
(Gardos) channel.
Also binds stomatin and activates the calcium ion–dependent potassium ion
Abundant antioxidant protein that is partially linked to band 3 on the membrane.
organization of lipid rafts.
4.1R and GPC/D molecules. Heavily palmitoylated. May contribute to
Links actin junctional complex to membrane via interactions with some protein
and probably resides in the ankyrin-linked band 3 complex.
ICAM that carries the LW blood group antigen. Interacts with RhAG/RhD/RhCE
Adhesion molecule that carries the Lu blood group antigen. Binds to a-spectrin.
function.
Functions in mature erythrocyte membranes
The Kx/Kell complex may reside in the actin-linked band 3 complex. Kell carries
Complex and unit cell‡
Oligomers in actin
junction complex
LUX
Ankyrin-1, erythrocyte ankyrin; BCAM, basal cell adhesion molecule; CAII, carbonic anhydrase II; CAIV, carbonic anhydrase IV; Cl , chloride ion; CO2, carbon dioxide; G3PD, glyceraldehyde-3-phosphate dehydrogenase; HCO32,
bicarbonate; ICAM4, intercellular adhesion molecule 4; LDH, lactic dehydrogenase; Lu, Lutheran; NH3/NH41, ammonia/ammonium ion; PFK, phosphofructokinase; PGK, phosphoglycerate kinase; PK, pyruvate kinase; TM, tropomyosin.
*Size of the mature protein chain without any signal peptide, propeptides, or initiator methionine, but not including posttranslational modifications.
†References for the numbers of copies of each protein are listed in the footnotes of Table 2 found in Lux6. It must be emphasized that the numbers of many red cell membrane proteins are roughly estimated but not accurately measured,
which affects the estimates of the composition of the ankyrin complex and actin junctional complex.
‡The unit cell is defined in Figure 8.
§Data shown are for full-length ankyrin (band 2.1), the major isoform, except for the number of copies per cell, which includes the smaller isoforms: bands 2.2, 2.3, and 2.6.
280.0
246.4
a-Spectrin
RhD
b-Spectrin
45.4
45.1
RhCE
44.2
85.0
PFK (liver)
RhAG
21.8
Peroxiredoxin 2
membrane protein 1
p55/Palmitoylated
52.3
50.9
Kx
ICAM4
82.8
Kell
Protein
Molecular
mass, kDa*
190
Table 1. (continued)
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BLOOD, 14 JANUARY 2016 x VOLUME 127, NUMBER 2
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BLOOD, 14 JANUARY 2016 x VOLUME 127, NUMBER 2
ANATOMY OF THE RED CELL MEMBRANE SKELETON
191
Figure 3. Spectrin. (A) Organization of erythrocyte spectrin and the dimer-tetramer equilibrium. Spectrin is a long, flexible, wormlike protein composed of 2 chains (a- and
b-spectrin). Each chain contains a tandem array of ;5.0-nm, ;106-amino acid, triple-helical spectrin-type repeats, with specialized functional domains for self-association and
ankyrin-1 binding at the head end, and for binding to actin, protein 4.1R, and other associated proteins at the tail end. Each spectrin repeat is formed by 3 a-helices (A, B, and C)
with short connecting loops that are folded like a flattened Z into a triple-helical bundle.12 a-Spectrin contains 21 numbered repeats (a1-a21), plus a partial repeat (a0) at the Nterminus that contains a single C-helix. One of the 21 is really an src homology 3 (SH3) domain (a10) but is numbered as a repeat by convention. b-Spectrin contains 16 true
repeats (b1-b16) plus a partial repeat (b17) at the C-terminus that contains just the A and B helices. Note that for the spectrin ab dimer to convert to the a2b2 tetramer, it must first
cleave the internal linkage between the partial spectrin repeats a0 and b17 and then unfold (open dimer).13 This is the rate-limiting step in the dimer-tetramer equilibrium.
Dimer-tetramer self-association occurs at the head end of the spectrin dimer where the adhesion protein Lutheran/basal cell adhesion molecule (Lu/BCAM) also attaches.14
Ankyrin-1 binds nearby to spectrin repeats b14-b15.15 These 2 reactions cooperate: ankyrin-binding favors spectrin tetramer formation, and vice versa.16 The isolated a- and
b-spectrin chains join to form spectrin heterodimers at a nucleation site near the tail end of spectrin (repeats a-21 pairs with b-1, and a-20 with b-2) and then zip together in a
cooperative manner.17 Actin and protein 4.1R bind to CH domains at the N-terminal end of b-spectrin, just beyond the nucleation site.18 Binding to the CH2 domain is activated by
phosphatidylinositol-4,5-bisphosphate (PIP2).18 Adducin binds in the same region.19 Protein 4.2 and calcium ion (Ca21) bind to a neighboring EF hand domain on a-spectrin.5 Both
the EF hand and CH domains are needed for full actin binding.20,21 PS denotes spectrin repeats that bind phosphatidyl serine.22 Blue stars mark repeats that are relatively unstable
at physiological temperatures.23 (B) Structure of spectrin repeats b8 and b9 (PDB 1S3524). Note that each repeat is formed by 3 a-helices (A, B, and C) in a Z configuration. Note
also that helix C in b8 and helix A in b9 form a continuous a-helix that spans the junction between the repeats. (C) Hypothetical model of the tail end of spectrin based on recent
structures of a-actinin.25,26 Note that the first actin-binding domain (CH1) binds to F-actin in an extended (open) conformation.26 The intimate relationship between the EF hand and
CH domains and the recent evidence that the EF hand domain is required for optimal spectrin-actin binding20,21 suggest that the EF hand domains regulate actin binding.
(Figure 2). Binding is magnesium ion dependent. The red cell TM
isoforms also bind to tropomodulin (see below). Red cell membranes that
lack TM are markedly more fragile than normal,40 and only endogenous
TM, not the longer muscle isoforms, can restore stability. Red cell TM is
just long enough (;34 nm) to cover the estimated 7 actin monomers in 1
strand of the F-actin protofilament, which supports the idea that TM both
serves as a molecular ruler (along with actin-capping proteins) in
assembling the filament41 and strengthens the filament after assembly.
In yeast, formins (a family of proteins that nucleate actin filaments)
direct which TM isoform associates with an actin filament.42 It will be
interesting to see whether that is also true in erythroid precursors and
whether formins play a role in protofilament formation.
Tropomodulin 1. Tropomodulin 1 (TMod1) has 2 functions: it
caps the pointed or slow-growing end of actin filaments (Figure 2) and
binds TM, which greatly strengthens actin capping.43 There are 30 000
copies per red cell, or 1 per actin protofilament. One TMod1 binds to both
actin strands and both TM dimers in the actin protofilament.43 Mice that
lack TMod1 in their red cells have variable actin protofilament lengths
and irregular holes in the membrane skeleton.44 These mice also have
increased tropomodulin 3 in their red cells, which may partially
compensate. Tropomodulin 3 is a protein that plays multiple roles in
definitive erythropoiesis, erythroblast binding to so-called nurse macrophages, and enucleation,45 but is not normally present in mature red cells.
Protein 4.1R. Protein 4.1R promotes spectrin-actin binding and
helps attach the membrane skeleton to the membrane. The first of these
functions is especially important. Under physiological conditions,
erythrocyte spectrin binds very weakly to F-actin. Protein 4.1R binds to
both spectrin and actin and generates a high-affinity ternary complex.46
The cofactor activity is contained in a 10-kDa spectrin-actin binding
domain (SABD) in the middle of the molecule47 (Figure 448,49). The
actin-binding site in the SABD is straddled by a 2-part spectrin-binding
site in a single linear peptide (Figure 4C).49 Red cells that lack protein
4.1R are very fragile, but their membrane strength is normalized by the
SABD peptide.50 It is unfortunate that the 3-dimensional structure of
the SABD within protein 4.1R is unknown. Solving the complete
structure of this important protein is a major need in the field.
The other important 4.1R domain is the N-terminal, globular
membrane binding domain. It binds glycophorin C, protein p55, band 3,
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BLOOD, 14 JANUARY 2016 x VOLUME 127, NUMBER 2
Figure 4. Protein 4.1R. (A) Domain map of protein
4.1R and the location of erythrocyte binding partners.
(B) Structure of the protein 4.1R membrane binding
domain (MBD).48 Subdomains where specific proteins
bind are colored and labeled. (C) Schematic representation of the protein 4.1R SABD (blue). Two spectrin
binding regions straddle an actin binding peptide.49 In
this hypothetical model, each of the spectrin binding
regions is assumed to interact with a different one of
the 2 CH domains of b-spectrin, and both CH domains
are assumed to interact with actin.49 Neither of these
assumptions has been proved. Ca21 , calcium ion;
CaM, calmodulin; PS/PIP 2 , phosphatidyl serine/
phosphatidylinositol-4,5-bisphosphate. Panel B adapted
from Han et al48 with permission.
and calmodulin in separate, well-defined areas48 (Figure 4B). These
interactions are discussed briefly in “The actin junctional complex.”
Adducin. Adducin is a complex multifunctional protein containing an a subunit, and either a b subunit or, less often, a related g
subunit.51 The adducin subunits contain a globular head domain and an
extended, flexible tail.51 The predominant form is probably the ab
heterodimer.52 There are 30 000 copies of the dimer per red cell.
As suggested by the number of copies, adducin caps the fast-growing
or “barbed” end of the actin protofilaments in red cells (Figure 2).52 It
also recruits spectrin to neighboring sites on actin,53 which enhances the
capping ;10-fold.50 These interrelated functions require a domain at the
end of the adducin tail.50 Both functions are regulated by calmodulin and
by several protein kinases.54 The effects of phosphorylation are
complex, and it is not clear whether they are physiologically important.55
Indeed, the importance of adducin itself is uncertain, because mice that
lack the protein have only mild hemolysis and spherocytosis.56
Adducin also helps attach the membrane skeleton to the lipid bilayer
through interactions with the anion-exchange channel (band 3)57 and
glucose transporter type 1 (Figure 2).58 The band 3 binding site is located
in the tails of a- and b-adducin.57 Whether adducin binds dimers or
tetramers of band 3 and whether it binds 1 or 2 of each is unknown.
Dematin. Dematin is an actin-binding protein with 2 actin
binding sites: one in its headpiece and one in its tail.59 The recombinant
protein is a monomer,59 but the native protein may be a trimer. Dematin
was first identified by its ability to bundle actin filaments into cables,60
but in mature red cells, where there are no such cables, dematin has 2
functions. First, unmodified dematin binds to spectrin and enhances
spectrin-actin binding.61 This function is lost when dematin is phosphorylated by protein kinase A.59,61 Second, dematin binds to the
glucose transporter58 and helps attach actin to the membrane.
How does spectrin bind to the
actin protofilament?
Surprisingly little is known about this central question. Actin and
protein 4.1 bind to 2 tandem CH domains at the N-terminal (or tail) end
of b-spectrin (Figure 3A). The CH1 domain is constitutively active but
the CH2 domain is incipient.18 It is unmasked by removal of a blocking
helix or by phosphatidylinositol-4,5-bisphosphate, a lipid involved
in many signaling pathways.18 It is not known how, or even if,
phosphatidylinositol-4,5-bisphosphate regulates the CH2 domain in the
native red cell.
The neighboring calmodulin-like EF hand domain of a-spectrin is
also important. Deletion of the last 13 amino acids of the domain in a
minispectrin or in the severely hemolytic sph1J mouse nearly ablates
actin binding.21
The relationships among the EF hand domain, the CH domains, and
F-actin are not known in spectrin but probably resemble those in better
studied spectrin family members such as a-actinin,25,26,62 utrophin,63
dystrophin, fimbrin, and plectin (Figure 3C). The problem is that these
proteins bind actin in different ways. Sometimes the CH domains are in
contact with each other (closed position), and sometimes they are
extended and separated from each other (open position). In some
analyses, only CH1 binds to actin26; in others, both CH1 and CH2
bind.25,62 The EF hand domain is always in contact with the CH
domains and not in contact with actin, so it presumably regulates the CH
domains. The fact that a-actinin CH domains adopt both open and
closed positions25 and utrophin also has 2 modes of binding63 suggests
that the CH domains in spectrin might also do so and that this might
be regulatory (convert weak binding to strong binding, for instance).
Perhaps protein 4.1R and/or the EF domain act by converting the CH
domains to a high-affinity conformation. Spectrin could occupy up to 2
actins in the actin filament, given the stoichiometry (6 spectrins per ;14
actin monomers; see Table 1), so a variety of arrangements is possible.
In any event, these are clearly key questions that need to be answered
going forward.
Another obvious problem that is rarely mentioned is that
spectrin is in a planar array, but the spectrin binding sites on the
helical actin protofilament must be in a helical array. Spectrin does
not hang down into the cytoplasm or stick up into the lipid bilayer,
so if all the spectrin binding sites on actin are saturated, either the
actin-binding end of spectrin must be flexible enough to curve
around the actin filament, or spectrin must be able to bind to actin
in more than 1 way.
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Figure 5. Negatively stained electron micrographs of red cell membrane skeletons and spectrin. (A) A membrane skeleton that was stretched during preparation. Note the
pseudohexagonal organization of the skeleton and the location of various proteins. Inset: example of a 37-nm long F-actin protofilament. Sp4, spectrin tetramer; Sp6, spectrin
hexamer; 2 Sp4, double spectrin tetramers. (B) Skeleton in situ in a red cell prepared by a minimally perturbing quick-freeze, deep-etch, rotary replication procedure. The dense
network of filaments averages 29 6 9 nm between intersections. (C-D) Spectrin from partially stretched skeletons. Some of the spectrin molecules show a helical periodic
substructure, as noted by the region between the arrowheads in panel C or by the single arrowhead in panel D. (E) Right-handed double-helical models of spectrin periodicity obtained
from the experiments in panel D by visual filtering of the periodic regions from multiple spectrin molecules. In this model, the springlike spectrins extend and contract by varying their
pitch and diameter. Native spectrin tetramer has ;10 turns, with a pitch of ;7 nm (70 Å) and a diameter of ;5.9 nm (59 Å). Panel A reprinted from Liu et al27; panel A inset from Byers
and Branton65; panel B from Ursitti et al66; panel C from Ursitti and Wade30; and panels D and E from McGough and Josephs,28 all with permission.
What is the structure of spectrin on
the membrane?
Electron micrographs of purified spectrin tetramers64 or spectrin
filaments in fully stretched skeletons27 (Figure 5A65) show a spaghettilike molecule with an end-to-end length of up to 200 nm. This
configuration is how spectrin is usually depicted in membrane models.
However, the filaments of native, unstretched skeletons are much more
compact (Figure 5B66), and simple calculations show that the average
distance between actin filaments (ie, the length of a spectrin tetramer) is
only 60 to 70 nm in vivo.6,67 This length corresponds roughly to that of
presumed spectrin filaments in native skeletons.30,31,66 It is not known
how spectrin folds in this resting state. The best evidence comes from
visually enhanced electron micrographs of spectrin in partially expanded
skeletons (Figure 5C-D), which suggest that a- and b-spectrin are coiled
about each other in a 2-start helix with ;10 turns per spectrin tetramer28
(Figure 5E). Some micrographs show relatively straight spectrin
Figure 6. Ankyrin. (A) Schematic of erythroid ankyrin structure.70 The membrane domain contains 24 ankyrin repeats, grouped functionally into 4 subdomains of 6 repeats. There
are 2 binding sites for band 3, one involving repeats 7 to 12 (domain D2), and one involving repeats 13 to 24 (D3/D4). The ankyrin binding loops on band 3 are predicted to interact with
ankyrin repeats 19 and 20 (light blue) of D4.71 The interaction site within D2 is not known. The spectrin domain contains 3 subdomains, of which ZU5A (light blue) contains the binding
site for spectrin.15,72 The C-terminal (regulatory) domain is thought to modulate the binding functions of the other 2 domains,70,73 and exists in numerous spliced variants of mostly
unknown function. The function of the conserved death domain (DD) is also a mystery. (B) Hypothetical model74 of the interaction between the membrane domain of ankyrin (green)
and a band 3 tetramer (red, blue, cyan, and purple subunits). Note how the ankyrin repeats form a large (9-nm diameter) twisted helical spiral. In this deduced model, the concave
surface of the D3/D4 region interacts with the red subunit in 1 band 3 dimer. One subunit in the second band 3 dimer contacts the concave surface of the D2 region, which contains a
second band 3 binding site. (C) Spectrin-ankyrin interaction. Note how ZU5A, the spectrin-binding subdomain within ankyrin, binds in the notch created by the sharp angle between
spectrin repeats b14 and b15. Panel B reprinted from Michaely et al74 and panel C from Ipsaro and Mondragón,15 both with permission.
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Attachment of the membrane skeleton
Ankyrin
Erythrocyte ankyrin links spectrin to a complex of band 3 and other
proteins in the lipid bilayer. The protein has 3 domains70 (Figure 670-73).
The membrane domain is composed entirely of ankyrin repeats. It is a
remarkably open, spiral structure, somewhat like a twisted sickle, that
encircles and binds band 3 tetramers (Figure 6B74). Whether band 3
spontaneously forms tetramers and then attracts ankyrin, stabilizing the
tetramers, or whether ankyrin, which has 2 binding sites for band 3
(Figure 6A), independently binds 2 band 3 dimers (ie, a “dimer of
dimers”), which then form a tetramer, is unresolved. The fact that a
stable subpopulation of band 3 tetramers can be isolated from nonionic
detergent extracts of ghosts75 argues for the former. But the structure of
a band 3 tetramer binding to the full membrane domain has not been
solved, and at least 1 recent paper argues that band 3 dimers must bind
independently,76 so the question remains.
The ankyrin-spectrin binding site is well defined. The ZU5A
subdomain of ankyrin binds to a special site created by the junction of
the 14th and 15th repeats within b-spectrin (Figure 6C). Only 1 ankyrin
binds per tetramer, perhaps for steric reasons. Ankyrin binding promotes spectrin tetramer and oligomer formation by ;10-fold, and vice
versa.16 Ankyrin attachment to band 3 also strengthens spectrin selfassociation.77 Presumably, both of these mechanisms help regulate the
relatively tenuous self-association interaction.
Figure 7. Organizational model of human erythrocyte band 3 (anion-exchange
protein). The protein contains 2 structurally and functionally distinct domains: a
cytoplasmic binding domain (amino acids 1-359) and a transmembrane domain (amino
acids 360-911) that forms the anion-exchange channel.78 In the cytoplasmic domain, the
glycolytic enzymes PFK, aldolase, and G3PD bind to amino acids 1 to 23 at the
N-terminus of band 3 and contact amino acids 356 to 384,79 which are nearby in the folded
protein. The enzymes are inactive when bound,80 but are displaced and activated by
deoxyhemoglobin (Hb), which also binds to the N-terminus,81 or by phosphorylation of 2
tyrosines (Tyr21-P and Tyr8-P) within the binding sites.80 Enolase, PK and LDH also
localize to the membrane and are displaced by deoxyhemoglobin, but do not bind to band
3.80 This fact suggests that many of the enzymes in the glycolytic pathway form a
functional complex (or “metabolon”) that efficiently generates adenosine triphosphate
(ATP), particularly under hypoxic conditions. Ankyrin also interacts with the N-terminus,82
but the main ankyrin sites are amino acids 63-73 and 175-185, which loop out from the
surface.71 Protein 4.1 binds to 2 sites with the (I/L)RRRY motif, near the end of the
domain.83 Ankyrin and protein 4.1R inhibit each other’s binding. The binding sites for
protein 4.2 and adducin have not been identified. In the membrane domain, the best
current model84 for the anion-exchange channel is based on the structure of the ClC
bacterial chloride channel. The intramembrane and transmembrane helices are lettered as
they are in the ClC channel. A complex carbohydrate structure is attached to Asn 642.
Carbonic anhydrase (CA)IV binds to the extracellular loop of band 3 between helices I and
J,85 and CAII may bind to the C-terminal segment86; both are perfectly positioned to create
bicarbonate from carbon dioxide and to shuttle the ions to or from the plasma through band
3. F-6-P, fructose 6-phosphate; Hb-O2, oxyhemoglobin; NAc, acetylated aminoterminus.
filaments28; others show some kinking.31 This difference may reflect
whether the filaments are under tension. In this model, spectrin molecules extend and contract along the helical axis by varying the pitch
and diameter of the double strand (Figure 5E).28 This springlike behavior is presumably critical to the elastic behavior of the membrane.
However, there is evidence for other models that propose that
spectrin molecules adopt a random wormlike configuration,68 or that
helical segments of the repeats melt and extend under tension,35 or
simply unfold when stressed.36 The fact that previously occluded cysteines can be labeled when red cells are deformed by shear is evidence
that some repeats unfold and that spectrin may even detach from
ankyrin or actin when stretched.69 Because the structure of spectrin
affects its mechanical properties, as well as the spatial relationships of
all the other proteins that bind to it, defining its structure in vivo, at rest,
and when deformed is a high priority.
Band 3
Erythrocyte band 3, or officially SLC4A1, is the major red cell
membrane protein, with ;1.2 million copies per cell. Functionally, it is
2 proteins78: (1) an N-terminal cytoplasmic, peripheral membrane
protein that is a key attachment site for the membrane skeleton, glycolytic enzymes, and deoxyhemoglobin; and (2) a C-terminal integral
membrane protein that forms the red cell anion-exchange channel and
aids carbon dioxide transport (Figure 778-86).
The glycolytic enzymes form a metabolic complex (metabolon)
extending from phosphofructokinase through lactic dehydrogenase.
Three of the enzymes bind to the N-terminus of band 3: phosphofructokinase, aldolase, and glyceraldehyde-3-phosphate dehydrogenase.
The others bind indirectly as part of the complex.80 The enzymes are
inactive when bound80 but are displaced and activated either by
deoxyhemoglobin, which also binds to the N-terminus,81 or by phosphorylation of 2 tyrosines within the binding sites80 (Figure 7). The
enzymes80 and their adenosine triphosphate product87 are located in
discrete clumps along the membrane, supporting the idea of a physical
metabolon. Conditions that displace the enzymes from the membrane
activate glycolytic fluxes in intact red cells by 45%, which supports the
idea of a functional metabalon.88
As noted earlier, ankyrin binds to band 3 tetramers. The acidic
N-terminus and 2 loops on the surface of band 3 are involved in the
binding69 (Figure 7). As with the glycolytic metabolon, ankyrin is
displaced from band 3 by deoxyhemoglobin.82 The affinity of
deoxyhemoglobin is weak, but the concentration of hemoglobin is so
high in red cells that approximately half of the band 3 molecules
are bound to deoxyhemoglobin when red cells are deoxygenated.82
Loosened ankyrin constraints could improve blood flow in hypoxic
areas, but with the risk that red cells subject to prolonged deoxygenation, such as those trapped in the spleen, might suffer membrane
vesiculation. (Could this be the long-sought mechanism for splenic
conditioning89 in hereditary spherocytosis?).
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Figure 8. Model of the unit cell of the membrane skeleton. (A) The membrane skeleton is a quasihexagonal lattice centered around ;40 000 F-actin protofilaments. Each
37-nm, double-helical protofilament is capped by adducin (A) and tropomodulin (T) and contains ;14 G-actin subunits. The red cell surface area is ;140 mm2, so each
hexagonal unit cell is 3500 nm2 and the average length of a spectrin dimer is ;32 nm, which is one-third its length when fully stretched. Available data suggest that the
spectrin chains are coiled about each other and expand and contract by changing their pitch and diameter28 (Figure 5E). They are shown here as straight, although they are
sometimes kinked.31 Spectrin oligomers are not shown in this model but exist in vivo. In addition, the skeleton is not as regularly arrayed in vivo as in this averaged model.
(B) A recent model of the ankyrin complex (Ank Cmplx; green ovals) assembled from known structures of the proteins by Burton and Bruce7 has a cross-sectional area of
17.5 nm 3 14 nm, assuming the proteins are closely packed. The cross-sectional area of the actin junctional complex is estimated to be ;20 nm 3 17 nm if it contains only a
single band 3 dimer7 (Actin Cmplx 1-Bd3; small tan ovals). Such a complex would presumably lie toward the end of the actin protofilament where adducin resides. This
position is shown in the 2 unit cells on the right. Alternatively, if the actin junctional complex contains 6 band 3 dimers (Actin Cmplx 6-Bd3) interacting with all 6 proteins 4.1R, it
would probably be more centered and approximate the area of the large tan oval in the unit cell on the left. Recent data favor the larger arrangement,96 but in either case, it is
likely that the ankyrin and actin junctional complexes would be close to each other and probably often touch, particularly during red cell deformation. Band 3 dimers that are not
associated with either the ankyrin or actin junctional complexes (small blue ovals) diffuse in the lipid bilayer within the spectrin corrals. There are 3 to 8 of these per unit cell,
depending on the number of band 3 dimers in the actin junctional complex. Reprinted from Lux6 with permission.
The ankyrin complex
Ankyrin and band 3 are part of the multiprotein ankyrin complex
(Figure 2). From the amounts of each protein in the red cell (Table 1),
the complex is estimated to contain 1 ankyrin, 1 band 3 tetramer, 2
glycophorin A or B dimers or heterodimers, 2 protein 4.2 molecules,
and 1 Rh complex (a trimer of RhAG combined with RhD and RhCE).
CD47, the Landsteiner-Wiener blood group antigen, and the proteins in
the glycolytic metabolon are also components, but are present in less
than stoichiometric amounts (Table 1) and so must contribute only to a
subset of complexes. One wonders how many such subsets there are.
Do they have constant composition or are they in a dynamic equilibrium? And are the rare complexes that contain CD47 and LandsteinerWiener blood group antigen localized to specific membrane
subdomains?
As detailed in Table 1 and Figure 2, there are multiple interactions
among proteins in the ankyrin complex. Some of these are critical; for
example, red cells lacking band 3 also lack protein 4.2 and glycophorin
A,90 and red cells lacking protein 4.2 nearly lack CD47.91 A consequence of this promiscuity is that missense mutations that interfere with
a single interaction within the ankyrin complex have little effect.
Hereditary spherocytosis is caused by defects in spectrin, ankyrin, band
3, or protein 4.2 that impair ankyrin complex formation, and almost all
the known mutations diminish the concentration of these proteins rather
than block their binding functions.
The actin junctional complex
The actin protofilament and its associated proteins are also attached to
the membrane. For many years, this attachment was believed to occur
exclusively via a ternary complex of protein 4.1R, p55, and glycophorin
C or D. Each of these proteins interacts with the other 2, and the
interaction sites are well mapped.48,92,93 However, if this was an
important skeleton attachment and the sole attachment at the actinbinding end of spectrin, it was never clear why patients with complete
absence of glycophorin C and D had, at most, very mild hereditary
elliptocytosis.
During the past few years, it has become clear that the actin junctional complex is more complex and, like the ankyrin complex, is
centered on band 3 (Figure 2). First, protein 4.1R and ankyrin compete
for binding to band 3,94 which suggests that they bind to separate band 3
populations. The interaction with protein 4.1R is known to be important
because zebrafish lacking band 3 can be rescued by expression of
mouse band 3 but only if the mouse band 3 contains the protein 4.1R
binding sites.95 Second, protein 4.2, which requires band 3 for
incorporation into the membrane, binds to the EF hand domain at the
actin-binding end of spectrin. Third, adducin binds to band 3,57 which
definitely localizes a subpopulation of band 3 to the neighborhood of
actin (Figure 2). The latter interaction is important because the membrane fragments when the connection is severed.57
The stoichiometry of the actin junctional complex is uncertain. On
average, 6 spectrins and 6 proteins 4.1R bind per actin (Figure 896), and
there is enough band 3, glycophorin A, glycophorin C/D, and probably
enough glucose transporter 1 and stomatin to supply 6 complexes per
actin filament (Table 1). However, there is only 1 copy of adducin per
actin filament and only 3 copies of dematin, if it is a monomer as recent
studies indicate,59 or one copy if it is a trimer. Similarly, whereas protein
4.1R, glycophorin C/D, and protein p55 are usually pictured as a 3-part
complex, there is only enough p55 for 1 copy per actin filament if it is a
dimer, as some data suggest.97 And there is only ;1 copy of protein 4.2
available if 240 000 copies (1 per band 3 dimer) are tied up in the
ankyrin complex. The actin junctional complex also contains enzymes
in the glycolytic metabolon,79 as well as the blood group proteins Kx/
Kell and DARC/Duffy,98 although the amounts of these proteins are
insufficient for even 1 copy per complex.
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Table 2. Some unanswered questions
General
Spectrin
What are the amounts and stoichiometries of the membrane proteins (only a few are accurately measured)?
What is the structure of spectrin in vivo? How does it change when the membrane is deformed?
How does spectrin bind to the actin protofilament? What is the stoichiometry? Where do the CH1 and CH2 domains and protein 4.1R bind on actin?
Is there .1 binding conformation? If so, how do these differ functionally and how are they regulated?
Why is the EF hand domain essential for normal spectrin-actin binding? How does it work?
How do spectrin molecules, which lie in a plane, accommodate different orientations of their binding sites on the helical actin protofilament?
Higher order spectrin oligomers (hexamers, octamers, etc) also serve as molecular junctions in the skeleton. Are there significant numbers of these
and do they have a unique functional role?
If the available spectrin repeat structures are correct and there is a continuous a-helix across the interrepeat junction, how is spectrin flexibility
achieved?
Why are the same repeats in different spectrins so similar? Do they have binding or mechanical functions that we do not yet know about?
What binds to the spectrin src homology 3 (SH3) domain? What are the consequences?
There is evidence that some of the spectrins in mature red cells contain the muscle isoform of b1-spectrin, which has a lipid-interacting pleckstrin
homology domain. Is this true, and if so, what is its function?
Actin
How are actin protofilaments formed? How do they achieve their uniform size? Do formins play a role?
Are the actin protofilaments stable or do they turn over, as the presence of a critical concentration of G-actin in the red cell cytoplasm suggests? If so,
how is this regulated?
How are the various proteins arranged on the actin protofilament? Is the arrangement the same in all the protofilaments? If there are open binding
sites on actin, do molecules like spectrin move from site to site with membrane deformation?
Do red cell myosin and caldesmon have a function in the membrane of mature red cells?
Ankyrin
Does ankyrin bind a band 3 tetramer or 2 dimers? If a tetramer, is it preformed before binding or do 2 dimers bind and convert to a tetramer on
ankyrin. What is the structure of the band 3 tetramer-ankyrin complex?
What is the structure of the ankyrin molecule? How do the various domains relate to each other spatially?
Where do protein 4.2 and RhAG bind?
What is the function of the death domain in ankyrin?
What are the functions of the many ankyrin spliceoforms?
Band 3
What is the overall structure of band 3?
Where do protein 4.2 and adducin bind on band 3?
Because the numbers of integral membrane proteins vary by orders of magnitude (Table 1), the band 3 multiprotein complexes must vary in
composition. How many such complexes are there? Do they have specific, stable compositions or are they in equilibrium and continually changing
their composition? Are the substoichiometric proteins (eg, CD44, CD47, LW, Kx/Kell, DARC) uniformly distributed or localized to lipid rafts or other
membrane lipid or protein subdomains?
How are the proteins in the band 3 multiprotein complexes arranged relative to each other?
Do protein interactions within the band 3 multiprotein complexes allosterically affect the interactions or functions of other proteins in the complexes?
Does protein 4.1R bind to band 3 in vivo? If so, does band 3 bind to each of the spectrin/actin/protein 4.1R complexes or only to a subset, such as the
subset that interacts with adducin (Figure 8)?
Are any band 3 molecules “unbound” (ie, not attached to the membrane skeleton directly or indirectly). If so, how many? Are they also part of
multiprotein complexes? Do they have a unique function?
Glycolytic metabolon
The enzymes that compose the proposed glycolytic metabolon vary in concentration by orders of magnitude. Are the estimates correct? If so, do
some complexes lack rare components (but then, how would they function?) or are they gigantic so as to include at least 1 copy of each enzyme?
Are other enzymes in the glycolytic pathway part of the complex?
How is the glycolytic metabolon regulated in vivo? What is the purpose of activating glycolysis by deoxyhemoglobin? Do products of glycolysis such
as adenosine triphosphate (ATP) cause vasodilation directly or indirectly? Does nitric oxide play a role? Is the rate of dissociation and activation of
the glycolytic metabolon rapid enough to be relevant during normal circulatory transits?
Protein 4.1R
What is the structure of intact protein 4.1R and the spectrin/actin/protein 4.1R complex?
Calmodulin binds to and regulates protein 4.1R. The EF hand domain is a calmodulin-like structure that resides near protein 4.1R in the actin
junctional complex. In addition, the EF domain binds calmodulin. Does protein 4.1R bind to the EF domain or do they bind each other through
calmodulin? If so, is this a regulatory interaction?
Does phosphatidyl-4,5-bisphosphate regulate protein 4.1R binding to actin or other proteins in vivo?
Because there is insufficient protein p55/palmitoylated membrane protein to make a 1:1:1 molar complex with protein 4.1R and glycophorin C/D, how
is the complex constructed?
Protein 4.2
What is the function of protein 4.2 when attached to the spectrin EF hand domain? Does it actually occupy the domain in vivo? If so, does it bind to
protein 4.1R?
What is the function of the ATP-binding site on protein 4.2?
Adducin
Is adducin a dimer or a tetramer?
What is the structure of adducin? How does the C-terminal tail relate to the head end?
Band 3, spectrin, and actin all bind to the C-terminal tail. What is the structure of this complex? How are the multiple interactions regulated? Can they
occur all at once on the same molecule or do some interactions interfere with others? When adducin binds spectrin and actin, does it also interact
with nearby proteins 4.1R or 4.2? Does adducin bind band 3 dimers or tetramers, and does it bind 1 or 2 molecules of each?
Dematin
How does dematin contribute to spectrin binding in vivo?
Is the ability of dematin to bundle actin filaments important in the mature red cell?
Where does dematin bind relative to spectrin and adducin on the actin protofilament? Does the number of dematins per protofilament vary?
Tropomyosin
Do the red cell isoforms of tropomyosin determine the unique structure of the short actin protofilaments? Do specific actin-nucleating formins play a
role in this process?
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Table 2. (continued)
Posttranslational
modifications
What do posttranslational modifications like phosphorylation, palmitoylation, myristoylation, hydroxylation, methylation, glycation, and ubiquitination
do? Are the effects important in vivo?
How are the multiple effects of calcium ion and calmodulin integrated in vivo?
Dynamics
Do the actin junctional complex and ankyrin complex contact each other in vivo? Do proteins like band 3, proteins 4.1R and 4.2, and adducin, which
have potential binding partners in both complexes, sometimes switch allegiances? If so, is this a regulatory process?
How labile are the individual bonds in the membrane skeleton? Which bonds, other than the spectrin dimer-tetramer interaction, break when the
skeleton is deformed? Are the rates of dissociation and reassociation such that the bonds dissociate during red cell deformation in the circulation?
How is the skeleton disassembled and reassembled during invasion by malaria and other parasites?
Lipids
What are the interactions between the membrane skeleton and the overlying lipids? Do these interactions have regulatory as well as structural
functions? Does the membrane skeleton vary in unique regions of the lipid bilayer such as lipid rafts?
Therefore, it is uncertain at the moment whether the actin junctional
complex contains only a single band 3 dimer, interacting with just 1 or 2 of
the 6 spectrin/protein 4.1R dyads, or whether it is a larger complex
containing 3 to 6 band 3 dimers that interact with all the spectrins and
proteins 4.1R. Both possibilities are depicted in Figure 8. Although the
data are not conclusive, recent evidence favors a larger complex.96 If so, it
is not clear whether all or just a subset of band 3 molecules bind to adducin.
In either case, as shown in Figure 8, an interesting and underappreciated fact is that the ankyrin and actin junctional complexes are quite close
to each other in situ on the membrane and must often collide, especially
during red cell deformation but perhaps even when the cell is at rest. This
knowledge raises the interesting possibility that proteins like band 3,
protein 4.1R, protein 4.2, and adducin, which have binding partners
in both complexes, may sometimes switch allegiances. Because some
proteins compete for the same binding sites (eg, as noted, protein 4.1R
displaces ankyrin from band 3),94 this process could be a regulatory one.
Distribution of band 3
Of the ;1.2 million band 3 monomers per red cell, 40% are tetramers
bound to ankyrin.96 Depending on whether the actin junctional complex contains an average of 1 or 6 band 3 dimers, an additional 7% to
40% of the band 3 dimers are located there. Recent experiments comparing normal mouse erythrocytes to a-adducin-deficient erythrocytes
suggest that roughly 33% of the band 3 molecules are immobilized by
adducin, which favors a larger complex.96 The remaining band 3 dimers
(25%-30%) are believed to be diffusing freely in the lipid bilayer,
constrained for the most part by the boundaries of the spectrin corrals,
which average ,100 nm.96 These percentages are remarkably similar
to those of freely diffusing band 3 dimers (25%-29%) measured in
normal red cells using different methods.96,99,100
It is important to remember that some of the minor proteins found in
band 3 complexes can exist only in a subset of the complexes (probably
multiple different subsets) and that the lipid bilayer is heterogeneous,
with areas like the lipid rafts where certain proteins concentrate.
Therefore, the band 3 complexes must be more heterogeneous than these
3 states of band 3 imply. Analyses of band 3 mobilities support this
conclusion.95
know how the skeleton is constructed during erythropoiesis. We know
very little about the dynamics of the skeleton: how labile individual
bonds are, how the skeleton responds to capillary deformation, how the
skeleton is disassembled and reassembled during invasion by malaria or
other parasites, and so forth. And as discussed earlier, we do not know
how spectrin is folded in the intact skeleton or how the proteins that
form the actin junctional complex are organized. Or how spectrin binds
to actin. We do not know whether band 3–binding proteins like protein
4.1R, protein 4.2, and adducin switch from one band 3 complex to
another, given the apparent proximity of the complexes at each end of
spectrin (Figure 8). We do not understand why the same repeats in
different spectrins are so similar if they just serve as spacers. Do they
have binding or mechanical functions that we do not yet know about?
We do not know what roles posttranslational changes like phosphorylation, fatty acylation, methylation, glycation, hydroxylation, and
ubiquitination play, or what roles calcium ion–binding and adenosine
triphosphate–binding play. And we do not understand the interactions
between the membrane skeleton and the overlying lipids, which will
likely have regulatory as well as structural functions. This is only a
partial list of questions; some others are itemized in Table 2. But I hope
my point is clear. Because the red cell membrane skeleton is the
paradigm for studies of spectrin-based membrane skeletons in all cells,
and because we now know that spectrin and other membrane skeletal
proteins invest internal organelles, some transport vesicles, and plasma
membranes, and even play a role in nuclear organization, it is more
important than ever to understand the structure and function of the red
cell membrane skeleton.
Acknowledgments
The author regrets that important papers from many talented individuals who have made significant contributions to red cell research
could not be discussed or cited due to space limitations. The author
gratefully acknowledges the camaraderie and friendship of a large
number of colleagues in the red cell membrane field who, over many
years, have enriched his career and aided his research.
The future
Authorship
A great deal has been learned about the structure of the red cell
membrane and the membrane skeleton in the 36 years since the first
model was published (Figure 1). I sometimes hear people say that the
red cell membrane is no longer an active area of hematology research,
but I disagree. We have the broad outlines of how the membrane is
organized (Figure 2) but know few of the details, and what we do know
is about a static structure. Many questions remain (Table 2). We do not
Contribution: S.E.L. wrote the review and designed the figures and
tables.
Conflict-of-interest disclosure: The author declares no competing
financial interests.
Correspondence: Samuel E. Lux, Boston Children’s Hospital,
300 Longwood Ave, Hunnewell 260.1, Boston, MA 02115; e-mail:
[email protected].
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198
LUX
BLOOD, 14 JANUARY 2016 x VOLUME 127, NUMBER 2
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2016 127: 187-199
doi:10.1182/blood-2014-12-512772 originally published
online November 4, 2015
Anatomy of the red cell membrane skeleton: unanswered questions
Samuel E. Lux IV
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