REVIEWS
ANNEXINS: LINKING Ca2+
SIGNALLING TO MEMBRANE
DYNAMICS
Volker Gerke*, Carl E. Creutz‡ and Stephen E. Moss§
Abstract | Eukaryotic cells contain various Ca2+-effector proteins that mediate cellular
responses to changes in intracellular Ca2+ levels. A unique class of these proteins — annexins
— can bind to certain membrane phospholipids in a Ca2+-dependent manner, providing a link
between Ca2+ signalling and membrane functions. By forming networks on the membrane
surface, annexins can function as organizers of membrane domains and membranerecruitment platforms for proteins with which they interact. These and related properties enable
annexins to participate in several otherwise unrelated events that range from membrane
dynamics to cell differentiation and migration.
*Institute of Medical
Biochemistry, Centre for
Molecular Biology of
Inflammation, University
of Münster, Germany.
‡
Department of
Pharmacology, University
of Virginia, Charlottesville,
Virginia, USA.
§
Division of Cell Biology,
Institute of Ophthalmology,
11–43 Bath Street, London
EC1V 9EL, UK.
Correspondence to V.G.
e-mail:
[email protected]
doi:10.1038/nrm1661
Ca2+ signalling has a pivotal role in the regulation of
many cellular processes in all eukaryotic organisms.
Proteins that are regulated by fluctuations in cellular
Ca2+ levels have evolved to mediate Ca2+-dependent
stimulus–response coupling BOX 1. Annexins are one
class of such Ca2+-regulated proteins. They are characterized by the unique architecture of their Ca2+binding sites, which enables them to peripherally
dock onto negatively charged membrane surfaces in
their Ca2+-bound conformation. This property links
annexins to many membrane-related events, such as
the regulated organization of membrane domains
and/or membrane–cytoskeleton linkages, certain
exocytic and endocytic transport steps and the
regulation of ion fluxes across membranes.
Although annexins are structurally well defined,
their precise functions have long been sought, and
considerable progress in this respect has been made
in recent years. In vitro systems that use atomic-force
or electron microscopy of membrane-bound annexins
have produced remarkable images of highly symmetric
annexin scaffolds that could enable these proteins to
organize membrane domains. In vivo, annexin knockout and knockdown approaches have identified several
processes that depend on annexins, including certain
membrane-trafficking steps, aspects of Ca2+ signalling
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
and extracellular events. In this review, we attempt to
organize our present understanding of annexin functions into a coherent view of these proteins as an important link between Ca2+ as an intracellular signal and the
regulation of the diverse functions of membranes.
Molecular structures of annexins
Most annexin functions are linked to their ability to
interact with cellular membranes in a reversible and
regulated manner. High-resolution structures of
several annexins have provided detailed models for
this membrane attachment1–3. They show that the
conserved membrane-binding domain — the annexin
core — has the shape of a slightly curved disc and uses
its more convex surface for peripheral membrane
binding (FIG. 1). Ca2+ that binds to this surface of the
annexin core forms the prime contact by simultaneously coordinating carbonyl and carboxyl groups of the
protein and phosphoryl moieties of the glycerol backbone of membrane phospholipids4. In the presence of
phospholipids, the Ca2+ affinity of these sites — also
known as annexin-type or type-II Ca2+-binding sites5,6
— is in the low micromolar range, although the exact
affinity varies substantially between different annexins
BOX 2. The annexin core can therefore be regarded as a
peripheral membrane binding module that docks onto
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REVIEWS
HYDRA ANNEXIN
Annexin B12 is the
predominant annexin in the
freshwater cnidarian Hydra
vulgaris, and has been the
prototype for biophysical
studies on annexin insertion
into phospholipid bilayers.
EFHAND SUPERFAMILY
The largest family of Ca2+binding proteins, which is
exemplified by calmodulin.
The family members share a
structural helix–loop–helix
motif — the EF hand — that
forms the Ca2+-binding site.
S100 PROTEIN
A family of 10–14-kDa, EFhand-containing Ca2+-binding
proteins, which transmit Ca2+dependent cell-regulatory
signals.
membranes through its Ca2+-bound convex surface. In
this configuration, the concave side faces the cytoplasm
and is available for further interactions that allow
annexins to assemble other interacting components at
membrane sites in a Ca2+-regulated manner.
In addition to Ca2+-mediated peripheral membrane
binding, some annexins can also interact with the
hydrocarbon chains of membrane lipids. This has been
shown for: annexin A5, which, in its Ca2+-bound conformation, exposes a tryptophan residue that can insert
into the membrane7,8; annexin A13, which becomes
attached to membranes in a Ca2+-independent manner
through N-terminal myristoylation9; and annexins A5
and B12 (B12 is a HYDRA ANNEXIN), which can adopt an
integral membrane configuration at low pH values or
in the presence of peroxide10,11.
The N-terminal domain of annexins is thought to
fold into a structurally separate unit, probably located
on the concave side of the core domains, opposite the
membrane-binding surface (FIG. 1a). This has been
shown in crystal structures of full-length annexins
that contain short N-terminal domains (for reviews,
see REFS 2,3,12), as well as in the structure of Ca2+bound annexin A1, which is characterized by an
N-terminal domain of intermediate length (40 residues).
Interestingly, the N-terminal domain in Ca2+-free
Box 1 | Ca2+-regulated proteins
Several Ca2+-regulated proteins have evolved to maintain cellular Ca2+ at low levels
(a prerequisite for signalling) and to couple changes in cytosolic Ca2+ to the wide
variety of physiological responses. They include Ca2+ channels and pumps, Ca2+transport and -buffering proteins, as well as Ca2+-effector proteins that are directly
involved in signalling downstream of a Ca2+ spike or sustained Ca2+ elevation.
Although functionally diverse, many of the Ca2+-effector proteins can be grouped
into different families on the basis of the fold of their Ca2+-binding site(s). The largest
of these families are the EF-hand proteins, the annexins and the C2-domain proteins.
EF hand denotes a Ca2+-binding motif that contains two α-helices orientated
almost perpendicular to one another, which flank a loop that is typically 12 residues
long. The bound Ca2+ ion is coordinated by seven oxygen ligands in a pentagonal
bipyramid arrangement, and these oxygens are provided by carboxyl and hydroxyl
side chains, as well as by carbonyl groups of the peptide backbone, all of which are
located in or next to the loop (for a review, see REF. 125). EF-hand proteins, which
include calmodulin, troponin C and the S100 proteins, typically function by binding
to, and thereby regulating, cellular targets in their Ca2+-bound conformation.
The Ca2+-binding sites of annexins differ fundamentally from the EF-hand
arrangement, because only five of the seven coordination sites are provided by
protein oxygens and because no EF-hand-type helix–loop–helix structure is
discernible. Instead the bound Ca2+ is coordinated by three carbonyl oxygens that
are in a short interhelical loop (between the A and B helices of an annexin repeat),
two carboxyl oxygens of an acidic residue that is located at the end of helix D (the
so-called ‘cap’ residue), and water molecules, which can be replaced by phosphoryl
moieties when the annexin binds lipid.
The C2 domain, another Ca2+- and phospholipid-binding motif, was first recognized
as the second conserved domain of protein kinase C, which is where the term ‘C2’
derives from. Although the chelation of Ca2+ by C2 domains also depends on
interactions with the phosphoryl groups of lipids and carboxyl side chains in
polypeptide loops, the core structure of the domain is based entirely on β-sheets
rather than on α-helices, which characterize the annexin structure. The annexin
Ca2+-binding sites and the C2 domains therefore seem to exemplify the convergent
evolution of an important characteristic for signalling proteins — the ability to bind
membranes in a Ca2+-regulated fashion — from two different structural backgrounds.
450 | JUNE 2005
| VOLUME 6
annexin A1 is buried in the core of the molecule and is
exposed only when the protein binds Ca2+ REF. 13. So,
in addition to mediating membrane binding, Ca2+ ions
can also induce a conformational switch that leads to
the presentation of the N-terminal domain.
Many cytosolic protein ligands bind to the N-terminal domain of annexins. In several cases, these ligands
are members of the EFHAND SUPERFAMILY of Ca2+-binding
proteins (for a review, see REF. 14). Annexins A1 and A2
interact with the S100 PROTEINS S100A11 (also known as
S100C) and S100A10 (also known as p11), which are
characterized by two consecutive EF hands; annexin A7
binds to the four-EF-hand-containing protein sorcin;
and annexin A11 binds to the two-EF-hand-containing protein S100A6 (calcyclin). In the case of annexins
A1 and A2, the structural nature of these interactions
has been elucidated. It was shown that the extreme
N-terminal sequences of these annexins adopt the
conformation of an amphiphatic α-helix, which
binds to a pocket formed by both subunits of the
S100 dimer15,16. This establishes a highly symmetrical
entity in which a central S100 dimer connects two
annexin A1 or A2 monomers17 (FIG. 1c). Such structures can form connections between two membrane
surfaces, as they harbour two membrane-binding
annexin cores that are bridged by an S100 dimer.
The only annexin that contains two annexin cores
within a single physical entity is annexin A6, which
arose from the duplication and fusion of the genes for
annexin A5 and A10 (for a review, see REF. 18). Crystal
structures of annexin A6 in solution and on membranes show that the two core modules can probably
orient themselves in a flexible manner relative to one
another, which allows the molecule to bind with its
core domains attached to one membrane or to two
separate membranes19,20 (FIG. 1d).
On the basis of the above information, we propose
the term ‘N-terminal interaction domain’ for the
unique N-terminal region of the annexin molecules.
However, it should be emphasized that, in addition to
forming protein-interaction sites, these domains are
also subject to post-translational modification. This
includes the myristoylation of annexin A13 and the
serine/threonine, as well as tyrosine, phosphorylation
of several annexins, most notably annexins A1, A2,
A4, A6 and A7. In the unphosphorylated state, the
phosphate-accepting residues are often tucked into
pockets in the core domain, such that they would
require considerable reorientation to be phosphorylated. Crystallographic data on phosphorylationmimicking mutations (T or S mutated to D or E) have
revealed the type of movements that would occur
within annexins A4 and A6 REFS 21,22. Tyrosine phosphorylation of the N-terminal domains of annexins
A1 and A2 — which is catalysed by the epidermal
growth factor (EGF) receptor kinase and Src-family
tyrosine kinases, respectively — has been studied
extensively in biochemical assays. It has been shown
to increase proteolytic sensitivity (for annexin A1) and
to lead to altered Ca2+ affinities in the core domain (for
annexin A2; for a review, see REF. 14).
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REVIEWS
a
c
Membrane
d
Ca2+
Protein core
domain
Annexin
repeat
N-terminal
domain
b
Membrane
C
N
Figure 1 | Annexin structure. a | A schematic drawing of an annexin that is peripherally attached to a membrane surface
through bound Ca2+ ions (blue). b | Structural model of an annexin core. A 310-amino-acid sequence that represents the
homologous core region of vertebrate annexins was derived from an alignment of 250 annexin sequences. It was threaded
through the crystal structure coordinates of closely related annexins including annexin A4 (Protein Data Bank (PDB) file 1ANN)
and annexin A5 (PDB file 1AVR)127. Each annexin repeat (coloured differently) contains five α-helices that are connected by short
loops or turns. The N and C termini are coloured black. Red spheres within stick-representation residues highlight atomic
oxygens that are involved in forming the type-II Ca2+-binding sites, whereas blue spheres denote nitrogen atoms of highly
conserved basic residues. Image courtesy of R. O. Morgan and M. P. Fernandez, University of Oviedo, Spain. c | Ribbon
representation of a model of the heterotetrameric (annexin A2)2–(S100A10)2 complex that was derived using crystal structures of
the individual subunits15,128. The annexin A2 core domains are depicted in red, the N-terminal α-helices that contact S100A10
are shown in green and yellow, and the S100A10 subunits are shown in two shades of blue. The model was kindly provided by
S. Rety, University of Paris, France, and was reproduced with permission from REF. 129 © (2000) Elsevier Science. d | Ribbon
representation of the annexin A6 crystal structure (PDB file 1AVC)19. Annexin A6 contains eight repeat segments, and the two
halves of the protein, which each consist of a four-annexin-repeat unit (core), are connected by a linker (depicted in dark green).
The flexibility of the linker enables the two halves to adopt different orientations with respect to one another20. Bound Ca2+ is
shown as blue spheres. Part d modified with permission from REF. 19 © (1998) Elsevier Science. In parts b–d, the membraneattachment sites of the annexin core units are indicated by green triangles.
Annexins as membrane scaffolds
Annexins not only bind to the cytosolic surfaces of
cellular membranes as single molecules or in complex
with protein ligands attached to their N-terminal interaction domain, they can also form lateral assemblies, at
least on model membranes. This is shown in cryo-electron and atomic-force microscopy images of annexin A5
bound to phosphatidylserine-containing bilayers23–26. In
such images, the protein forms two-dimensional (2D)
crystals on the bilayer, with annexin A5 trimers representing the principal building block (FIG. 2a,b). Efficient
trimer formation depends on Ca2+ and membrane
binding, and is probably stabilized by lateral contacts
between membrane-bound annexin A5 cores. Moreover,
establishment of the 2D annexin A5 network can induce
membrane indentation and subsequent inward vesicle
budding if phosphatidylserine is exposed on the surface
of the membrane27. Given the abundance of annexin A5
in some cells and its localization to the plasma membrane,
2D assemblies of the protein might function in stabilizing
certain plasma-membrane structures and/or in changing
membrane curvature and therefore cell shape.
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
Annexins A1 and A2 seem to associate into different types of assembly. Atomic-force-microscopy images
of annexins A1 and A2 bound to bilayers that contain
phosphatidylserine/phosphatidylcholine mixtures
reveal more amorphous, monolayered protein clusters28,29 (FIG. 2c). Interestingly, the establishment of the
annexin assembly seems to be accompanied by the
segregation of membrane lipids, with certain negatively
charged phospholipids accumulating underneath the
annexin clusters. This has been shown for annexins A2
and A4, which were bound to artificial membranes of
simple phospholipid mixtures29,30. Such an activity
could be responsible for an annexin-mediated and/or
-supported formation of certain phospholipid domains
in cells. Indeed, it has been observed that annexin A2 can
bind directly and specifically to phosphatidylinositol-4,5bisphosphate (PtdIns(4,5)P2), and this binding has
been linked to the formation or stabilization of actin
assembly sites at cellular membranes (see later). It still
needs to be established whether the annexin association is a prerequisite for the segregation of certain lipids
or whether it simply follows the formation of lipid
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Box 2 | Biochemical properties that define annexins
Proteins of the annexin family share unique Ca2+- and lipid-binding properties,
which are considered to be their biochemical hallmark — that is, they can associate
with negatively charged phospholipids in a Ca2+-dependent and reversible manner.
Structurally this interaction is mediated through Ca2+ that is bound at the protein–
phospholipid interface4,6 (FIG. 1). In vitro assays using phospholipid liposomes in
Ca2+-dependent co-pelleting experiments are typically used to record this canonical
annexin property. They have shown that acidic phospholipids are the preferred
binding partners, in particular, phosphatidylserine, phosphatidylinositol and
phosphatidic acid. In line with the cytosolic localization of annexins, these lipids are
enriched in the cytoplasmic leaflets of cellular membranes. Some annexins also show
further, more specific interactions with membrane lipids — for example, annexin A2
binds phosphatidylinositol-4,5-bisphosphate, annexins A3, A4, A5 and A6 can
interact with phosphatidylethanolamine, and annexin A5 has been reported to bind
to phosphatidylcholine (for reviews, see REFS 48,95). The free Ca2+ concentrations
required to initiate phospholipid binding differ markedly between different annexins
and different headgroups. They range from 20 µM for the binding of annexin A5
to phosphatidylserine-containing liposomes, through submicromolar Ca2+
concentrations for the phosphatidylserine- and phosphatidic-acid-binding of
annexins A1 and A2, to less than 100 nM for the interaction of the annexin-A2–
S100A10 complex with phosphatidylserine-containing membranes (for more
information, see REF. 95). This indicates that the annexin family as a whole can
respond to a large spectrum of stimulus-induced Ca2+ changes with cytosol-tomembrane translocation. However, individual family members are precisely tuned to
react only to stimuli of a certain amplitude.
PLECKSTRINHOMOLOGY
DOMAIN PROTEIN
Proteins that contain a
pleckstrin-homology domain,
which is a conserved motif that
is most frequently associated
with binding to inositol
phospholipids.
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domains through specific binding to certain headgroups, for example, to PtdIns(4,5)P2 in the case of
annexin A2.
In addition to peripherally binding to membranes
of a certain composition, some annexins — most notably annexins A1, A2, A4, A6 and A7 — can aggregate
membranes in a Ca2+-dependent manner. The mechanism by which annexins link two membranes, which
we refer to as ‘bivalent’ annexin activity, is not fully
understood. One model proposes that amphipathic
helices in the N-terminal domain of annexins can
interact directly with a second membrane while the
core domain is bound to a first membrane13. However,
annexin core domains in isolation can aggregate membranes, so the N-terminal domain is not essential for
this function in vitro31,32. At least under these in vitro
conditions, it seems that two or more annexin cores
that are facing different membranes interact to promote contact between membranes, or that another
membrane-binding surface must be present in a single
annexin core (for a review, see REF. 14). In the case of the
annexin-A2–S100A10 complex and probably also
the annexin-A1–S100A11 complex, membrane aggregation can be explained by the configuration of the
heterotetrameric complex, with the central S100 dimer
providing a bridge between two membrane-bound
annexin cores (FIG. 1c). Cryo-electron microscopy
images of membrane vesicles that have been aggregated by annexin-A2–S100A10 indeed show highly
symmetric junctions that probably comprise arrays of
annexin-A2–S100A10 complexes that connect two
opposing membranes33.
Provocative evidence that annexins can promote
intermembrane contacts in vivo comes from studies of
the membrane complexes of the spermathecal valve of
Caenorhabditis elegans34. This structure is an opening
between the spermathecal chamber, where fertilization
occurs, and the uterus. After fertilization, the egg must
pass through this opening. However, the diameter of
the egg is five times the diameter of the opening, so the
opening must expand to allow the egg to pass. After
passage of the egg, the opening must close and the
extensive membrane that lined the opening has to be
folded up in an accordion-like fashion. The cytoplasmic surfaces of the membrane folds are richly endowed
with the main nematode annexin NEX-1 REFS 34,35.
This places the annexin in a perfect position to promote the folding of the membrane by initiating membrane contacts, and this idea is consistent with in vitro
studies of annexins that show them to be membrane
aggregators rather than fusogens.
Phosphorylation of the N-terminal domain has
a marked effect on the ‘bivalent’ activity of several
annexins. For annexins A1, A2 and A4, Ca2+-dependent
membrane aggregation is strongly inhibited by phosphorylation22,36,37, whereas for annexin A7 aggregation
is activated38. As these phosphorylation events do not
greatly influence the ‘monovalent’ ability of annexins
to bind to a single membrane, they probably alter
other structural features that are essential to bring two
membranes together.
Annexins in membrane organization and traffic
Annexins and membrane domains. In addition to
triggering and/or supporting membrane-domain
formation in vitro, annexins have been shown to
participate in such events in vivo. This has been most
extensively studied for annexin A2, which is implicated
in the organization of membrane lipids at sites of actin
cytoskeleton attachment (FIG. 3). Evidence for such a
role came from studies that identified annexin A2 in
two distinct settings. First, annexin A2 was shown
to be a component of the filamentous (F)-actin-rich
comet tails that propel newly formed endocytic vesicles
from the plasma membrane to the cell interior, and a
dominant-negative mutant of annexin A2 was shown to
abrogate actin-dependent vesicle rocketing39,40. Second,
annexin A2 was observed to be a component of the
F-actin pedestals that form at the membrane-attachment
sites of enteropathogenic Escherichia coli41.
Despite these being unrelated biological processes,
they are characterized by the dynamic reorganization of actin at membrane sites that are enriched
in PtdIns(4,5)P2. Biochemical studies and various
imaging strategies showed that annexin A2 binds to
PtdIns(4,5)P2 in vivo, and that binding occurs with a
similar affinity to that for many PLECKSTRINHOMOLOGY
DOMAIN PROTEINS, but with an unusually high specificity
for this phosphoinositide42,43. Together with studies
showing that annexin A2 can bind to membranes
containing cholesterol in a Ca2+-independent manner
(for a review, see REF. 44), these observations reinforce
the idea that annexin A2 has a role in the organization of lipid-raft-like membrane constituents at sites
of actin recruitment. Interestingly, annexin A2 is an
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REVIEWS
LIPID MICRODOMAIN
A localized membrane region
that differs from the
surrounding membrane in its
lipid composition and order.
LIPID RAFT
Lateral lipid aggregates that are
rich in cholesterol and
sphingolipids, and are thought
to occur in cellular membranes.
These lipid microdomains are
resistant to solubilization by
non-ionic detergents and
probably resemble the liquidordered domains that are found
in model membranes.
actin-binding protein itself and might function directly
as an F-actin interaction platform. The organization
of ordered LIPID MICRODOMAINS by annexin A2 might be
important not only for cellular signalling, but also in
the stabilization of cell–cell contact sites in endothelial
and epithelial monolayers (for a review, see REF. 45).
Furthermore, in smooth muscle cells that undergo
cycles of contraction and relaxation, annexin A2
has been shown to be involved in the Ca2+-dependent
organization of LIPID RAFTS, whereas annexin A6 is
involved in membrane–actomyosin interactions46.
A different process is affected by annexin A11,
which has been shown to be involved in CYTOKINESIS.
Annexin A11 is a MIDBODY protein and therefore probably functions in the terminal phase of cytokinesis when
abscission occurs and the two daughter cells separate.
EM
a AFM
EM
CYTOKINESIS
The final stage of the celldivision cycle, in which two
daughter cells become separated
by the central spindle.
MIDBODY
A dense protein matrix that
forms at the midpoint of the
central spindle during
cytokinesis. Midbody proteins,
of which annexin A11 is one,
are required for cleavage-furrow
formation and the final
separation of daughter cells
by abscission.
10 nm
10 nm
b
c
10 nm
Annexin A5 in solution
P6 OR P3
Space groups that define crystal
lattices with sixfold and
threefold axes of symmetry,
respectively.
Ca2+
Annexin A5 on membrane
0 nm
Annexin A5 trimers
p6 2D crystals
p3 2D crystals
Membrane containing negatively
charged phospholipids
5 µm
Acidic
phospholipids
Annexin-A2–S100A10
Figure 2 | Annexin assemblies on membranes. a | Annexin A5 arrays. Following its Ca2+-dependent binding to model
membranes that contain negatively charged phospholipids, annexin A5 self-assembles into two-dimensional (2D) ordered
arrays, which can be visualized by atomic-force microscopy (AFM) and electron microscopy (EM)23,25,26. The red and blue
colouring in the EM image highlights two annexin A5 trimers, which represent the building blocks of the 2D crystals. The
individual annexin repeats, which are labelled I–IV (one core domain is depicted in red for clarity), are easily discernible. Two types
of 2D crystal can be identified that have P6 OR P3 symmetry (these images show the p6 symmetry; blue and green rectangles in
the AFM image point to areas with defects in the 2D assembly). The p3 crystals are typically found on membranes that have
a higher phosphatidylserine content or at higher Ca2+ concentrations. Images courtesy of A. Brisson, University of Bordeaux,
France. The AFM image was modified with permission from REF. 26 © (1998) Elsevier Science, and the EM image was
modified with permission from REF. 130 © (2001) Elsevier Science. b | Model for the formation of the ordered annexin A5 arrays.
c | Annexin A2 in complex with S100A10 forms more amorphous structures on membranes that contain a mixture of
phosphatidylcholine and phosphatidylserine. AFM analysis shows a height difference of 4.3 nm between the lipid bilayer and the
protein layer29. This indicates the formation of a monolayered protein network. The image shown in part c was kindly provided by
C. Steinem, University of Regensburg, Germany.
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
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EPEC
Exocytic/secretory
vesicle
Endocytic
vesicle
Nucleus
SHP2
Rac1
Annexin A2
F-actin
Figure 3 | The regulation of membrane–actin interactions by annexin A2. Several
annexins have been shown to be involved in membrane–cytoskeletal organization, and
this schematic figure represents one particularly well-studied theme. Annexin A2 is
involved in diverse cellular activities that involve the coordination of actin polymerization
with membrane dynamics. In two instances, annexin A2 has been shown to bind
phosphatidylinositol-4,5-bisphosphate at sites where actin polymers form membrane
contacts — namely the attachment points for enteropathogenic Escherichia coli (EPEC)41
and on rocketing endocytic vesicles39,40. Annexin A2 is also required for the actindependent transport of glucose transporter-4 (GLUT4)-containing secretory vesicles in
adipocytes and exocytic vesicles in polarized epithelial cells (this is not considered further in
this article, so for further information, see REF. 44). Last, annexin A2 is a component of
mature cholesterol-rich junctions in endothelial cells. Here, it associates with the tyrosine
phosphatase SHP2 (SH2-domain containing phosphatase-2) and exists in complex with
Rac1 at cell–cell contact sites between polarized epithelial cells and, in both instances,
annexin A2 is concentrated at sites where filamentous (F)-actin is coordinated with the
plasma membrane. This role is also not considered further in this article, so for further
information, see REFS 44,45.
Consistent with this idea, depletion of annexin A11
leads to a failure in midbody formation and to apoptosis of the two daughter cells47. One possible role for
annexin A11 in cytokinesis is in the trafficking, or insertion and fusion, of the new membrane that is known to
be required for abscission.
NUCLEOPLASM
The part of the nucleus that is
contained by, but is distinct
from, the nuclear envelope.
HYDROXYAPATITE
A crystalline form of calcium
phosphate that is present in the
matrix of bone.
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Annexin transport. The biochemical properties of
annexins indicate they should be soluble and freely
distributed in the cytoplasm of cells at resting Ca2+ levels,
and that they should move to membranes only when
Ca2+ levels become elevated. However, there are many
exceptions to this expected pattern, which indicates that
other factors influence annexin transport and localization in cells (for a review, see REF. 14). The localization
of annexins to particular membrane systems, rather
than to all phospholipid-containing surfaces in a cell,
might reflect non-homogeneous concentrations of
Ca2+ in different regions of a cell (see later; also see
REF. 48 for an overview of annexin localization).
Moreover, the interaction of annexins with particular
membrane lipids (see above) and/or other proteins
could result in specific subcellular localizations.
Localization studies have also shown that some
annexins move from the cytoplasmic compartment into
the nucleus. Annexin A2 has a nuclear export signal
in its N-terminal domain that is a substrate for the
leptomycin-sensitive nuclear transport machinery49. As
a consequence, this annexin is generally excluded from
the nucleus. However, tyrosine phosphorylation might
cause conformational changes near this export sequence
that allow the protein to enter the nucleus, because
increased levels of nuclear annexin A2 are detected in
the presence of tyrosine-phosphatase inhibitors, and
lower levels are seen in the presence of a tyrosine-kinase
inhibitor49. Annexin A5 also seems to enter the nucleus
after serum stimulation and tyrosine-kinase signalling50,
and in response to oxidative stress51. Furthermore,
annexin A11 has been detected as a nuclear protein52
that translocates from the NUCLEOPLASM to the nuclear
envelope in cells at prophase53. The physiological
importance of these nuclear localizations remains to
be elucidated, but it should be noted that annexin A2
has been reported to bind certain species of RNA54,55,
which indicates a possible role in RNA transport or
export from the nucleus.
Annexins have also been found outside cells in
the context of multicellular organisms, and this has
raised the question of whether extracellular annexins
are the product of cell lysis or whether there are specific transport systems that mediate annexin export
from cells. Non-classical secretion pathways have
been proposed (see, for example, REFS 5658), but the
export process might involve a mechanism similar to
the one that underlies mineral-content release from
matrix vesicles during bone formation. Annexins are
abundant proteins in bone matrix vesicles, and it has
been speculated that they have a role in Ca2+ entry into
the vesicles and in the formation of HYDROXYAPATITE59.
Matrix vesicles pinch off from the plasma membrane
of bone-forming cells, and then the vesicle membranes break down and release their contents. Release
by such a ‘blebbing’ process could be a more general
phenomenon that provides a mechanism for annexin
export in other contexts. To be selective for annexins,
such a release process would have to depend on Ca2+
influxes to concentrate the annexins in the nascent
vesicles. It is an interesting, but unexplored, possibility that the bivalent activity of annexins might be
involved in closing of the neck of the matrix vesicle,
or other bleb structure, as it pinches off from the cell.
It has recently been reported that the externalization
of annexin A2 from vascular endothelial cells seems
to require the phosphorylation of a tyrosine residue
in the N-terminal domain, because a Y→A mutant
protein cannot be released60. Perhaps phosphorylation is necessary for the annexin to be appropriately
localized and concentrated in a release vesicle, or to
escape through another, as yet undefined, transport
machinery that might specifically recognize the phosphorylated tyrosine.
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CHROMAFFIN GRANULES
The secretory vesicles of the
adrenal medulla. They contain
noradrenaline or adrenaline, a
number of biologically active
peptides and high
concentrations of ATP and
ascorbic acid. The name is
derived from the histological
observation that the vesicles are
readily stained by chromium
salts.
ARACHIDONIC ACID
A highly unsaturated, longchain fatty acid (20 carbon
atoms: 4 double bonds) that is
often found at the sn-2 position
of the glycerol backbone of
membrane phospholipids.
It is typically released by
phospholipase action in
stimulated cells, which allows it
to function as a membrane
fusogen or as the precursor of
active signalling molecules such
as the prostaglandins and
leukotrienes.
Exocytosis. A recurring theme in annexin research
has been the possible involvement of these proteins in promoting membrane fusion during Ca2+regulated exocytosis61 BOX 3. The first annexin to
be characterized, annexin A7, was discovered in a
search for proteins that promote the contact and
fusion of CHROMAFFIN GRANULES, the secretory vesicles
of the adrenal medulla 62,63 . Subsequently, several
other annexins were found to be key components
of the ‘chromobindin’ fraction of chromaffin cells
— that is, proteins that bind to chromaffin-granule
membranes in the presence of Ca2+ REF. 64. However,
in vitro studies and electron-microscopy examination
of the contacts formed between chromaffin granules
by annexin A7 made it clear that this annexin is not a
fusogenic protein — it promotes only the close attachment of membranes to one another62. If annexin A7
initiates membrane contacts in exocytosis, something
else then has to happen to promote membrane fusion.
In vitro, low concentrations of free ARACHIDONIC ACID
were found to specifically promote the fusion of membranes that had been brought together by annexin
A7 REF. 65. However, in vivo, it now seems probable
that essential fusogenic proteins — such as the
66
SNARE PROTEINS — take the place of arachidonic acid
in driving the fusion of membranes that have been
aggregated by an annexin.
Several studies have also indicated that annexin A2
is involved in Ca2+-regulated exocytosis. In permeabilized chromaffin cells, the time-dependent loss of
Box 3 | Ca2+-regulated exocytosis
The terminal step in the secretion pathway of proteins, polypeptide hormones and
amine neurotransmitters is the fusion of the secretory-vesicle membrane with the
plasma membrane. Frequently, this is a point of interruption in the secretory pathway,
and mature secretory vesicles accumulate in the cell until the stimulus-dependent entry
of Ca2+ into the cytoplasm precipitates the final membrane-contact and -fusion steps.
Some of the underlying proteins that mediate membrane fusion have been well defined
on the basis of genetic studies, the unravelling of the functional mechanisms of toxins
that block exocytosis and the in vitro reconstitution of membrane fusion in cell-free
systems. Specific integral membrane proteins on the vesicle, so-called v-SNAREs, and
on the plasma membrane (target- or t-SNAREs) intertwine with one another and
rearrange the relevant lipid bilayers so that fusion ensues (for a recent review, see
REF. 126). However, it is much less clear how the absence of Ca2+ interrupts this process,
or how the presence of Ca2+ accelerates it.
A wide range of Ca2+-binding proteins is present on the relevant membranes and in
the cytoplasm of typical secretory cells. These proteins might function at different
sites in the overall process of exocytosis, such as during membrane–membrane
approach, docking, fusion or during the recovery of the vesicle membrane for re-use.
Important candidate proteins include EF-hand proteins such as calmodulin and
the S100 proteins, and C2-domain-containing proteins such as synaptotagmin,
rabphilin, RIM (Rab3-interacting molecule) and MUNC13 (mammalian homologue
of Caenorhabditis elegans UNC-13). Because of their ‘bivalent’ ability to link two
membranes, annexins are further obvious candidates that might have a role in this
process. However, it remains an open question whether the contacts formed by
annexins between membranes are crucial precursors for biological membrane
fusion, or indeed whether annexins might hinder the fusion process by obstructing
SNARE interactions. Both roles seem possible and the net effect of annexins in
secretory cells might depend on the relative abundance of different annexin species
at the membrane-fusion site.
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
secretory capacity could be blocked by the addition
of annexin A2 to the culture medium67. The activity of
annexin A2 in this system depends on the proteinkinase-C-mediated phosphorylation of S25 in its
N-terminal domain (protein kinase C is activated in
chromaffin cells in response to the nicotinic stimulation of secretion)68,69. However, arguing against a
general function of annexin A2 in Ca2+-regulated exocytosis, a dominant-negative annexin A2 mutant did
not affect exocytosis in the neuroendocrine PC12 cell
line70. More recent studies in endothelial cells — which
are non-excitable and contain different types of secretory granule — have shown that the downregulation
of annexin A2 using small interfering RNAs, and the
disruption of the annexin-A2–S100A10 complex by
the injection of peptide competitors, can block the
Ca2+-evoked exocytosis of Weibel–Palade bodies without affecting other Ca2+-regulated exocytic routes71,72.
Annexin A2, in complex with S100A10, therefore
seems to be involved in only a subset of Ca2+-dependent exocytosis events, possibly those that involve especially large organelles such as Weibel–Palade bodies
and chromaffin granules (FIG. 4a).
A specialized function for an annexin in exocytic
membrane traffic has also been found for annexin A13,
which is expressed in polarized epithelial cells. The
annexin A13b splice variant associates specifically with
sphingolipid- and cholesterol-rich membrane domains
of the trans-Golgi network, and N-terminally myristoylated annexin A13b is required for the budding of
these domains, which are subsequently delivered to the
apical plasma membrane73 (FIG. 4a).
If annexins do have a role in regulating membrane fusion in cells, they probably work together
with the SNARE proteins, as mentioned above. A
minimal lipid vesicle membrane-fusion assay has
been established that shows that the SNAREs alone
can promote vesicle fusion66. The rate of fusion in
this system can be accelerated by the addition of Ca2+
and the soluble portion of synaptotagmin, a protein
that binds membranes through the action of two
Ca2+-dependent C2 domains74. Interestingly, this fragment of synaptotagmin has also been shown to have
a bivalent activity that is similar to that of annexins,
in that it promotes the Ca 2+ -dependent aggregation and fatty-acid-dependent fusion of chromaffin
granules in vitro75. It would be interesting to further
investigate this SNARE-based membrane-fusion
model by adding annexins and Ca2+ to determine if
they also accelerate the fusion process by improving
the kinetics of initial membrane-contact formation. However, it is possible that the annexins might
instead inhibit fusion, as they are known to inhibit
other membrane-active proteins, such as phospholipases and protein kinase C, by shielding membrane
surfaces. The results of such an experiment — the
acceleration or inhibition of SNARE-dependent
fusion — might determine which of the two different
roles the ‘annexin membrane shield’ has; is it a facilitator of, or a protective inhibitor against, biological
membrane fusion?
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REVIEWS
a Exocytosis
b Endocytosis
Annexin A2
Plasma
membrane
Apical delivery
in polarized
epithelial cells
Annexin A6
Cholesteryl ester
internalization/
transport
Certain clathrindependent
budding events
Ca2+-induced
exocytosis of
certain secretory
granules
Annexin A1
Annexin-A2–
S100A10
Cytosol
Chromaffin granule,
Weibel–Palade body
Trans-Golgi
network
Sorting
endosome
Inward vesicle
budding
Receptor
recycling
Annexin A2
Annexin A13b
Biogenesis of
multivesicular
endosomes
Budding of apical
transport vesicles
Annexin-A2–S100A10
Recycling
endosome
Multivesicular
endosome
Figure 4 | Annexins in membrane organization and trafficking. A schematic representation of various membrane-trafficking
steps showing the involvement of annexins. a | In the biosynthetic pathway, annexin A2 in complex with S100A10 has been
shown to participate in the Ca2+-evoked exocytosis of chromaffin granules and endothelial Weibel–Palade bodies. The complex
probably functions at the level of the plasma membrane, possibly by linking the large secretory vesicles to the plasma membrane
or by organizing plasma-membrane domains so that efficient fusion can take place. Annexin A13b is required for the budding of
sphingolipid- and cholesterol-rich membrane domains at the trans-Golgi network, and therefore the delivery of such material to
the apical plasma membrane in polarized epithelial cells. b | In the endocytic pathway, annexin A6 has been proposed to be
involved in clathrin-coated-pit budding events that depend on the activity of a cysteine protease that is required to modulate the
spectrin membrane skeleton. Annexin A2, which can associate with caveolae, has been shown to form a lipid–protein complex
with acylated caveolin and cholesteryl esters that seems to be involved in the internalization/transport of cholesteryl esters from
caveolae to internal membranes. Annexin A2 is also found on early endosomes, where it is required, in complex with S100A10,
to maintain the correct morphology of perinuclear recycling endosomes. Moreover, its depletion can interfere with the proper
biogenesis of multivesicular endosomes from early endosomes. Annexin A1 also seems to function in multivesicular endosome
biogenesis, more specifically, in the process of inward vesicle budding. For further details, and the relevant references, please
refer to the main text.
SNARE PROTEINS
(soluble N-ethylmaleimidesensitive fusion protein (NSF)
attachment protein receptor
proteins). Integral membrane
proteins in vesicle or cellsurface membranes that interact
with one another during
membrane fusion. The name is
derived from the role of these
proteins as receptors for a
cytosolic protein, NSF, that is
essential for organelle
trafficking steps that involve
membrane fusion.
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| VOLUME 6
Endocytosis. Several annexins, in particular annexins
A1, A2 and A6, are present on endosomal compartments, and unique endosome targeting sequences have
been identified in the N-terminal interaction domain
of annexins A1 and A2 REFS 7679. More importantly,
interfering with annexin function by overexpressing
dominant interfering mutants, and by using knockdown or knockout approaches, has revealed direct
effects on endosome morphology and endocytic
transport. A defect that was observed following the
small-interfering-RNA-mediated downregulation of
annexin A2 was the inhibition of the lysosomal transport of fluid-phase tracers and internalized EGF receptors. This correlated with an improper detachment of
multivesicular endosomes from the early endosomal
compartment80. These observations led to the hypothesis that two sequential steps are essential for multivesicular endosome biogenesis, one that requires the
action of HRS (hepatocyte-growth-factor-regulated
tyrosine-kinase substrate) and ESCRT (endosomal
sorting complex required for transport) proteins for
receptor sorting and inward vesiculation, and a second
that depends on an annexin A2 scaffold on early endosomes that facilitates the efficient detachment of the
multivesicular regions of early endosomes81 (FIG. 4b).
Similar scaffolding functions of annexin A2 are probably also responsible for another endosomal phenotype
that has been documented in annexin A2-depleted
cells. A separate study showed that cells deprived of
the endosomal and cytosolic annexin-A2–S100A10
complex are characterized by an aberrant morphology
of perinuclear Rab11-positive recycling endosomes82.
These endosomes appear more condensed, their characteristic tubules are often bent to form circles and they
contain an increased number of clathrin-positive buds.
A picture is therefore emerging in which annexin A2
associates with endosomes at specific sites (possibly
those that are characterized by a high membrane cholesterol content80,83,84) to provide a membrane scaffold
that is required for the formation and maintenance of
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elongated endosomal tubules and the detachment/biogenesis of certain regions (FIG. 4b).
Annexin A1 has been implicated in the inward
vesiculation that is seen in multivesicular endosomes
(FIG. 4b). It is a substrate of the EGF receptor kinase,
and it becomes phosphorylated in preparations of multivesicular endosomes that contain internalized EGF
receptors. This phosphorylation alters the sensitivity
of annexin A1 to Ca2+ and proteolysis, and has been
linked to the apposition of membranes during the
process of inward vesicle budding85. Recent work using
fibroblasts from annexin A1-knockout mice shows that
multivesicular endosomes are formed in the absence of
annexin A1, but that these endosomes contain fewer
internal vesicles (C. Futter, personal communication),
which is a phenotype reminiscent of that observed in
HRS-depleted cells86.
By using a truncated annexin A6 mutant, Kamal
et al.87 have shown that a cysteine-protease-dependent
type of budding of clathrin-coated pits requires annexin
A6 and its association with NONERYTHROID SPECTRIN
(FIG. 4b). The mutant also interferes with the trafficking
of low-density-lipoprotein-containing vesicles to degradative endosomal compartments88,89. However, the
functional mechanism of such a mutant is not clear, as a
similar mutant that was expressed in the hearts of transgenic mice caused changes in Ca2+ signalling and
stimulus–response coupling90. Although these studies
might argue for the participation of annexin A6 in
remodelling the membrane cytoskeleton during specific
budding events, it is evident that such annexin A6dependent mechanisms do not operate in all cells — for
example, not in A431 cells that lack annexin A6 REF. 91.
The uptake of cholesteryl esters is a unique type
of internalization that requires plasma-membrane
caveolae and their principal protein caveolin-1 REF. 92.
Annexin A2 has been identified as part of a chaperone
complex that contains caveolin-1 and cholesteryl esters,
which has been implicated in cholesteryl-ester transport from caveolae to internal membranes93. Complex
formation requires the association of annexin A2 with
acylated caveolin, which is evident not only in cultured
cells but also in the intestines of zebrafish and mice.
In zebrafish, the downregulation of annexin A2 using
antisense oligonucleotides prevented complex formation and cholesterol processing, probably by inhibiting
intestinal sterol transport94.
So, several endocytic processes involve annexins
and, in most cases, these proteins seem to provide a
structural framework on the respective membranes
that is required for organelle organization and
efficient transport.
NONERYTHROID SPECTRIN
An isoform of spectrin that is
sometimes also called fodrin
and is expressed in cells other
than erythrocytes.
Ion channel regulation. One of the more contentious
issues in annexin biology has been the proposal that
annexins function as Ca2+ channels. This subject has
been extensively reviewed (see, for example, REFS 12,95),
so here it is addressed only briefly. The idea that annexins could be Ca2+ channels originates from studies on
recombinant annexins in artificial lipid bilayers. In
this setting, annexins show many ion-channel-like
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
properties, with selectivity for Ca2+, voltage dependency and single channel conductance values that are
broadly similar to those of other Ca2+ channels. The
Ca2+-channel hypothesis was boosted by the elucidation
of the first annexin crystal structure. This structure has
a central pore that lies at the core of the protein and is
lined by residues that were shown to be determinants
of ion selectivity and voltage sensitivity in mutational
studies96. So, on the basis of structural and biophysical properties, annexins seem to satisfy a minimum
set of essential criteria that indicate they function as
Ca2+ channels.
However, evidence for annexins functioning as Ca2+
channels in vivo is scarce, and conceptual problems have
obstructed acceptance of the Ca2+-channel hypothesis.
There are questions as to how peripherally associated
annexins could conduct Ca2+ across the membrane. In
addition, the diameter of the central pore is inconsistent with the observed conductance values, and channel
openings are infrequent and require extreme degrees of
hyperpolarization. Arguments about the mechanism
of Ca2+ conductance have been countered by two models. In one, theoretical calculations predict that annexin
A5 could sufficiently perturb the organization of lipids
in the bilayer at the site of Ca2+-dependent attachment
to effectively electroporate the membrane and therefore
permit Ca2+ entry97. In the second, which is supported
by experimental evidence, extensive mutagenesis
of the Hydra annexin B12 in conjunction with spinlabelling showed that, at a mildly acidic pH, the protein
undergoes a considerable conformational change that
is accompanied by membrane insertion. Despite the
thermodynamic considerations of such a structural
metamorphosis, the hypothetical membrane-inserted
annexin is proposed to have seven transmembrane
domains and would therefore adopt the topology of a
more conventional channel10.
Data concerning the regulation of ion channels
by annexins are less controversial. Roles for annexins
A2, A4 and A6 as modulators of plasma-membrane
Cl– channels and sarcoplasmic reticulum Ca2+-release
channels have recently been reviewed (see REF. 14). An
interesting aspect of the possible role of annexin A2 as
a Cl–-channel regulator is the observation that its effect
seems to be dependent on complex formation with
S100A10 REF. 98. In this context, S100A10 has itself
been identified in several reports as being an interacting partner of the NaV1.8 Na+ channel99, the two-pore
acid-sensitive K+ channel-1 (TASK1)100, and the transient receptor potential-V5 (TRPV5) and TRPV6
epithelial Ca2+ channels101. The theme that emerges
from these studies is that S100A10, usually in complex
with annexin A2, is required for the trafficking of these
ion channels from their intracellular sites (on the biosynthetic or endosomal route) to the plasma membrane. These observations exemplify the functional
importance of annexin A2 in intracellular transport,
particularly in polarized cells, and also raise the possibility that the annexin-A2–S100A10 complex has a
secondary function in tethering ion channels to the
cytoskeleton underlying the plasma membrane.
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REVIEWS
Vascular
endothelium
Lumen
Plasminogen
tPA
N
Annexin-A2–
S100A10
Plasmin
Annexin A1
N
Neutrophil/
monocyte
N
FPR/FPRL
Fibrin
cleavage
Glucocorticoids
Inhibition of
extravasation
Figure 5 | Annexins have specialized extracellular roles. It is a remarkable characteristic of
certain annexins that they have functionally distinct roles inside and outside cells. Extracellular
roles for annexins are exemplified in this schematic figure by annexins A1 and A2. Annexin A1
translocates to the surface of neutrophils and monocytes following their exposure to
glucocorticoids and, once extracellular, annexin A1 and/or its proteolytically removed N terminus
can bind to chemoattractant receptors of the formyl peptide receptor (FPR) family (FPR and FPRlike (FPRL) receptors). Through mechanisms that involve receptor desensitization, but that are
not fully understood, this annexin-A1/annexin-A1-peptide binding inhibits neutrophil and
monocyte extravasation, and therefore moderates the accumulation of pro-inflammatory
leukocytes at sites of injury or infection. By contrast, annexin A2, in complex with S100A10, can
be resident on the surface of vascular endothelial cells, where it functions as a receptor for tissue
plasminogen activator (tPA) and plasminogen. It can therefore mediate the generation of plasmin,
which, in turn, is crucial for the degradation of fibrin. Loss of annexin A2 function owing to
modification of its N-terminal domain by systemic stress factors, such as glutathione and
homocysteine, might therefore lead to increased thrombus formation.
The functions of annexins outside cells
GLUCOCORTICOIDS
A class of steroid hormones
with a potent antiinflammatory activity.
NEUTROPHIL EXTRAVASATION
The processs by which
neutrophils
(polymorphonuclear
leukocytes) leave a blood vessel.
PERITONITIS
Inflammation of the
peritoneum (the membrane
that lines the abdominal cavity
and digestive organs of
vertebrates).
ACUTEPHASE REACTION
The defense reaction of an
organism to infectious or toxic
agents, which helps to restrict
organ damage through the
cytokine-induced production of
protective acute-phase proteins
such as complement-reactive
and serum-amyloid protein.
458 | JUNE 2005
| VOLUME 6
Annexin A1 in inflammation and apoptosis. Extracellular
occurrence has been shown consistently for several
annexins, and different means of an unconventional
secretion have been reported (see earlier). Annexin
A1 is the most prominent of these examples. It has
long been known to occur extracellularly under conditions of inflammation, and it shows potent antiinflammatory activities when applied exogenously in
animal models of inflammation. As the expression
and secretion of annexin A1 can be stimulated by
GLUCOCORTICOIDS, and as its anti-inflammatory effects
mimic those elicited by glucocorticoids, the protein is
considered to be a mediator of glucocorticoid actions
in inflammatory scenarios (for reviews, see REFS 102105).
In vitro and in vivo models both show that exogenously administered annexin A1 inhibits NEUTROPHIL
EXTRAVASATION and thereby limits the degree of inflammation. This activity is retained in N-terminal annexin
A1 peptides, which are probably generated by proteolysis at sites of inflammation and interact with specific
receptors on leukocytes (FIG. 5).
The annexin A1 receptors on leukocytes have been
identified as members of the formyl peptide receptor
(FPR) family — FPR and the FPR-like receptors FPRL1
and FPRL2 REFS 106109. FPR, the founding member
of the family, is a G-protein-coupled chemoattractant receptor, which can sense gradients of bacterial
peptides of the prototype formylmethionine-leucinephenylalanine (fMLF) and thereby direct leukocytes
towards sites of bacterial infection (for a review, see
REF. 110). In a dose-dependent manner and by triggering FPR/FPRLs, annexin A1 can stimulate several
responses in leukocytes. These include the induction
of L-selectin shedding and the resulting detachment
of adherent leukocytes from activated endothelium,
as well as the desensitization of the receptor towards
the fMLF stimulus106,111. As a consequence, leukocyte
extravasation is severely inhibited and the extent of the
inflammatory response is downregulated (FIG. 5).
Evidence for this and for a physiological function
for annexin A1 in regulating leukocyte trafficking in
inflammatory scenarios comes from the analysis of
annexin A1-null mice, which show increased neutrophil
extravasation following zymosan-induced PERITONITIS
and an exacerbation of antigen-induced arthritis112,113.
Interestingly, the active N-terminal annexin A1 peptide
also triggers FPR-mediated processes — in particular,
chemotaxis and ACUTEPHASE REACTIONS — in cells of
non-myeloid origin114. This indicates that, through
interactions with FPR and FPR-like receptors, annexin
A1 and its N-terminal peptide can regulate many
cellular responses that result in the complex control of
cell-migration processes.
In addition to affecting the migration of leukocytes
through FPR activation, extracellular annexin A1 has
been implicated in different aspects of apoptosis. For
neutrophils, it can trigger pro-apoptotic responses115,
whereas in JURKAT TLYMPHOCYTES, it can function as an
engulfment ligand that is presented on the surface
when cells become apoptotic. Cell-surface annexin A1
seems to be required for the clearance of the apoptotic
cells, which can be mediated by phosphatidylserine
receptors on the engulfing cells that possibly recognize
annexin A1 or annexin A1–phosphatidylserine complexes116. Annexin A1 has also been identified as a
selective surface marker of the vascular endothelium
in several solid tumours, and radioimmunotherapy
using anti-annexin A1 antibodies has been shown to
destroy such annexin A1-positive tumours specifically117. Together, these data show that extracellular
annexin A1 can have several functions, which mainly
involve cells of the vasculature and are often linked
to certain pathophysiological responses.
Annexin A2 and fibrinolysis. Annexin A2, like annexin
A1, exhibits the remarkable characteristic of having
unrelated intracellular and extracellular roles. The
presence of annexin A2 in an extracellular compartment seems to be restricted to the vascular endothelium, where it was discovered by investigators seeking
the endothelial cell-surface receptor for plasminogen.
Cell-surface annexin A2 that functions as a co-receptor for tissue plasminogen activator and plasminogen
can mediate the generation of plasmin, which, in turn,
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REVIEWS
JURKAT TLYMPHOCYTES
A commonly used cell line that
is derived from an acute
lymphoblastic leukaemia of
T-cell origin.
is crucial for the degradation of fibrin and therefore
for the maintenance of fibrinolytic homeostasis (for a
review, see REF. 118) FIG. 5). Consistent with this, mice
that lack annexin A2 are essentially normal, but histopathological examination reveals extensive deposition
of fibrin in their tissues119.
Although the accumulation of fibrin in annexin
A2-null mice is not associated with any overt disease
phenotype, the loss of function of endothelial cellsurface annexin A2 in humans might contribute to
disease pathology in the presence of systemic risk factors. It might therefore be significant that annexin A2
has been shown to be highly affected by several risk
factors that are linked with cardiovascular disease and
diabetes. For example, oxidative stress is associated
with elevated levels of cellular glutathione and the activation of nitric oxide synthase. Annexin A2 has been
shown to be glutathionylated in HeLa cells120 and nitrotyrosylated in lung epithelial cells121. It is also susceptible
to the incorporation of the prothrombotic amino acid
homocysteine122 and to modification by non-enzymatic
glycation under conditions of high glucose123. In all
these examples, the stress-associated modification
of annexin A2 considerably altered the properties
of the protein. Given that annexin A2 regulates both
intracellular activities (the Ca2+-regulated exocytosis of
von Willebrand factor and P-selectin) and extracellular
activities (plasmin generation) in vascular endothelial
cells, insights into the effects of disease risk factors on
annexin A2 function might lead to a better understanding of the associated cellular pathophysiology.
Annexin A5 and coagulation. Anticoagulant roles
have been proposed for extracellular annexin A5.
The model for annexin A5 function in this context
proposes that the protein forms a 2D crystalline
shield on cell-surface phospholipids, which effectively
sequesters them from procoagulant factors that use
phospholipids in the clotting cascade. In antiphospholipid syndrome, which is linked to recurrent pregnancy loss and to an increased risk of thrombosis, the
serum of patients frequently contains autoantibodies
to phospholipid, β2-glycoprotein-I and annexin A5.
The precise role of these antibodies in the pathogenicity of antiphospholipid syndrome is a subject of intense
research and much speculation, and there are widely
varying reports as to the functional significance of antiannexin A5 antibodies in these patients. Nevertheless,
1.
2.
3.
4.
Huber, R., Römisch, J. & Paques, E. P. The crystal and
molecular structure of human annexin V, an anticoagulant
calcium, membrane binding protein. EMBO J. 9,
3867–3874 (1990).
The first crystal structure of an annexin (annexin A5)
shows the characteristic fold of the annexin core.
Liemann, S. & Lewit-Bentley, A. Annexins: a novel family of
calcium- and membrane-binding proteins in search of a
function. Structure 3, 233–237 (1995).
Swairjo, M. A. & Seaton, B. A. Annexin structure and
membrane interactions: a molecular perspective. Annu.
Rev. Biophys. Biomol. Struct. 23, 193–213 (1994).
Swairjo, M. A., Concha, N. O., Kaetzel, M. A.,
Dedman, J. R. & Seaton, B. A. Ca2+-bridging mechanism
and phospholipid head group recognition in the
NATURE REVIEWS | MOLECULAR CELL BIOLOGY
5.
6.
7.
8.
in recurrent fetal loss, a prevailing theory is that antibodies specific for annexin A5 effectively unmask the
procoagulant surface of the placental syncytiotrophoblast to create a prothrombotic microenvironment (for
a review, see REF. 124). Clearly, the idea that annexin A5
forms a protective shield on the syncytiotrophoblast
would be enhanced by a better understanding of how
annexin A5 gets into the extracellular milieu and how
it binds to the cell surface.
Conclusion
As summarized in this review, annexins are involved in
a wide range of functions both inside and outside cells.
This seems befitting of a class of abundant proteins that
interact both with an important signalling ion (Ca2+)
and with important components of the intracellular
face of all membranes (acidic phospholipids). Cellular
annexin knockdowns and mouse knockout models
have revealed processes that are affected by the loss of
an annexin. As expected, these events are often linked
to Ca2+ signalling and membrane function, although in
some cases extracellular functions have been revealed
— for example, in the regulation of inflammatory reactions and fibrinolytic homeostasis. However, it seems
that the annexins often function as modulators of these
processes, rather than as essential effectors. In several
models, though, it might be the case that crucial annexin
functions were concealed by possible redundancies in
the family. Future studies that knockout several annexins at a time will be needed to address this point.
The high degree of conservation of the Ca2+-binding
sites in most annexins assures us that Ca2+ has a central
role in annexin biology, in particular, in the regulation of their intracellular activities. Therefore, when
studying annexin functions in vivo, emphasis should be
placed on the role of Ca2+ in regulating the biological
activities under investigation. The trafficking of endosomes, to take just one example, is not usually thought
to be significantly regulated by Ca2+. However, annexins that possess highly conserved Ca2+-binding sites
associate with endosomes and are involved in aspects
of endosomal membrane dynamics. So, in endocytosis,
and in many other cellular activities, the presence of
annexins indicates that we should look more closely for
the potential involvement of Ca2+ regulation. In each
case, hundreds of millions of years of conservation
indicate that there is something important waiting to
be uncovered.
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REVIEWS
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of recycling endosomes and in the biogenesis of
multivesicular endosomes, respectively.
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101. van de Graaf, S. F. et al. Functional expression of the
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An excellent review by two key investigators who
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This paper shows for the first time that annexin A1
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107. Perretti, M., Getting, S. J., Solito, E., Murphy, P. M. &
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109. Ernst, S. et al. An annexin 1 N-terminal peptide activates
leukocytes by triggering different members of the formyl
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References 108 and 109 show that annexin A1 also
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FPRL1 and FPRL2.
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112. Hannon, R. et al. Aberrant inflammation and resistance
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Reveals aberrant inflammatory reactions and an
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114. Rescher, U., Danielczyk, A., Markoff, A. & Gerke, V.
Functional activation of the formyl peptide receptor
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Acknowledgements
We thank our colleagues who provided unpublished information
and materials that were used in the figures, and apologize to all
those researchers whose work could not be discussed owing to
space limitations. Work in the authors’ laboratories is supported
by: the Deutsche Forschungsgemeinschaft, the Interdisciplinary
Center for Clinical Research of the Münster Medical School, and
the European Union (V.G.); the National Institutes of Health
(C.E.C.); and the Wellcome Trust, the Medical Research Council
and Fight for Sight (S.E.M.).
Competing interests statement
The authors declare no competing financial interests.
Online links
DATABASES
The following terms in this article are linked online to:
Prosite: http://us.expasy.org/prosite/
C2-domain | EF-hand
Swiss-Prot: http://us.expasy.org/sprot/
annexin A1 | annexin A2 | annexin A4 | annexin A5 | annexin A6 |
annexin A7 | annexin A10 | annexin A11 | annexin A13 |
annexin B12 | NEX-1 | sorcin | S100A6 | S100A10 | S100A11
FURTHER INFORMATION
Volker Gerke’s laboratory: http://zmbe.uni-muenster.de/
institu/imbmain.htm
Access to this interactive links box is free online.
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