Annexins and membrane dynamics

Biochimica et Biophysica Acta 1357 Ž1997. 129–154

Review

Annexins and membrane dynamics Volker Gerke a

a,)

, Stephen E. Moss

b

Institute for Medical Biochemistry, ZMBE, UniÕersity of Munster, Õon-Esmarch-Str. 56, D-48149 Munster, Germany ¨ ¨ b Department of Physiology, UniÕersity College London, London WC1E 6BT, UK Received 21 November 1996; revised 18 February 1997; accepted 28 February 1997

Keywords: Calcium; Endocytosis; Exocytosis; Ion channel

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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3. Annexin ion channels . . . . . . . . . . . . . . . . . . . . 3.1. Evidence for and against the annexin ionchannels 3.2. Is there a physiological annexin ion channel? . . . 3.3. Annexins as ion channel regulators . . . . . . . . .

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4. Gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Annexin gene regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Cell growthrdifferentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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5. Involvement in membrane traffic and membraneorganization . . . . . . . . . . . . . . 5.1. Intracellular localization and potential targetmembranes for individual annexins 5.2. Annexins in exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Annexins in endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Annexins in membrane organization . . . . . . . . . . . . . . . . . . . . . . . . . .

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6. Conclusions and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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2. Biochemical properties and three-dimensionalfolding 2.1. General biochemistry . . . . . . . . . . . . . . . . 2.2. Molecular structure . . . . . . . . . . . . . . . . . 2.3. Annexin protein ligands . . . . . . . . . . . . . . .

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Corresponding author. Fax: q49 251 836748.

0167-4889r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 8 9 Ž 9 7 . 0 0 0 3 8 - 4

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V. Gerke, S.E. Moss r Biochimica et Biophysica Acta 1357 (1997) 129–154

1. Introduction Intracellular Ca2q signalling and Ca2q homeostasis are among the central problems in cell biology that have received major attention for a number of decades. Fascinating and intricate signalling pathways and modes of regulation have emerged from research in this area, boosting among other things our understanding of the regulation of intracellular Ca2q levels through specific channels and pumps located in the plasma membrane and in the membranes of intracellular organelles. Conceptually, proteins involved directly in Ca2q signalling and Ca2q homeostasis should be able to bind the divalent cation and considerable effort has been put into the identification of such Ca2q effector proteins. In fact, a vast number of intracellular Ca2q binding proteins are known to date, only a few of them, however, having a clear function assigned to them. Among the Ca2q binding proteins whose function still is enigmatic are the annexins, a discrete multigene family of Ca2q binding proteins characterized by their ability to interact Ca2q dependently with membrane phospholipids. Properties and potential functions of annexins will be discussed in this review which will put a particular emphasis on recent developments and the putative roles of annexins in cellular physiology and membrane biology. A typical annexin protein is characterized by two distinct features: it exhibits Ca2q-dependent binding to phospholipids, preferentially acidic phospholipids containing one to several negative charges, and it contains as a conserved sequence element the annexin repeat, a segment of some 70 amino acids which is repeated four or eight times in a given member of the family. Thus, biochemical and gene cloning approaches have led to the formulation of the annexin characteristics and to the classification of a given protein or gene as an annexin. Moreover, such approaches have proven very helpful to identify annexins, which were initially described in higher vertebrates, in a larger number of other organisms ranging from lower vertebrates to insects, nematodes and plants. While very distinct structural and biochemical criteria define an annexin, proteins of this multigene family have been implicated in a wide range of biological processes often but not always related to membranes. These include the regulation of mem-

brane organization, membrane traffic, membrane– cytoskeleton linkage, and ion conductance across membranes. Based on biochemical properties displayed by at least some annexins, e.g. the inhibition of phospholipase A 2 , the inhibition of blood coagulation, and the binding to certain matrix or extracellular components, members of the family are also proposed to function as anti-inflammatory and anticoagulant compounds and as mediators or regulators of certain cell–cell and cell–matrix interactions. This diversity in putative function reflects itself in the number of different names initially given to proteins of the annexin family, e.g. lipocortins, chromobindins, calcimedins, anticoagulant proteins, anchorins. The nomenclature tangle has meanwhile been resolved with the introduction of the universal name annexin w1x. Several excellent reviews of the recent past have discussed in detail the diversities in proposed functions and names and the general biochemical and structural properties of the annexins and we recommend this literature to the interested reader w2–5x. The emphasis of this review, thus, does not lie in a complete survey of the annexin literature but in discussing some recent findings relating annexins to certain aspects of membrane organization, membrane traffic, and cellular physiology.

2. Biochemical properties and three-dimensional folding 2.1. General biochemistry The Ca2q-dependent binding of annexin to negatively charged membrane phospholipids is well documented and regarded as a biochemical hallmark of the family. A detailed comparison as to the Ca2q affinities and phospholipid specificities of different annexin is given in a recent review w5x and the general picture emerging from this comparison is that annexins preferentially bind to membranes containing negatively charged phospholipid Žphosphatidic acid, phosphatidylserine, phosphatidylinositol. . This peripheral membrane binding is strictly Ca2q-dependent and is often accompanied by a Ca2q-dependent aggregation of membrane surfaces, e.g. of phospholipid vesicles. The Ca2q concentrations required by different annexins for half-maximal membrane binding and

V. Gerke, S.E. Moss r Biochimica et Biophysica Acta 1357 (1997) 129–154

lipid vesicle aggregation cover a wide range with annexins I and II requiring the least Žsub-micromolar to a few micromolar Ca2q for half-maximal binding. and annexin V requiring the most Ca2q Ž10 to ) 100 mM Ca2q for half-maximal binding, depending on the phospholipid and the conditions employed. w5x. Recently, this picture of a strictly ionic interaction of annexins with acidic phospholipids has become more complex. It was shown, for example, that annexins can also bind to non-charged phospholipids, e.g. phosphatidylethanolamine and phosphatidylcholine, albeit requiring high Ca2q concentrations for such interactions w6–9x. Moreover, a direct non-ionic interaction between a hydrophobic amino acid side-chain of the protein and the lipid phase of the bilayer has been observed for Ca2q-bound annexin V w10–14x. Finally, several annexins, e.g. annexin II, V and VI, also appear to associate with biological membranes in a mode not regulated by Ca2q Ž see below.. The molecular mechanism underlying the vesicle aggregation is not completely understood. It remains possible that each annexin polypeptide contains two spatially separated lipid binding sites and experimental support for this view has been provided, e.g., in the case of annexin I w7,15,16x. On the other hand, there is increasing evidence that at least for some annexins vesicle aggregation requires the self-association of two annexin molecules bound to two separate membranes Žfor review see w2,5x.. However, such a scenario is likely to be different in the case of annexins II and VI which clearly contain two distinct lipid binding domains per physical entity. This is the result of a duplication of the principal annexin unit Žthe annexin core domain. either through cross-linking via a protein ligand Ž p11 in the case of the annexin II 2 p11 2 complex. or through gene duplication Ž in annexin VI, Fig. 1.. Annexin-mediated aggregation of membrane vesicles, i.e. the cross-linking of two membrane surfaces, is a prerequisite for the membrane fusion activity displayed by annexins. However, in a simple system consisting of membrane vesicles, an annexin protein and Ca2q, the rate of fusion is very slow, indicating that annexins are not fusogenic proteins per se w2x. Interestingly, certain components, in particular cis-unsaturated fatty acids, when added to the vesicle–annexin–Ca2q mixture can markedly increase the rate of fusion, suggesting that annexin-mediated membrane fusion might be

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Fig. 1. Structural organization of different annexins. Known vertebrate annexins are classified according to structural characteristics of their N-terminal domains. The four annexin repeats Žeight in the case of annexin VI. are schematically represented by rectangles with a highly conserved sequence of 17 amino acid, the endonexin fold, shown in grey. The preceding N-terminal domains vary in length between 12 and 19 Žannexins III, IV, V, VIII. and close to 200 residues Žannexin XI. and show certain structural features. In annexins I and II these include amphipathic a-helices Žsmall rectangles. representing binding sites for S100 protein ligands Žp11 in the case of annexin II., phosphorylation sites for serine ŽS.rthreonine ŽT. and tyrosine ŽY. specific protein kinases and a glutamine ŽQ. residue at position 18 of the annexin I chain which is the site for a transglutaminase cross-link. In annexins VII and XI the long N-terminal domain is rich in glycine ŽG., tyrosine and proline ŽP. residues which have been proposed to form a pro-beta helix. N-terminal splice variants have been reported for different annexins, e.g. annexin XIII Ža and b. which is the only annexin described so far to become N-terminally myristoylated. See text for references.

physiologically meaningful when elevated concentrations of free polyunsaturated fatty acids occur within cells w2,17x. While vesicle fusion following aggregation under the above-mentioned circumstances should be an activity principally displayed by all annexins remarkable differences are observed when individual members of the family are compared, e.g. the rate of

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Ca2q-dependent fusion between phospholipid vesicles and neutrophil plasma membrane preparations is substantially increased by annexin I but inhibited by annexin V w18x. Collectively these data show that different annexins albeit all capable of binding phospholipids in a Ca2q-dependent manner display profound differences in their Ca2q threshold for lipid binding and vesicle aggregation and their ability to catalyze fusion of membrane bilayers. 2.2. Molecular structure The binding of annexins to the common ligands, Ca2q and phospholipid, is mediated through a structurally well-conserved domain, the annexin protein core. This core was initially defined through its characteristic resistance towards limited proteolytic treatment w19–22x. Sequence analysis, initially reported for annexins I and II w23–26x, revealed that the characteristic annexin core comprises four tandemly repeated segments, each 70–80 amino acids in length. The segments, termed annexin repeats, show intraand intermolecular sequence homologies Žsequence comparisons and homology values are given in w27x.. Crystal structure analysis, initially carried out for annexin V w11,28,29x and later also for annexins I, II, III, VI and XII w30–35x, described a very characteristic and highly homologues folding for the different annexin cores which most likely can serve as a blueprint for the entire family, thus defining an annexin structurally. The cores, typically some 300 residues in length, have the shape of a compact, slightly curved disc with the four annexin repeats forming four domains. These are arranged in a cyclic array with repeats 1 and 4, and 2 and 3, respectively, building tight modules stabilized through hydrophobic side-chain interactions Ž for review see w36–38x.. The centre of the molecule, separating the repeat 1r4 and repeat 2r3 modules, is a hydrophilic pore proposed to represent an ion conductance pathway and thus the structural basis for the Ca2q channelling activity displayed by some annexins in vitro Žfor review see w39,40x; see below for details. . In the folded molecule each annexin repeat comprises five a-helices Ža–e. wound into a right-handed superhelix. The helices are connected through short loops or turns and loops extending from the more convex side of the curved disc are involved in Ca2q

complexation w41x. Two types of Ca2q binding sites have been identified in annexin crystals. They have been termed type II and type III binding sites to distinguish them from the long-known and well-characterized EF hand or ‘‘type I’’ Ca2q binding site found in proteins like parvalbumin and troponin C w30x. In the type II site the Ca2q ion coordinates to three carbonyl oxygens of the peptide bonds located in the loop connecting helices a and b and to the carboxyl oxygens of an aspartate or glutamate residue Žthe so-called cap residue. found in or close to the loop connecting helices d and e of an annexin repeat. The type III site offers only three proteinaceous oxygen ligands for Ca2q complexation, usually one carbonyl and two nearby carboxylate oxygens. Crystallization studies and electron microscopical analyses have been carried out to describe the structural basis of annexin–monolayer interactions. These data reveal that in the case of a four repeat annexin Žannexin V. the convex surface of the folded annexin is orientated towards the membrane. The Ca2q ions bound to this convex side serve a bridging function and coordinate not only to the protein but also to phosphoryl moieties of the membrane phospholipids. Moreover, it appears that in the four repeat annexins the conformation of the molecule is not grossly altered upon membrane binding w39,42–49x. The latter is however different in annexin VI which comprises eight annexin repeats folded into two similar halves each representing a typical annexin core. Upon membrane binding these two halves are rotated about 908 relative to one another to assume a coplanar arrangement with the membrane w34,35x. The principles governing Ca2q binding and Ca2q-mediated phospholipid binding established for the crystalized annexins most likely also apply to the proteins in solution. Using site-directed mutagenesis approaches the cap residues of individual type II Ca2q sites as well as residues in or close to the loop between helices a and b were shown to be crucial for high-affinity Ca2q– lipid binding w50–54x. Although the general folding of the protein core domain is highly similar for the different annexin analyzed so far the number and exact location of the individual Ca2q sites appears to differ between members of the family w30–35,41x. This could be the basis of the different Ca2q sensitivities and phospholipid specificities observed for different annexins.

V. Gerke, S.E. Moss r Biochimica et Biophysica Acta 1357 (1997) 129–154

While the common structural principles and biochemical properties of annexins reside in the wellconserved protein core domain, specificity with respect to the function of individual annexins is thought to be conferred through the N-terminal domain preceding the protein core. This domain, also called head or tail, is sensitive to limited proteolysis and differs in length between 11 to 19 Žannexins III, IV, V, VI, X, XII, XIII. and more than 100 residues Žannexins VII and XI. Žfor review see w5x; a schematic overview is given in Fig. 1.. The sequences of the N-terminal domains are highly variable and with the exception of the short-tail annexins III, V and XII w28,31,32x their three dimensional folding and location with respect to the protein core is not known. N-terminal domains of the short-tail annexins appear to be restricted to the concave side of the proteins and are possibly involved in the regulation of ion conductance through the central hydrophilic pore w32,36x. More complex modes of regulation can be discussed for the longer N-terminal domains of the larger annexins. Through direct intramolecular interactions they could affect the Ca2q and phospholipid binding sites located on the convex side of the core domain. Likewise, properties of the core domain could be altered through conformational changes induced by the N-terminal tail domain. Such modulating activity of the N-terminal domain has been demonstrated in vitro and in vivo for several longertail annexins, in particular annexins I and II. In phospholipid binding experiments, for example, removal of the N-terminal domain in annexins I and II leads to an increased affinity towards Ca2qrphospholipid w55–57x. An increase in the Ca2q affinity of annexin I is also observed in the presence of antibodies directed against the tail domain w58x whereas such antibodies and certain cleavages in the N-terminal domain have an inhibitory effect on the vesicle aggregation property displayed by this annexin w15,59x. More directly, a modulating effect of the N-terminal domain of annexin I has been confirmed by analyzing the lipid vesicle binding and aggregation properties of chimeric annexin proteins consisting of an Nterminal portion of annexin I and C-terminal parts of annexin V w12,60,61x. In vivo, the importance of an N-terminal annexin domain has been documented through the ectopic expression in baby hamster kidney Ž BHK. cells of N-terminally truncated versions

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of annexin I. Here, residues 13–26 of the N-terminal domain have been shown to be involved in specifying the early endosomal localization of the protein w62x. The regulatory importance of the N-terminal domain is also underscored by the fact that it often harbours phosphorylation sites for signal transducing protein kinases Žfor reviews see w3,5x.. In the case of annexins I and II consequences of such regulatory phosphorylations have been documented in phospholipid vesicle binding and aggregation experiments. Phosphorylation by the EGF receptor kinase at Tyr-20 of annexin I, for example, renders the protein more susceptible to proteolytic degradation thus altering in turn its Ca2qrphospholipid binding and vesicle aggregation properties Žfor review see w5,63x.. In mutagenesis experiments, a substitution of Tyr-20 by a negatively charged amino acid mimicking to some extent the phosphorylation by the EGF receptor kinase results in an enhanced Ca2q requirement of the mutant protein for promoting vesicle aggregation w59x. Similar effects are observed when Ser-26, a site for phosphorylation by protein kinase C, is replaced by aspartate or glutamate w16,59x. Annexin II also contains a tyrosine residue in its N-terminal domain which functions as a phosphorylation site in this case for pp60 src Žfor review see w64,65x.. Phosphorylation at this Tyr-23 decreases the affinity of annexin II for phospholipids in liposome binding experiments and abolishes the chromaffin granule aggregation activity displayed by this annexin at micromolar Ca2q levels when complexed with its cellular protein ligand p11 w55,66x. Protein kinase C phosphorylation of annexin II occurring in the N-terminal domain, on the other hand, has no effect on the Ca2q-dependent lipid binding of the protein but significantly decreases the rate and extent of the annexin II-mediated lipid vesicle aggregation w67x. This finding, however, appears to contradict the observation that protein kinase Cmediated phosphorylation of Ser-25 of annexin II is a prerequisite for an activity of the annexin II–p11 complex in the Ca2q-evoked secretion in permeabilized chromaffin cells w68x Ž see below.. While all in vitro data strongly favour the notion that the N-terminal domain of an annexin modulates properties displayed by the conserved core domain and that this modulation can be regulated by phosphorylation, direct in vivo observations supporting this view are still lacking. Subcellular fractionation and immunoisola-

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tion of certain endocytotic structures, the multivesicular bodies ŽMVBs., indicate that within 3T3 cells overexpressing the human EGF receptor annexin I is associated with MVBs in a Ca2q-independent manner and that phosphorylation of annexin I by the EGF receptor kinase occurring in the MVBs converts it into a form requiring Ca2q for the membrane association w69x. However, this might not be a general phenomenon since the membrane association of annexin I in BHK cells appears to be strictly Ca2q-dependent w62x. The N-terminal domains of annexins also harbour other sites for posttranslational modifications likely to be of regulatory importance. These include a glutamine residue at position 18 of the annexin I tail which is the site for a transglutaminase cross-link to another annexin I chain. The resulting annexin I homodimer has an increased Ca2q sensitivity for phospholipid binding and associates with A431 cell membranes in an EGTA-resistant, i.e. Ca2q-independent manner w70,71x. In the case of annexin XIIIa, Ca2q-insensitive membrane association is achieved through N-terminal myristoylation, a property most likely shared by different N-terminal splice variants of this protein w72,73x. Whether other annexins are also modified by myristoylation or some other type of fatty acid attachment remains to be determined. 2.3. Annexin protein ligands Several protein ligands which appear to be unique for individual annexins and affect properties displayed by the proteins in vitro have been identified. As can be expected from the modular structure of the annexins most ligand binding sites have been mapped to the unique N-terminal domain of the respective annexin. Interestingly, several members of the S-100 family of EF-hand type Ca2q-binding proteins form complexes with different annexins. The prototype of such complexes identified more than 10 years ago is the annexin II–p11 heterotetramer in which a dimer of the S100 protein p11 ŽS100A10. links two annexin II molecules w74–76x. p11-induced complex formation is of regulatory importance as it markedly reduces the Ca2q requirement for phospholipid binding and chromaffin granule aggregation by annexin II w55,77x. Moreover, the annexin II–p11 complex has an altered subcellular localization as compared to

monomeric annexin II and complex formation appears to be a prerequisite for tightly anchoring this annexin in the cortical cytoskeleton of fibroblasts and adrenal chromaffin cells w78–80x. Hydrophobic forces predominantly contribute to the stabilization of the annexin II–p11 interaction and the hydrophobic amino acid side-chains involved have been mapped to the N-terminal domain of the annexin and the unique C-terminal extension of the S100 protein w81,82x. In the case of annexin II, the entire p11 binding site is contained within the N-terminal 14 amino acid residues and synthetic peptides of this sequence compete effectively with full-length annexin II for p11 binding w81,83x. These structural informations have led to the introduction of synthetic peptides and fusion proteins containing the N-terminal annexin II sequence in functional studies Žsee below.. The other two annexin–S100 pairs identified more recently are the annexin I–S100C and the annexin XI–calcyclin ŽS100A6. complexes w84–87x. In both cases, the S100 binding site has again been mapped to the N-terminal domain of the respective annexin w86–88x. However, it is not known whether binding of the dimeric S100 protein leads to the formation of a heterotetrameric unit and what the physiological consequences of the complex formations are. Regulation by Ca2q binding to the S100 protein marks a difference in the case of the annexin I–S100C and annexin XI–calcyclin complexes when compared to the annexin II–p11 heterotetramer. While the former complex formations are strictly Ca2q-dependent the latter occurs in the absence of Ca2q. This is due to the deletion and substitution of Ca2q-coordinating residues in the EF-hand loops of the p11 molecule which render the protein inactive in Ca2q binding and most likely freeze it in a conformation competent for binding to its ligand annexin II w89x. Using an affinity chromatography approach another Ca2q binding protein of the EF-hand superfamily has recently been identified as an annexin ligand. Sorcin, a 22-kDa protein containing four EF-hand motifs Žin contrast to the two EF-hand sites in S100 proteins. which is homologous to the calpain light chain, binds to the long N-terminal domain of annexin VII w90x. This N-terminal domain in annexin VII is similar to the one found in annexin XI ŽFig. 1. and composed of repetitive sequence stretches rich in glycine ŽG., tyro-

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sine ŽY. and proline ŽP. residues which have been proposed to form a pro-beta helix w91x. Other proteins shown mainly by biochemical approaches to bind to members of the annexin family include a number of cytoskeletal or cytoskeletonassociated proteins. Annexins I, II and VI, for example, bind in a Ca2q-regulated manner to filamentous actin w20,74,92,93x. This interaction has been studied most extensively in the case of annexin II where a 9 amino acid sequence in the third annexin repeat was reported to represent the actin binding site w94x. Annexin II in its heterotetrameric complex with the p11 dimer is also capable of bundling actin filaments at micromolar Ca2q concentrations and this bundling as well as the F-actin binding is inhibited by pp60 srccatalyzed tyrosine phosphorylation of annexin II w66,74x. The physiological significance of the bundling activity is, however, questionable since annexin II appears to be excluded from stress fibers in fibroblasts w95x. In contrast the binding of annexin II to F-actin is consistent with the localization of both proteins to the cortical cytoskeleton of a variety of cells Žfor review see w64x.. Members of the nonerythroid spectrin family are also found in this submembraneous structure and represent in vitro ligands for annexin II and annexin VI w74,96,97x, indicating that at least some annexins might serve a structural function in this cortical region of the cell Ž see below.. While cytoskeletal binding partners for annexins, in particular annexin II, generally appear to be components of the microfilament system, exceptions from this rule exist, e.g. the Ca2q-dependent binding of annexin II to the intermediate filament protein GFAP Žglial fibrillary acid protein. observed in vitro w98x. The above does not represent a complete list of all intracellular binding proteins identified to date Žthis would go beyond the scope of this review. but it illustrates two features widespread among the annexins. First, proteins of this family often represent targets for regulation through S100 protein binding. Second, annexins or annexin–S100 complexes can associate not only with membranes but also with cytoskeletal proteins, in particular with those found in cytoskeletal structures underlying membranes. In addition to the examples discussed above, proteins or structures found on the external surface of cells have also been identified as annexin ligands. These include proteins of the extracellular matrix

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such as collagen, which binds annexin V w99x, and tenascin C, which was reported to interact with annexin II exposed on the surface of endothelial cells w100x and whose mitogenic stimulation of endothelial cells is blocked by antibodies against annexin II w101x. Endothelial annexin II also was shown to act as a receptor for tissue plasminogen activator Žt-PA. and to enhance the t-PA dependent plasminogen activation w102,103x. Another plasma protein interacting with annexins is apolipoprotein A1 which binds to annexins I and VII, but not annexins IV and VI, in a Ca2q-dependent manner w104x. In some cases, annexins have been implicated in intercellular interactions. These include annexins II and VI, which were identified as lymphoma cell membrane components participating in the divalent cation-dependent endothelial cell adhesion w105,106x, and annexin I, which was shown to bind to leukocytes w107x and was proposed to act anti-inflammatory by interfering with the trans-endothelial migration of leukocytes w108x. Cell surface receptors for these annexins have not been identified but it is interesting to note in this respect that annexin VI binds to carbohydrate structures w109x and that annexins I and II appear to be choline binding proteins w110x. Annexin binding to the extracellular structures listed above as well as the anti-coagulant and anti-inflammatory activities reported for several annexins Žfor review see w5x. critically depend on the extracellular presentation andror secretion of annexins. The experimental data for a specific secretion of certain annexins are still rather scarce with the most convincing evidence coming from the quantification of annexin concentrations in the seminal plasma from the human prostate gland. While annexins I, IV and V are expressed at comparable levels in the ductal epithelium of the gland, only annexins I and V, but not annexin IV, are found at considerable levels in the extracellular fluid w111x. However, we are still in need for more clear cut evidence for the active release of annexins from cells, e.g. through pulsechase labelling of cultured cells and a careful analysis of the released proteins. Moreover, there is so far no satisfactory model providing a mechanistic basis for a specific release of the proteins Žannexin cDNAs, for example, do not contain sequences encoding hydrophobic signal peptides. . Conceptually, annexins could be released after membrane breakdown, a sce-

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nario possibly met in the case of matrix vesicles present in the extracellular matrix of mineralizing tissues. Matrix vesicles which are derived from chondrocyte microvilli contain several annexins and are also enriched in sphingomyelin, cholesterol and phosphatidylserine, i.e. lipids annexins are known to interact with directly or when present in certain membrane domains w112,113x Žsee also below.. Upon break down of the matrix vesicles occurring during bone formation, annexins present in the vesicles could be released into the extracellular milieu.

3. Annexin ion channels Arguments have continued about the functions of annexins since it was first proposed that annexin I is a mediator of the anti-inflammatory response. Claim is followed by counter-claim and the functional questions remain largely unanswered. One of the most recent and provocative suggestions is that members of the annexin family are ion channels. Here, we review the evidence both for and against this role and consider the physiological context in which such ion channels might operate. The first report of ion channel activity for any annexin was by Rojas and Pollard w114x who showed that purified annexin VII Žsynexin. formed capacitative gating currents in acidic phospholipid bilayers. The same group then demonstrated that annexin VII formed highly selective, voltagesensitive Ca2q channels in artificial membranes w115x. This work was extended to show that purified and recombinant annexin V had similar activity w116x. At the same time, Huber et al. published the crystal structure of annexin V w28x. This crystal structure Žsee above. revealed features consistent with annexin V being an ion channel. First, the centre of the molecule forms a pore which was suggested to be the ion conductance pathway. Second, the fact that the pore is lined by the side-chains of acidic amino acids explains why the annexin V channel exhibits selectivity for cations. Finally, the pore was oriented perpendicular to the convex surface of the annexin V molecule on which the Ca2q-binding sites are located and which is the surface that interacts with phospholipids. Huber et al. proposed a model in which the convex surface of annexin V lies in close apposition to the inner leaflet of the plasma membrane — this is

consistent with the known phospholipid-binding preferences of annexin V — with the hydrophilic channel providing a cation-selective gateway to the cell interior w39x Žsee Fig. 2 for a model. . 3.1. EÕidence for and against the annexin ion channels Despite the electrophysiological data and the supporting structural studies, which clearly show that annexin V has ion channel activity, several important questions have yet to be answered if the ion channel theory is to gain acceptance. First, the idea of an ion channel that does not span the plasma membrane presents obvious conceptual problems. Karshikov et al. w117x countered this by proposing a mechanism based on hypothetical calculations, in which annexin V could electroporate the membrane causing a localised area of increased permeability through which extracellular ions would flow. The selectivity of the channel for calcium would ensure the exclusion of other ions. A more direct answer to the question of membrane spanning came with the description of a hexameric form of annexin XII w31x. Here, the six annexin XII monomers are arranged as a sandwich of two trimers. An unexpected feature of this structure was that the convex faces of the six annexin units were all buried at the intermolecular face of the two trimers. In addition to providing a functional unit with the necessary molecular dimensions to span the plasma membrane, the hexamer also had a central pore lined with the side-chains of charged amino acids ŽFig. 2.. In the case of annexin XII these residues are basic, suggesting an anion rather than a cation channel. A hexameric annexin is also structurally more acceptable than a monomeric annexin because of the larger size of the central pore. In the hexamer the ˚ in diameter as most constricted part of the pore is 8 A ˚ opposed to 1 A for monomeric annexin V. The larger pore is more consistent with the known conductance values for annexin V. Interestingly, a theoretical hexameric model of annexin V forms a structure with a central pore lined with acidic residues, but different ones from those that line the pore in monomeric annexin V. As yet, no annexin V hexamer has been described although annexin V trimers are well documented w47x. If annexin V does function as a Ca2q-

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channel is it as a hexamer which is structurally acceptable but may not exist, or a monomer, which exists but which less readily conforms to the general perception of an ion channel. In favour of the latter model, Huber et al. showed that point mutation of a glutamic acid residue in the central pore of monomeric annexin V changes the ion selectivity from Ca2q to Naq w10x. Other mutational studies demonstrated the importance of specific amino acids in voltage sensing and ion permeability, which together lend weight to the idea that monomeric annexin V can form a functional Ca2q-channel. Another concern is that as more annexins have their structures solved Žthe list now includes annexins I, II, III, V, VI and XII. it is becoming clear that almost all display ion channel activity in vitro. This is not unlike the position ten years ago, when after the excitement of discovering that annexin I was an inhibitor of phospholipase A 2 , it gradually became clear that there were at least nine other annexins with identical activity. This does not rule out the possibility that annexin I is an inhibitor of phospholipase A 2 or that annexin V is an ion channel, but it does raise questions about why members of such an expansive gene family should all be doing the same thing. In

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fact, the anti-inflammatory role for annexins is not a strictly accurate historical precedent because there are important differences between annexins with respect to their ion channel properties. Annexin III has little channel activity and as mentioned earlier, annexin XII lacks detectable cation channel activity and is more likely to form an anion channel. An interesting observation emerging from structure-function studies on the annexin ion channels is that channel activity appears to depend not only on the central pore, but also on the N-terminal domains. Removal of these domains in annexins II and V virtually abolishes channel activity w33,118x and replacement of a tryptophan residue with an alanine in the N-terminus of annexin III enhances calcium channel activity ŽB. Favier-Perron, F. Russo-Marie, A. Lewit-Bentley and R. Huber, pers. commun... These are further examples of how annexin N-termini are likely to be important regulatory domains. 3.2. Is there a physiological annexin ion channel? A third criticism is that in in vitro systems molecules that are clearly not physiological ion channels can integrate into a phospholipid bilayer and

Fig. 2. Ion conductance by annexins. The figure shows two models for annexin ion channels. On the left, a monomeric annexin binds Ca2q-dependently to the inner leaflet of the plasma membrane. It is proposed that the convex surface of the annexin generates a localised region in which the phospholipid bilayer is disturbed and that under conditions of membrane depolarization or hyperpolarization, the membrane becomes leaky w117x. The central pore of the annexin acts as an ion filter, in the case of annexin V the filter is selectively permeable to Ca2q. On the right, an annexin homohexamer forms a membrane-spanning structure in which two trimers are joined Ca2q-dependently at their convex faces. A pore is formed at the centre of the hexamer, which is suggested to be the ion conductance pathway w31x.

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generate ion channel activity. Thus, a 21 amino acid synthetic peptide consisting of only serine and leucine will form a cation-permeable pore in a lipid bilayer w119x. The channel activity of annexins may also be artefactual, although the observations that: Ž 1. despite their overall similarity not all annexins form channels; and Ž2. mutation of specific residues in the central pore of annexin V switches the ion selectivity from Ca2q to Naq, tend to argue against this. Nevertheless, the challenge now is to demonstrate that annexins form functional ion channels in living cells. Virtually all cells use Ca2q as a second messenger and tight regulation of free intracellular Ca2q concentration wCa2q x i is critical for normal cell function. The transient opening of Ca2q channels to either intracellular stores andror the extracellular milieu leads to increases in wCa2q x i , which contribute to a variety of cellular responses including contraction, exocytosis and growth depending on the cell type and the initiating stimulus. Several of the key players in this process have now been identified, for example, the ligand-gated ryanodine receptors and IP3 receptors that regulate release of Ca2q from intracellular stores w120x, and the voltage-gated L-, N- and T-type channels that conduct Ca2q across the plasma membrane in excitable cells such as neurones and muscle fibres. Calcium channels in non-excitable cells appear in various guises, as calcium-release activated channels ŽCRACs., store-operated calcium channels ŽSOCs. and receptor-operated calcium channels ŽROCs. w121x. These are the channels that mediate calcium entry in T lymphocytes, hepatocytes, smooth muscle cells, platelets, neutrophils, endothelial cells and fibroblasts. Ca2q entry in these non-excitable cells is poorly understood and for reasons outlined below, it is here that annexins can be included with certain other molecules as candidate Ca2q channels. The best characterised of the potential CRACs, ROCs and SOCs are the transient receptor potential Ž trp . and trp-like genes in Drosophila for which mammalian homologues have recently been discovered w122x, and which enhance Ca2q entry when transiently expressed in COS cells or block Ca2q entry when expressed in antisense orientation. The two features of trp that make it a good candidate for a Ca2q entry channel are that it is gated by depletion of the intracellular Ca2q stores and is inhibited by La3q but is insensi-

tive to the dihydropyridine blockers such as nifedipine that block the classic voltage-gated Ca2q channels. Annexins are candidates for similar reasons. First, they tend to be much more abundant in non-excitable cells such as lymphocytes, epithelial cells and hepatocytes than in neurones. Second, Ca2q entry in nonexcitable cells is usually driven by membrane hyperpolarisation and the annexin V channel conducts Ca2q in response to both depolarising and hyperpolarising potentials w117x. Third, Ca2q channels in non-excitable cells are inhibited by La3q but are insensitive to dihydropyridine blockers, again this is identical to the known pharmacology of the annexin V channel. Finally, annexin V has a Ca2q conductance similar to those of electrophysiologically characterised channels in non-excitable cells w121x. These may be coincidences but because the Ca2q channels in question have yet to be identified at the molecular level, annexin V and trps both remain reasonable candidates. 3.3. Annexins as ion channel regulators To add further confusion to an already complicated picture, it appears that in addition to having their own intrinsic channel activity, annexins may also modulate the activity of other ion channels. This was first demonstrated by Diaz-Munoz et al. w123x who showed that purified annexin VI increased the mean open time and opening probability of sarcoplasmic reticulum Ca2q channels in an isolated membrane preparation. Although this effect was shown to be specific to annexin VI, it required the presence of the protein on the lumenal side of the membrane, and annexin VI like other annexins is generally thought to be a cytosolic protein. Recently, Huber et al. solved the crystal structure of annexin VI and demonstrated that the purified protein, like annexins II and V, has Ca2q channel activity w34x. This raises the possibility that the apparent increase in channel activity observed by Diaz-Munoz et al. Žabove. was in fact due to annexin VI itself and not the consequence of an interaction between annexin VI and the sarcoplasmic reticulum Ca2q channels. To address the question of whether or not annexin VI is involved in regulation of Ca2q homeostasis in vivo, Dedman’s group recently generated the first

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annexin transgenic mice, in which annexin VI was over-expressed in the heart under the control of the myosin heavy chain gene promoter w124x. These mice exhibited various cardiac problems and those with highest levels of expression died of heart failure shortly after birth. However, using Ca2q-sensitive fluorescent dyes and isolated cardiac myocytes from these transgenic mice, it was shown that cells with increased levels of annexin VI had a lower resting Ca2q level and generated smaller Ca2q spikes on electrical stimulation. The mechanism of annexin VI action in cardiac myocytes is not understood but if the function of annexin VI is to suppress Ca2q mobilisation then this implies that annexin VI might inhibit exocytosis in secretory cells or reduce the growth rate of proliferating cells. In this context it is interesting that purified annexin VI inhibits annexin II-mediated chromaffin granule aggregation w125,126x and that heterologous expression of annexin VI in A431 carcinoma cells reduces their growth rate w127,128x. Annexin IV is expressed in columnar epithelial cells of various tissues including intestine, lung, stomach, trachea, kidney, fallopian tube and uterus. Together with the preferential localisation of annexin IV to the apical membranes of polarised secretory epithelial cells in the intestine this suggests a possible role in fluid secretion. As chloride ions are the major secreted anion, and the apical membrane is the primary site for regulation of chloride secretion, Kaetzel et al. w129x examined whether or not annexin IV might be a modulator of chloride channel activity. They showed that introduction of purified exogenous annexin IV into a colonic cell line through a patch pipette specifically blocked Ca2q-dependent Cly current activation. This effect was reversed both by an affinity-purified antibody to annexin IV and by an antisense oligonucleotide complementary to the annexin IV mRNA. The same group then showed that annexin IV may exert its inhibitory role on Ca2q-dependent Cly current activation by blocking phosphorylation of the Cly channel by calmodulin-dependent protein kinase II ŽCaMKII. w130x. It will be some time before we have a clear picture of how, or indeed if, annexins function either as ion channels or ion channel regulators in vivo. However, it must be hoped that further identification of annexins in cellular pathways involved in the regulation of

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ion channel activity, will provide new therapeutic targets for diseases such as cystic fibrosis in which normal ion channel function is compromised. 4. Gene expression Annexins are often described as ‘‘ubiquitous’’, and as a family this is true, but individual annexins have discrete patterns of expression, presumably reflecting singular mechanisms of gene regulation related to cell-type specific functions. Some family members are widely expressed, for example annexins I, II and VI, whereas others such as annexins VIII and XIII are expressed in very few cell types. Any one cell type might express up to ten different annexins, so it is possible that each cell type has its own unique annexin ‘‘fingerprint’’. In this section we review the current perception of annexin gene regulation and discuss how this correlates with ideas about annexin function. 4.1. Annexin gene regulation Several annexin genes have been cloned and the close structural relationship of the annexin proteins is mirrored by strikingly conserved patterns of genomic organisation. The positions of intron–exon boundaries within the regions of annexin genes corresponding to the protein repeats, are at identical locations for all mammalian annexins. The relationship between genomic organisation and protein structure has been reviewed elsewhere w131x. In contrast, there is no similarity in the genomic organisation of the first coding exons that specify the divergent annexin Nterminal domains. However, examination of the genomic structures of annexins can provide clues as to functional domains. This is exemplified by the annexin II gene in which the first exon encodes the p11-binding domain w132x and the second exon encodes a domain recently demonstrated to mediate binding to endosomes w133x. Relatively little is known about annexin gene regulation in terms of defining active regions within promoters. Genomic sequences upstream of the transcription start sites ŽTSS. have been described for human annexins I, II, V and VI w134–137x and most of these contain recognisable elements in keeping with these domains being functional gene promoters.

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Consistent with annexins having distinct expression patterns, the putative promoters have little if any sequence similarity. Annexins I, II, V and VI all have CAAT and TATA boxes within these upstream sequences although in the annexin VI promoter they lie somewhat distal to the TSS and in deletion experiments appear to be functionally redundant ŽS.R. Donnelly and S.E. Moss, unpublished observations.. In contrast, in the annexin I gene promoter, analysis of the activity of a series of 5 deletions revealed that the region containing the CAAT and TATA boxes possesses full promoter activity ŽS.R. Donnelly and S.E. Moss, unpublished observations.. Imai and Kohsaka w138x recently described two promoters for the rat annexin V gene, one of which is very GC-rich and lies proximal to the TSS and a second promoter several kilobases upstream which contains putative binding sites for early response genes such as AP1. This is consistent with the findings of Braselmann et al. w139x who discovered annexins V and II as fos-inducible genes. It is not known why annexin V has two separate promoters, but sequencing of alternatively spliced transcripts showed that both are functional and that transcription from the distal promoter splices out the exon lying immediately downstream of the proximal promoter. Imai and Kohsaka w138x also showed that alternative splicing of transcripts originating from the distal promoter could generate an mRNA in which the usual first coding exon was absent and from which an annexin V variant lacking the N-terminal 25 amino acids would be produced. An intriguing possibility is that following cell activation and c-fos induction, cells start to synthesise the N-terminally truncated annexin isoform. If annexins function as multimers then this truncated form could act as a dominant interfering mutant, blocking the activity of the existing cellular pool of annexin V. In support of such a model, Berendes et al. w118x have already shown that an annexin V mutant lacking the N-terminal domain does not have Ca2q-channel activity. The most detailed study of an annexin promoter to date is for the pigeon Annexin Icp35 gene w140x. Horseman and co-workers initially discovered Annexin Icp35 when searching for genes induced by prolactin in the pigeon cropsac. In pigeons there are at least two annexin I genes, one of which closely resembles mammalian annexin I, is constitutively

expressed and like its mammalian counterpart is insensitive to prolactin, and Annexin Icp35 which is also very similar to mammalian annexin I but lacks the tyrosine phosphorylation site for the epidermal growth factor receptor and which is absolutely reliant on prolactin for expression w141x. A region of the Annexin Icp35 promoter close to the TATA has been identified which binds to putative transcription factors only in prolactin-stimulated cropsac. Molecular cloning of these factors will provide new insight into both annexin gene regulation and the mechanism of prolactin-induced gene expression. The question as to why pigeon cropsac should have two annexin I genes remains unanswered, but again there is the possibility that the inducible form lacking the tyrosine phosphorylation site Ž Annexin Icp35 . somehow interferes with the activity of the constitutively expressed form. 4.2. Cell growthr differentiation The discovery that some annexins are fos-inducible raises the possibility that they have roles in cell growth andror differentiation. There are several instances where cell differentiation is accompanied by an increase in annexin expression, for example, annexin I is up-regulated during differentiation of HL60 and U937 myelomonocytic cells w142,143x, annexin II is up-regulated during nerve growth factor-induced differentiation of PC12 phaeochromocytoma cells w144,145x and annexin VI is up-regulated during Band T-lymphocyte development w146x. As a general rule, annexins tend to be more highly expressed in terminally differentiated cells, examples of annexin down-regulation are rare and normally linked to acquisition of a specialist phenotype, for example, down-regulation of annexin VI in the ductal epithelial cells of lactating Žas opposed to non-lactating. breast tissue w146x. Annexin expression is also variable and dependent on cell growth state. Schlaepfer and Haigler w144x showed that the levels of annexins I and V change in fibroblasts according to factors such as cell density and renewal of growth medium. The question is whether or not changes in annexin expression that occur during cell differentiation or proliferation are a consequence of these processes, or do annexins actually contribute some important activity or driving force. At present, there is little evidence to support or

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repudiate either argument. Most studies of annexins in development have been descriptive and whilst these may suggest certain roles, proof has not yet been forthcoming. Carter et al. w147x showed that annexin II is up-regulated during embryonic avian limb development, being virtually undetectable at day 5 of development but strongly expressed by day 14. If cells were dissociated at day 5 and grown in culture then annexin II expression was rapidly induced, but only if the cells were allowed to attach to a substratum. This reinforces the view that cell culture conditions are an important determinant of annexin expression. Similar developmental changes in annexin expression have been reported in the fish embryo w148x, the embryonic rodent nervous system w149–151x and diverse other tissues. In cultured cells the causal relationship problem is the same, but perhaps a little less intractable since annexin expression can, at least to some extent, be manipulated. Masiakowski and Shooter w152x showed that transfection of p11, the cellular ligand of annexin II, into PC12 cells stimulated differentiation into cells resembling sympathetic neurones. Whether this was accompanied by increased expression of annexin II Žas seen with NGF-induced PC12 differentiation. is not known. Puisieux et al. w153x reported that in cells expressing p11 mRNA, p11 protein was undetectable unless annexin II was also expressed, suggesting that p11 probably cannot function alone and that annexin II contributes to PC12 cell differentiation. In contrast, F9 mouse teratocarcinoma cells show an expression of p11 both at the mRNA and at the protein level whereas annexin II appears to be absent w154,155x. In other cell types annexins may function to moderate or inhibit cell proliferation. This was first shown for A549 lung adenocarcinoma cells which dramatically reduce their growth rate when cultured in the presence of recombinant annexin I w156x. Growth arrest was also observed when A549 cells were cultured in the presence of dexamethasone, which coincidentally stimulates the appearance of annexin I on the cell surface. Subsequently, Croxtall et al. w157x showed that inhibition of A549 cell proliferation could be mimicked by N-terminal peptide fragments of annexin I. The active fragments incorporated amino acids 13–25 and 21–33 whereas a peptide corresponding to amino acids 1–12 Žthe S100C-binding domain. was inactive. The squamous epithelial carci-

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noma cell line, A431, was also shown to be growthinhibited by annexin VI w127x. These cells do not normally express annexin VI but when stably transfected clones expressing annexin VI were examined in proliferation assays, they were found to grow more slowly in a serum concentration-dependent manner. Thus, at low serum concentrations Ž1–2%., wild-type A431 cells grow normally whereas A431 cells expressing annexin VI become growth-arrested and accumulate in the G1 phase of the cell cycle. Theobald et al. w128x then showed that despite appearing to have partially reverted to a non-transformed phenotype, annexin VIq A431 cells were still tumorigenic in immunodeficient mice, although the tumours grew signficantly more slowly than those derived from control transfected cells. These data argue that annexins are not just ‘‘innocent bystanders’’ in important cellular processes such as growth regulation and differentiation and that they probably have pro-active roles which remain to be elucidated.

5. Involvement in membrane traffic and membrane organization The common property displayed by annexins in vitro, i.e. the Ca2q-dependent binding to acidic phospholipids found preferentially in the cytosolic leaflet of cellular membranes, is highly suggestive of a function of the proteins in intracellular membrane-related events. To some extent this has been a dogma in the field since already the first annexin described in the literature, annexin VII Žat the time known as synexin., was isolated as a protein mediating membrane contact and fusion w158x. While these are indeed properties shared by several annexins detailed analyses in the years following the initial discovery of annexin VII revealed that in most cases the Ca2q concentration required to achieve vesicle aggregation Žand fusion. are above the cytosolic free Ca2q levels experienced in resting and even stimulated cells Žsee above.. This still remains to be a main conceptual problem in the annexin field. However, at least annexins I and II appear to be able to associate with membranes in vitro at micromolar or even submicromolar Ca2q levels and several members of the family have been localized in situ to intracellular membranes andror the plasma membrane at the light and elec-

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tron microscope level. Moreover, recent studies have provided evidence for Ca2q-independent annexin– membrane interactions and an involvement of annexins in specific membrane trafficking events. Here we discuss these aspects of annexin biology in light of the intracellular distribution of individual members of the family. 5.1. Intracellular localization and potential target membranes for indiÕidual annexins A clear view with respect to the intracellular distribution and the specific allocation of individual annexins to one or more membranous structures is obscured by the different cell types, fixation methods and antibodies used in a substantial and ever increasing number of immunocytochemical studies on annexins. Below we summarize some of these data for the major mammalian annexins trying to obtain a conclusive picture for the individual proteins. We do not intend to present a complete literature survey and we do not include data which contradict the general picture obtained for a given annexin and appear only once in the literature. Taking this into account membrane-bound annexin I appears to reside on the plasma membrane and on the membranes of certain endocytotic and phagocytic structures. The latter is particularly evident in macrophages, where annexin I was localized to early endosomal and phagosomal membranes at the ultrastructural level ŽDiakonova, M., V. Gerke, J. Ernst, G. van der Vusse, G. Griffiths, submitted for publication., and in neutrophils, where annexin I was shown to translocate from the cytosol to the phagosomal and the plasma membrane when the cells are stimulated with opsonized zymosan or take up opsonized yeast w159,160x. In baby hamster kidney Ž BHK. and Madin-Darby canine kidney Ž MDCK. cells annexin I is present on the plasma membrane and on the membrane of early endosomes w62x, whereas in NIH 3T3 cells overexpressing the EGF receptor annexin I is found on multivesicular endosomes w69x. Under certain conditions or in some cell types annexin I may also associate with other structures as it is present on insulin-containing granules in pancreatic beta-cells and appears to interact to some extent with cytoskeletal structures in human keratinocytes w161,162x. A similar but not overlapping subcellular distribu-

tion is often found for annexin II although these two annexins Ž I and II. are only co-expressed in a limited number of cell types Žfor a recent comparison of the tissue distributions of annexins I and II, see Dreier, R., K.W. Schmid, V. Gerke, K. Riehemann, submitted for publication.. Annexin II is a general constituent of the submembraneous cytoskeleton Žfor review see w64x. and has also been identified on early endosomes after immunoisolation of these structures and by immuno-electron microscopy of cryosectioned BHK, MDCK and J774 cells Žw163,164x; Diakonova, M., V. Gerke, J. Ernst, G. van der Vusse, G. Griffiths, submitted for publication.. Interestingly the submembranous localization of the protein, both at the level of the plasma membrane and the early endosomal membrane, is not uniform. This is evident in polarized cells Ženterocytes, filter-grown MDCK cells, mammary epithelial cells., where annexin II appears concentrated underneath the apical membrane w74,165–167x, and in J774 macrophages, where the protein is enriched in plasma membrane areas showing ruffles, microvilli and membrane folds as compared to more smoother parts of the membrane ŽDiakonova, M., V. Gerke, J. Ernst, G. van der Vusse, G. Griffiths, submitted for publication.. Likewise the early endosome-associated annexin II appears not uniformly distributed over the endosomal membrane but is concentrated in discrete regions which in some cases have also filamentous material attached to them w164x. These results are indicative of a role of annexin II in organizing membranes, possibly by linking certain membrane domains to the underlying cytoskeleton Žsee below.. In addition to the plasma membranerendosome localization annexin II is also present on vesicles of the secretory transport route, at least in certain types of cells. Such cell types include adrenal chromaffin cells, where the protein is a major constituent of the chromaffin granules w77,168,169x, and secretory cells of the anterior pituitary where some annexin II appears to reside on the secretory granules w170,171x. In quick-freeze, deep-etch analyses of these secretory cells it even appears that annexin II can form physical connections or bridges between the plasma membrane and the membrane of the respective secretory vesicles w169,171x. Finally, annexin II has also been identified in phagosome preparations from J774 macrophages w172x.

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While annexin II appears to serve a structural role in the cells and systems discussed above its association with the different membranes appears to be regulated by different means. In MDCK cells, for example, treatment with trifluoperazine or cultivation in a low-Ca2q medium leads to a translocation of annexin II from the plasma membrane to the cytoplasm w173,174x. This could be a direct consequence of a drop in the intracellular Ca2q levels since Ca2q binding is a prerequisite for tightly anchoring annexin II in the cortical cytoskeleton of fibroblasts w79x. Cellular differentiation often represents another stimulus regulating the annexin II association with certain membranes. Examples are mammary epithelial cells, which show a striking appearance of annexin II on the apical, secretory membrane only after parturition, and cultivated MDCK cells, in which translocation of cytosolic annexin II to the apical and basolateral membranes is observed after establishment of a polarized cell morphology w166,173x. Finally, the stimulation of certain membrane transport events appears to correlate with the regulated association of annexin II with specific membranes. This is evident in hepatocytes, where the transcytosis of cholestatic bile salts leads to extensive redistributions of annexin II from the basolateral membrane to the perinuclear region and finally to the apical membrane w175x and in nicotine-stimulated chromaffin cells, where catecholamine secretion is accompanied by a translocation of annexin II from the cytosol to the plasma membrane w80x. It seems likely that in addition to changes in the intracellular Ca2q levels other signals are involved in regulating the annexin II–membrane interactions. These could include phosphorylation at one ore more of the sites found in the N-terminal domain of annexin II since a peptide corresponding to this N-terminal sequence interferes with the above-mentioned translocation in nicotine-stimulated chromaffin cells w80x and since phosphorylation by protein kinase C of Ser-11 of annexin II interferes with p11 binding w21,176x which itself is prerequisite for anchoring annexin II tightly in the submembraneous cytoskeleton w79x. The subcellular distribution of annexin III has m ainly been studied in neutrophils and monocytesrmacrophages. Here, the protein is found on the plasma membrane and on the membranes of intracellular granules and phagosomes w177x ŽDi-

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akonova, M., V. Gerke, J. Ernst, G. van der Vusse, G. Griffiths, submitted for publication.. As also observed for annexin I, a translocation of annexin III to the phagosomal and the plasma membrane is induced upon phagocytosis and upon stimulation of the cells with phorbol ester w159,177x. Annexin IV, which is most abundantly expressed in epithelial cells, is found associated with the plasma membrane although different domains of this membrane appear to be the prime site of annexin IV-association in different types of polarized epithelial cells. In enterocytes and epithelial cells of the lung annexin IV is enriched on the basolateral plasma membrane domain w178,179x, whereas the protein has been localized to the apical w129x and the basolateral membrane domain w180x in renal epithelial cells and to the apical plasma membrane in epithelial cells of the uterus w181,182x. In addition to the plasma membrane-localized pool some endosome-associated annexin IV has been identified in J774 macrophages and BHK cells ŽDiakonova, M., V. Gerke, J. Ernst, G. van der Vusse, G. Griffiths, submitted for publication. w133x. Annexin V is another member of the family which has been identified on the plasma membrane and on endocytotic membranes in a variety of cells w183,184x ŽDiakonova, M., V. Gerke, J. Ernst, G. van der Vusse, G. Griffiths, submitted for publication.. In certain types of cells some annexin V is also found associated with the endoplasmic reticulum Ž glial cells, w185x. and the sarcoplasmic reticulum Ž skeletal muscle, w184x.. As observed for other annexins certain stimuli lead to a translocation of cytosolic annexin V to membranes. In platelets thrombin induces such relocation resulting in a tight, Ca2q-insensitive association of annexin V with the membrane w186x and membrane depolarization has been shown to lead to a membrane translocation of annexin V in neuroblastoma cells w187x. Finally, in human dendritic cells endocytosis of fluorescently labelled albumin is accompanied by a re-distribution of annexin V which colocalizes with the albumin-containing vesicles w188x. The intracellular localization of annexin VI is somewhat more controversial. While it has been identified on the plasma membrane in a variety of cell types including lymphocytes w189x and hepatocytes w167,190x, a codistribution with mitochondria of liver

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cells w191x, the sarcoplasmic reticulum of skeletal muscle and structures most likely resembling the endoplasmic reticulum of non-muscle cells w192x has also been observed. The latter can be correlated with the modulating activity annexin VI exerts on the sarcoplasmic Ca2q release channel in vitro w123x Žsee also discussion above. . Using subcellular fractionation annexin VI has also been localized to phagosomes of J774 macrophages w172x and to endosomal membranes of rat liver w193x although a specific endosomal enrichment of annexin VI has not been observed in BHK cells w62x. Collectively, these data suggest that annexin VI could act on a variety of target membranes and that these interactions are regulated differently in different types of cells. Annexin VII, the protein initially described as the granule aggregating activity of adrenal chromaffin cells Žsee above., is present in the cytosol of these cells but appears concentrated around the granule membrane in the granular cell area w194x. Whereas the protein is also located in the cytosol of cultured muscle cells it is found preferentially on the plasma membrane and on transverse tubules in striated muscle w195x. While only very scarce or no information is available about the subcellular distribution of annexin VIII and the Drosophila annexins IX and X, several reports have described the localization of annexin XI. Interestingly, in cultured fibroblasts this annexin is localized to the nucleus and this localization depends on the presence of the long N-terminal domain w196,197x. In contrast, a 42-kDa fragment of this protein appears to be cytosolic protein in neutrophils which translocates to the membrane of the neutrophil granules in a Ca2q-dependent manner w198x. Annexin XIII is the last mammalian identified so far. It was initially described as an intestinal specific annexin and was shown to be particularly enriched in the apical brush border region of different enterocytes although some annexin XIII was also found associated with the basolateral membranes w72x. A splice variant of this protein containing a 41 amino acid sequence inserted in the N-terminal domain, annexin XIIIb, was identified as a component of apical exocytotic carrier vesicles purified from polarized MDCK cells and was specifically localized to these vesicular structures and the apical plasma membrane of such cells by immunocytochemical techniques w73x. The

mechanisms specifying and regulating these and other distinct membrane interactions of individual annexins are not known but are likely to require the unique N-terminal domains of the molecules and, in the case of annexin XIIIb, the spliced-in exon. The examples discussed above stress the importance of annexin–membrane interactions and this is supported by the finding that the membranes of essentially all intracellular compartments are stained with a pan-annexin antibody on cryosections of BHK cells w199x. However, one should keep in mind that most annexins are not permanently associated with the respective target membranes. Certain cellular condition, in most cases an elevated Ca2q concentration appears to be of crucial importance, need to be met for an annexin to bind to membranes. Thus, annexin proteins are likely to shuttle between the cytosol and their target membraneŽs. thereby participating in membrane-related processes in a regulated manner. The recent documentations of Ca2q-independent annexin–membrane interactions, however, seem to suggest that a non-regulated binding to membranes can occur in the case of some annexins or certain isoforms of individual annexins. Examples are here the Ca2q-independent associations of myristoylated annexin XIIIb with apical transport vesicles w73x, of annexin II with certain domains of the early endosomal and the plasma membranes w133,164x and of non-phosphorylated annexin I with multivesicular bodies w69x. A striking example for the membrane localization of annexins has recently also been provided in the case of a lower eucaryotic species, the nematode Caenorhabditis elegans. Here, the product of the nex-1 annexin gene is present in high concentrations on membrane systems in the spermathecal valve. These membranes unfold and then refold in an accordeon-like fashion as eggs pass through the valve, suggesting that the nex-1 annexin could participate in mediating this type of membrane–membrane interactions w200x. 5.2. Annexins in exocytosis The first annexin described in the literature, annexin VII, was purified as a component capable of aggregating chromaffin granules in a Ca2q-dependent manner and ever since annexins have been implicated

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in exocytotic events, in particular in the Ca2q-regulated secretion Ž for review see w2x.. Although direct in vivo data for such a role of annexins are still not available, some interesting approaches in the recent years have provided further evidence for the involvement of annexins in transport along the biosynthetic pathway, in particular in late exocytotic steps occurring at or close to the plasma membrane. Annexin I, for example, was implicated in the glucose-induced insulin secretion in rat pancreatic islets since it specifically locates to insulin-containing granules during this process. Interestingly, the secretion process correlates with a phosphorylation of annexin I on Ža. serine residueŽs. w162x. A function of annexin I in exocytotic events is also in line with the finding that the protein represents a major fusogenic activity of the cytosol from human neutrophils w201x. Various studies using adrenal chromaffin cells and their exocytotic granulae have focused on annexin II and the annexin II 2 p11 2 complex as a potential mediator of Ca2q-regulated secretion of the granule content. Annexin II was identified as a prominent granule protein Žfor review seew2x., it was localized to the site of the granule–plasma membrane contact w169x and was shown to be capable of aggregating isolated chromaffin granules at Ca2q concentrations occurring within the stimulated cells w77x. Furthermore, in permeabilized chromaffin cell systems the progressive loss of secretory responsiveness to Ca2q stimulation can be slowed down by adding purified annexin II w202x. This effect of annexin II depends at least in part on the annexin II–p11 complex formation since monomeric annexin II is less efficient in the assay than the heterotetrameric complex. Moreover, protein kinase C-catalyzed phosphorylation of annexin II appears to be required for the stimulatory activity of the protein, correlating well with the activation of the enzyme during nicotine stimulation of adrenal chromaffin cells w68x. In vitro studies also revealed that phosphorylation of chromaffin granule-bound annexin II by protein kinase C induced a fusion of the aggregated chromaffin granule membranes w203x. Finally, a careful analysis of the annexin II localization in chromaffin cells revealed that monomeric annexin II translocates from the cytosol to the subplasmalemmal region upon nicotinic stimulation of catecholamin secretion w80x. This is a Ca2q-triggered process which appears to be accompanied by annexin

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II–p11 complex formation since p11 is restricted to the cortical region even of unstimulated cells. However, apart from the annexin II–p11 interaction other modes of regulation are likely to be involved in inducing this translocation since a synthetic annexin II peptide covering amino acids 15–26, i.e. a region of the N-terminal domain containing phosphorylation sites but not the p11 binding site, blocks the nicotine-induced recruitment of annexin II to the cell periphery and concomitantly inhibits the Ca2q-triggered catecholamine secretion w80x. The proposed role of annexin II in Ca2q-regulated secretion is most likely not restricted to adrenal chromaffin cells since exogenously added annexin II also stimulates the Ca2q-induced exocytosis of lamellar bodies in permeabilized alveolar epithelial cells w204x. Subcellular localization and the use of a permeabilized cell system have also helped to identify another annexin most likely involved in transport steps along the exocytotic pathway. Annexin XIIIb, an N-terminal splice variant of the intestine-specific annexin XIIIa, is found specifically enriched on apical transport vesicles of polarized MDCK cells. Moreover, antibodies directed against the unique exon in annexin XIIIb, interfere with vesicle transport to the apical but not the basolateral plasma membrane w73x. This was shown in permeabilized MDCK cells after the apical and basolateral plasma membranes had been perforated separately by treatment of sheets of polarized cells with streptolysin O ŽSLO. administered either from the apical or the basolateral side. In these cells transport of exocytotic vesicles from the trans-Golgi network to the apical and the basolateral plasma membrane Žthe one not treated with SLO. can be restored by adding cytosol and ATP, and antibodies to annexin XIIIb had a specific inhibitory effect on the apical route. Interestingly, the apical vesicle traffic in this system is not affected by targeting the SNARE–SNAP–NSF-dependent docking–fusion machinery through the use of antibodies or bacterial toxin whereas the basolateral transport is clearly inhibited by these agents w205x. In contrast, transcytosis of pre-internalized IgA receptor to the apical surface of polarized, SLO-permeabilized MDCK cells is sensitive to N-ethylmaleimide and Botulinum neurotoxin, indicating that this transport route is SNARE– SNAP–NSF-dependent w206x. These findings indicate that annexin XIIIb could be part of a certain special-

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ized exocytotic machinery acting independently of the known NSF, SNAPs and SNAREs. It remains to be seen whether annexins are also involved directly or indirectly in other apparently NSF-independent vesicle transportrfusion pathways, e.g. a certain exocytotic route in BHK and hamster CHO cells w207x and the homotypic fusion of ER membranes Žw208x; for review see w209x.. Localization studies have also provided circumstantial evidence for an involvement of annexins or annexin-like proteins in exocytotic processes in lower organisms. In Paramecium, several proteins were tentatively identified as annexins by immunological and biochemical criteria. Antibodies recognizing these proteins revealed a dynamic labelling of cortical structures during trichocyst exocytosis, suggesting that annexin-like proteins are involved in the sitespecific positioning and attachment of secretory organelles at the surface of Paramecium cells w210x. One of the annexins of C. elegans, the product of the so-called nex-1 gene, is specifically associated with several membrane systems of different cell types of the adult nematode. Among them are secretory gland cells of the pharynx, a finding providing further circumstantial evidence for a possible participation of annexins in exocytotic events in simple organisms w200x. The mechanistic aspects of the annexin action in the exocytotic processes discussed above are not understood. Since the proteins appear not to be fusogenic per se Žan exception could be phosphorylated annexin II bound to chromaffin granules, see above. they might need physiologically occurring cofactors, e.g. cis-unsaturated fatty acids, to exert such activities Žfor review see w2x.. Alternatively, annexins may serve a structural role by physically linking secretory vesicles to one another andror to the plasma membrane w169x. This could hold the vesicle in place for the actual fusion event to occur. Moreover, certain annexins might also provide a connection between the vesicle membrane and cytoskeletal elements thereby aiding vesicle traffic andror contact with the plasma membrane. However, a major conceptual problem with all models discussed concerns the specificity. Based on the central biochemical activity displayed by annexins, i.e. the Ca2q-dependent binding to acidic phospholipids, it is not entirely clear why individual annexins interact preferentially with a

certain target membrane, e.g. the annexins mentioned above with that of the exocytotic vesicle or the plasma membrane. A certain degree of specificity could stem from the observed preference of a given annexin for a certain phospholipid headgroup Ž see above. and the unequal distribution of the different acidic phospholipids over the different intracellular membranes. Alternatively, specific membrane proteins or certain subdomains of membrane lipidsrproteins might exist which act as receptors for a unique domain or structure within a given annexin, e.g. the alternatively spliced exon in the N-terminal domain of annexin XIIIb. Some experimental evidence for the existence and structural basis of such specific annexin–membrane interactions exists in the case of annexins I and II and their association with endocytotic membranes Žsee below.. 5.3. Annexins in endocytosis In addition to the immunocytochemical localizations Žsee above. data from in vitro as well as in vivo approaches have implicated several annexins in transport andror sorting events along the endocytotic pathway. Annexin I is a major substrate for the EGF receptor kinase which becomes phosphorylated on Tyr-20 in the N-terminal domain upon internalization of the receptor w211–213x. Using NIH 3T3 cells overexpressing the EGF receptor Futter et al. w69x could show that annexin I phosphorylation by the receptor kinase occurs in multivesicular bodies ŽMVBs., endocytotic compartments involved in sorting receptors destined for degradation such as the EGF receptor away from recycling receptors which return to the plasma membrane. In the NIH 3T3 cells this sorting process, i.e. the removal of EGF receptors to the internal vesicles of the MVBs, correlates with the tyrosine phosphorylation of annexin I which somehow alters the mechanism by which annexin I associates with the MVB membrane. While unphosphorylated annexin I appears to interact at least to some extent with both, the plasma membrane and the membrane of MVBs, in a Ca2q-independent manner, phosphorylation by the EGF receptor kinase converts it to a form requiring Ca2q for membrane interactions w69x. Collectively, these findings suggest that phosphorylation of annexin I provides a signal for the sorting of the EGF receptor in the MVBs and that

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phosphorylated annexin I could participate in the inward vesiculation process w69x. However, by subcellular fractionation and immunocytochemical localization on cryosections of BHK and MDCK cells the bulk of membrane-bound annexin I was identified on the plasma membrane and on the membrane of early endosomes but not on that of late and multivesicular endosomes w62x. Moreover, it appears that in this case the membrane association is strictly dependent on the presence of micromolar Ca2q concentrations. While the basis of this discrepancy is not known it seems reasonable to assume that the cell system used, in particular the overexpression of the EGF receptor in the case of the NIH 3T3 cells, could be of importance. Overexpression of the receptor and the resulting increase in internalization could alter to some extent the intracellular distribution of one of its substrates, annexin I, shifting it from early to multivesicular endosomes. Subcellular fractionation in combination with the ectopic expression of mutant annexin I derivatives also revealed that in BHK cells the specific association of this annexin with its target membranes, the plasma membrane and that of early endosomes, is mediated through the unique N-terminal domain. While a truncation of residues 1 to 13, i.e. the region representing the S100C binding site in annexin I w86,87x does not affect the early endosomal association of the molecule, a further deletion of an additional 13 residues Ž 13–26. abolishes this co-fractionation w62x. The region of interest in the annexin I molecule Žresidues 13–26. harbours phosphorylation sites for the EGF receptor kinase and protein kinase C. However, phosphorylation at these sites appears not to be directly involved in specifying the early endosomal membrane interaction as annexin I derivatives with mutated phosphorylation sites continue to co-fractionate with early endosomes ŽJ. Seemann and V. Gerke, unpublished observation.. The findings therefore provide evidence for the existence of an early endosomal receptor Žprotein or certain membrane-lipid structure. which interacts with the region spanning residues 13–26 in the N-terminal domain of annexin I. Likewise, it remains possible that this N-terminal sequence specifically affects the membrane binding properties of annexin I through some sort of intramolecular communication. Annexin II is another annexin implicated in early

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endocytotic events. It is specifically localized to certain areas of the plasma membrane and to cisternal and tubular regions of early endosomes in different types of cells w163,164,173x ŽDiakonova, M., V. Gerke, J. Ernst, G. van der Vusse, G. Griffiths, submitted for publication.. Moreover, in vitro systems reproducing the homotypic fusion between early endosomal membranes revealed that annexin II is one of the few proteins transferred efficiently from a donor to an acceptor endosomal membrane w163x and that antibodies against annexin II interfere with a Ca2q-dependent endosomal fusion event w214x. In vivo evidence for the participation of this annexin in endocytosis stems from the transient expression of a trans-dominant annexin II mutant. This mutant molecule, in which the N-terminal 18 amino acids of annexin II are fused to an entire p11 chain, leads to the formation of annexin IIrp11 aggregates absorbing the entire intracellular annexin II and p11. In polarized MDCK cells, where the annexin II–p11 complex is restricted to the cell periphery, the mutant-induced annexin IIrp11 aggregation leads to a parallel translocation of transferrin receptor-positive early endosomes from the cell periphery to the sites of the annexin IIrp11 aggregates w173x. This indicates that in such polarized cells the annexin II–p11 complex is involved in establishing andror stabilizing a peripheral localization of early endosomes possibly by linking endosomal membranes to the cortical cytoskeleton. It remains to be seen whether such a structural role of the annexin II–p11 complex is regulated by Ca2q, in particular since annexin II can interact with endosomal membranes in a Ca2q-independent manner. This was revealed by subcellular fractionation of BHK cell membranes in the presence of a Ca2qchelating agent and subsequent analysis of the annexin II content in the early endosomal fraction w133,164x. Moreover, an ectopically expressed annexin II mutant with inactivated type II Ca2q binding sites continues to co-fractionate with early endosomal membranes w133x. Interestingly, the endosomal association is not affected by deleting the p11 binding domain of the molecule Žresidues 1–14. but is sensitive to a deletion of residues 14–26 in N-terminal domain of annexin II w133x. This supports the idea of a membrane receptor Žlipid or protein. which is present on early endosomes and interacts in a Ca2q-inde-

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pendent manner with a unique sequence in the annexin II molecule. A Ca2q-independent association of annexin VI with cellular membranes isolated from BHK cells has also been observed although a specific enrichment on individual compartments is not evident in this case w62x. This contrasts the situation in rat liver cells where annexin VI specifically co-fractionates with early endosomes w193x. An in vitro assay measuring the loss of clathrin from immobilized plasma membranes has directly implicated this annexin in coated pit budding. The budding Žs loss of clathrin. was dependent on cytosol, Ca2q and ATP, was inhibited when annexin VI-depleted cytosol was used and was restored upon supplementing the depleted cytosol with purified annexin VI w215x. The view that annexin VI has a general role in the coated vesicle formation during receptor mediated endocytosis has, however, been challenged by the finding that the protein is not expressed in every cell type carrying out receptor-mediated endocytosis. This is the case in human A431 cells which do not express annexin VI and where rate and extent of receptor-mediated endocytosis are not affected by ectopically expressing the protein w216x. Thus, annexin VI either is required for the clathrin-coated vesicle budding only in certain cell types or exercises its budding activity only in the particular in vitro system and not in living cells. More in vivo information is required to settle this point in particular and to explain mechanistic aspects of the annexin functions in exocytosis and endocytosis in general. 5.4. Annexins in membrane organization The proposed functions of annexins in exocytotic and endocytotic membrane transport events would be in accord, at least to a large extent, with a role of the proteins in organizing and stabilizing certain membrane domains. Such membrane organization would be required for cytoskeletal attachment and lipid segregation and would in turn affect vesicular transport from a donor to an acceptor compartment. At the level of the plasma membrane, for example, domain organization reflects itself in coated pits, where membrane receptors destined for internalization are concentrated, and in cholesterol-rich membrane rafts, where certain lipids Ž e.g. glycosphingolipids. and

proteins Že.g. caveolin, GPI anchored proteins, CD44. are enriched Žfor review see w217,218x.. Likewise, a membrane organization is probably required in the early endosomal compartment, e.g. to separate molecules destined for transport to late endosomes from those recycling back to the plasma membrane Žfor review see w219x., and in the trans-Golgi network where a sorting of vesicles carrying material to different plasma membrane domains and to lysosomes occurs Ž for review see w220,221x.. Several structural components localized at the cytosolic faces or in the cytosolic leaflets of the respective membranes are known to be involved in generating andror stabilizing membrane domains. These include adaptor molecules of the respective vesicle coats, e.g. AP1 and AP2 complexes of clathrin coats, and the cholesterol binding protein VIP 21rcaveolin. However, less is known about a possible linkage of these or other membrane structuresrdomains to the submembraneous, actin-dominated cytoskeleton. Based on their biochemical properties and their discrete localization to certain membrane domains, e.g. of the plasma membrane of macrophages ŽDiakonova, M., V. Gerke, J. Ernst, G. van der Vusse, G. Griffiths, submitted for publication., it remains possible that some annexins participate in anchoring certain membrane domains at the cortical cytoskeleton or in establishing a certain lipid segregation per se. Experimental evidence for such models is severalfold. Due to the preferential, Ca2q-bridged interaction of annexins with phospholipids containing negatively charged headgroups a binding of the proteins could induce a clustering of such acidic phospholipids in the plane of the cytosolic leaflet of the membrane. In the case of annexin IV, for example, binding to mixed lipid membranes containing phosphatidylglycerol and phosphatidylcholine induces a segregation of the lipids w222x. The annexin II–p11 complex, a prominent component of the cortical cytoskeleton of many cells, has long been implicated in providing a membrane–cytoskeleton contacts Žfor review see w64x.. Recently, cholesterol-rich membrane domains have been identified as a discrete site for annexin II attachment. Moreover, cholesterol-clustering agents, like filipin and digitonin, selectively release from membrane fractions containing an EGTA-resistant form of annexin II a limited set of proteins, namely

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annexin II itself, actin and the actin binding proteins a-actinin, ezrin and moesin w164x. Since these components from a multiprotein complex in the presence of cholesterol-containing membranes it appears that annexin II could participate in specifically linking cholesterol-rich membrane domains to the actin cytoskeleton w164x. Such a structural role of annexin II might also be the mechanistic basis of the proposed function of the annexin II–p11 complex in sensing or transmitting mechanical signals. In endothelial cells, the mechanical activation Žthrough shear stress or cell volume changes. of a chloride channel in the plasma membrane is sensitive to disrupting the annexin II– p11 complex. This has been shown by an electrophysiological analysis of cells loaded via the patch pipette with a peptide comprising the N-terminal 14

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residues of annexin II. This peptide harbours the entire p11 binding site and efficiently interferes with annexin II–p11 complex formation w81x. The peptide-induced disruption of the annexin II–p11 complex leads to a significant decrease in the amplitude of the volume-activated chloride current. Moreover this effect is highly specific since a mutant peptide, which contains a single amino acid replacement resulting in a more than 1000 times reduced affinity towards p11, has no effect w223x. As depicted schematically in Fig. 3, these data suggest that the annexin II–p11 complex could be involved in stabilizing certain membrane domains Žthose having a high cholesterol content. through a complexation with the underlying cytoskeleton and that this organization is required to provide the structural rigidity necessary

Fig. 3. Putative role of the annexin II–p11 complex in organizing membrane domains. The schematic illustration depicts a lipid bilayer containing different domains Žblack and cross-hatched. linked at certain points to an underlying cortical cytoskeleton. This organization leads to a restricted diffusion and thus a segregation of certain membrane lipids and proteins resulting, e.g., in a different membrane composition of buds forming at a donor membrane. A putative protein complex connecting membrane domains to the cortical cytoskeleton is shown enlarged. It contains annexin IIrp11 together with one or more additional actin-binding proteins and a membrane receptor specifically interacting with cholesterol-rich membrane domains Žsee text for discussion.. Such a structure depends on the formation of the heterotetrameric annexin II–p11 complex which itself can be regulated by phosphorylation w176x. Disruption the annexin II–p11 complex with a specific p11 binding peptide could therefore lead to some uncoupling of membrane–cytoskeleton contacts resulting, e.g., in a reduced cellular response to mechanical shear stress activation w223x.

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for sensing mechanical alterations at the level of the plasma membrane.

points and have to establish the mechanistic basis for the function of annexins in membrane dynamics.

6. Conclusions and outlook

Acknowledgements

Due to their thoroughly studied biochemical properties annexins have long been known as Ca2q-regulated phospholipid and membrane binding proteins. Nevertheless it has not been Žand to a large extent still is not. clear how these well established properties translate into and correlate with the physiological roles of these proteins. Moreover, it has been enigmatic for a long time why such a complex multigene family has evolved with their individual members showing strikingly similar biochemical properties. The recent findings discussed in this review contribute to our understanding towards this end. Structural work, detailed intracellular localizations and functional attempts using permeabilized cell systems and the overexpression of annexin mutant proteins provide increasing evidence that annexins indeed function in membrane related events. These approaches also reveal that different annexins act on different target membranes and most likely display different biological activities thus explaining the need for a complex multigene family. The general theme emerging is that the two structurally defined domains of an annexin, the C-terminal protein core and the N-terminal tail, are designed to perform different functions. The conserved protein core is a building block mediating Ca2q-regulated membrane association and the highly divergent N-terminal domain is an entity specifying membrane contact and modulating the activity of a membrane bound annexin core, e.g. through the interaction with specific protein ligands. The latter most likely involves the establishment of membrane–membrane and membrane–cytoskeleton interactions and the organization of membrane domains, activities required for membrane sorting and vesicle traffic along the exocytotic and the endocytotic routes. Possibly, target membrane selection and intra- as well as intermolecular regulation could also specify that a certain annexin once docked at the appropriate membrane can also affect ion currents across this membrane. Future in vivo work, e.g. annexin gene knock outs in cell lines and in animals which are currently under way, have to settle these

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