Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers

Vascular anticoagulant alpha (VAC alpha, annexin V) is a member of the family of calcium and phospholipid binding proteins, the annexins. The binding properties of VAC alpha to phospholipid bilayers were studied by ellipsometry. Adsorption was calcium-dependent and completely reversible upon calcium depletion. Half-maximal adsorptions to phospholipid bilayers consisting of 100, 20, 5, and 1% dioleoyl-phosphatidylserine (DOPS) supplemented with dioleoyl-phosphatidylcholine (DOPC) were reached at Ca2+ concentrations of 0.04, 0.22, 1.5, and 8.6 mM. These surfaces all showed the same maximal adsorption of 0.22 +/- 0.01 micrograms of VAC alpha/cm2 (mean +/- S.D.). The adsorption to bilayers containing more than 10% DOPS was independent of VAC alpha concentrations in the range of 0.5-100 nM. Dissociation constants for VAC alpha binding to these surfaces were estimated to be below 2 x 10(-10) M. No adsorption was observed on pure DOPC bilayers at a Ca2+ concentration of 3 mM. The ability to mediate VAC alpha binding to 20% DOPS/80% DOPC bilayers was highly specific for Ca2+. The use of other divalent cations resulted in decreased binding in the order Cd2+ greater than Zn2+ greater than Mn2+ greater than Co2+ greater than Ba2+ greater than Mg2+. Zinc ions had a synergistic effect on Ca2(+)-dependent VAC alpha binding. The Ca2+ concentration needed for half-maximal binding to cardiolipin, dioleoyl-phosphatidylglycerol, DOPS, phosphatidylinositol, phosphatidic acid, dioleoyl-phosphatidylethanolamine, and sphingomyelin increased in that order. Adsorption was independent of the overall surface charge of the phospholipid membrane.     

Structure of membrane-bound annexin A5 trimers: a hybrid cryo-EM - X-ray crystallography study

Annexins constitute a family of phospholipid- and Ca(2+)-binding proteins involved in a variety of membrane-related processes. The property of several annexins, including annexin A5, to self-organize at the surface of lipid membranes into 2D ordered arrays has been proposed to be functionally relevant in cellular contexts. To further address this question, we investigated the high-resolution structure of annexin A5 trimers in membrane-bound 2D crystals by cryo-electron microscopy (Cryo-EM). A new 2D crystal form was discovered, with p32(1) symmetry, which is significantly better ordered than the 2D crystals reported before. A 2D projection map was obtained at 6.5 A resolution, revealing protein densities within each of the four domains characteristic of annexins. A quantitative comparison was performed between this structure and models generated from the structure of the soluble form of annexin A5 in pseudo-R3 3D crystals. This analysis indicated that both structures are essentially identical, except for small local changes attributed to membrane binding. As a consequence, and contrary to the common view, annexin A5 molecules maintain their bent shape and do not flatten upon membrane binding, which implies either that the four putative Ca(2+) and membrane-binding loops present different types of interaction with the membrane surface, or that the membrane surface is locally perturbed. We propose that the trimerization of annexin A5 molecules is the relevant structural change occurring upon membrane binding. The evidence that 2D arrays of annexin A5 trimers are responsible for its in vitro property of blood coagulation inhibition supports this conclusion.     

Membrane composition affects the reversibility of annexin A2t binding to solid supported membranes: a QCM study

By means of the quartz crystal microbalance (QCM) technique, we investigated the interaction of porcine heterotetrametric annexin A2t with solid supported lipid membranes. Dissociation and rate constants of annexin A2t binding to various lipid mixtures were determined as a function of Ca2+ concentrations in solution. In contrast to what has been observed for annexin A1, the binding affinity and kinetics of annexin A2t binding are not influenced by cholesterol. In the experimental setup chosen, the annexin A2t binding is strictly Ca2+-dependent and only affected by the amount of phosphatidylserine (PS) in the membrane and the Ca2+ concentration in solution. By Ca2+-titration experiments at constant annexin A2t concentration, we investigated the reversibility of annexin A2t adsorption and desorption. Surprisingly, Ca2+-titration curves display a significant hysteresis. Protein desorption curves starting from annexin A2t bound to the membrane at 1 mM CaCl2 exhibit high cooperativity with half-maximum Ca2+ concentrations in the submicromolar range. However, protein adsorption curves starting from an EGTA-containing solution with soluble annexin A2t always show two inflection points upon addition of Ca2+ ions. These two inflection points may be indicative of two protein populations differently bound to the solid-supported membrane. The ratio of these two annexin A2t populations depends on the amount of PS molecules and cholesterol in the membrane as well as on the Ca2+ concentration. We propose a model discussing the results obtained in terms of two binding sites differing in their affinity due to lipid rearrangement.     

Phospholipid binding of annexin V: effects of calcium and membrane phosphatidylserine content.

We studied the binding of fluorescein-labeled annexin V (placental anticoagulant protein I) to small unilamellar phospholipid vesicles at 0.15 M ionic strength as a function of calcium concentration and membrane phosphatidylserine (PS) content. As the mole percentage of PS in the membrane increased from 10 to 50%, the stoichiometry of binding decreased hyperbolically from 1100 mol phospholipid/mol annexin V to a limiting value of 84 mol/mol for measurements made at 1.2 mM CaCl2. Over the same range of PS content, Kd remained approximately constant at 0.036 +/- 0.011 nM. A similar hyperbolic decrease in stoichiometry was observed with vesicles containing 10 or 20% PS when the calcium concentration was increased from 0.4 to 10 mM. Thus, the density of membrane binding sites is strongly dependent on the membrane PS content and calcium concentration. The effect of calcium on annexin V-membrane binding is proposed to be due to the formation of phospholipid-calcium complexes, to which the protein binds, rather than to an allosteric effect of calcium on protein-phospholipid affinity.     

Following the formation of supported lipid bilayers on mica: a study combining AFM, QCM-D, and ellipsometry

Supported lipid bilayers (SLBs) are popular models of cell membranes with potential biotechnological applications and an understanding of the mechanisms of SLB formation is now emerging. Here we characterize, by combining atomic force microscopy, quartz crystal microbalance with dissipation monitoring, and ellipsometry, the formation of SLBs on mica from sonicated unilamellar vesicles using mixtures of zwitterionic, negatively and positively charged lipids. The results are compared with those we reported previously on silica. As on silica, electrostatic interactions were found to determine the pathway of lipid deposition. However, fundamental differences in the stability of surface-bound vesicles and the mobility of SLB patches were observed, and point out the determining role of the solid support in the SLB-formation process. The presence of calcium was found to have a much more pronounced influence on the lipid deposition process on mica than on silica. Our results indicate a specific calcium-mediated interaction between dioleoylphosphatidylserine molecules and mica. In addition, we show that the use of PLL-g-PEG modified tips considerably improves the AFM imaging of surface-bound vesicles and bilayer patches and evaluate the effects of the AFM tip on the apparent size and shape of these soft structures.     

Binding of 1alpha,25-dihydroxyvitamin D(3) to annexin II: effect of vitamin D metabolites and calcium

We have recently reported that annexin II serves as a membrane receptor for 1alpha,25-(OH)(2)D(3) and mediates the rapid effect of the hormone on intracellular calcium. The purpose of these studies was to characterize the binding of the hormone to annexin II, determine the specificity of binding, and assess the effect of calcium on binding. The binding of [(14)C]-1alpha,25-(OH)(2)D(3) bromoacetate to purified annexin II was inhibited by 1alpha, 25-(OH)(2)D(3) in a concentration-dependent manner. Binding of the radiolabeled ligand to annexin II was markedly diminished by 1alpha, 25-(OH)(2)D(3) at 24 microM, 18 microM, and 12 microM and blunted by 6 microM and 3 microM. At a concentration of 12 microM, 1beta, 25-(OH)(2)D(3) also diminished the binding of [(14)C]-1alpha, 25-(OH)(2)D(3) bromoacetate to annexin II, but cholecalciferol, 25-(OH)D(3), and 24,25-(OH)(2)D(3) did not. Saturation analyses of the binding of [(3)H]-1alpha,25-(OH)(2)D(3) to purified annexin II showed a K(D) of 5.5 x 10(-9) M, whereas [(3)H]-1beta,25-(OH)(2)D(3) exhibited a K(D) of 6.0 x 10(-9) M. Calcium, which binds to the carboxy terminal domain of annexin II, had a concentration-dependent effect on [(14)C]-1alpha,25-(OH)(2)D(3) bromoacetate binding to annexin II, with 600 nM calcium being able to inhibit binding of the radiolabeled analog. The inhibitory effect of calcium was prevented by EDTA. Homocysteine, which binds to the amino terminal domain of annexin II, had no effect on the binding of the bromoacetate analog to the protein. The data indicate that 1alpha,25-(OH)(2)D(3) binding to annexin II is specific and suggest that the binding site may be located on the carboxy terminal domain of the protein. The ability of 1beta,25-(OH)(2)D(3) to inhibit the binding of [(14)C]-1alpha, 25(OH)(2)D(3) bromoacetate to annexin II provides a biochemical explanation for the ability of the 1beta-epimer to inhibit the rapid actions of the hormone in vitro.     

Phosphatidylserine membrane domain clustering induced by annexin A2/S100A10 heterotetramer

By means of scanning force and fluorescence microscopy of artificial membranes immobilized on mica surfaces, the lateral organization of the annexin A2/S100A10 heterotetramer (annexin A2t) and its influence on the lateral organization of the lipids within the membrane have been elucidated. Planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) were prepared on atomically flat mica surfaces by the spreading of unilamellar vesicles. Fluorescence images of fluorescently labeled annexin A2t and scanning force microscopy images of nonlabeled protein bound to POPC/POPS bilayers show the formation of micrometer-sized lateral protein domains in the presence of 1 mM CaCl2. By means of scanning force microscopy, not only protein domains became discernible but also small membrane domains, which were attributed to POPS-enriched areas. A depletion of these POPS domains was observed in the vicinity of annexin A2t protein domains. These results indicate that annexin A2t is a peripheral membrane-binding complex capable of inducing lipid segregation.     

Key role of the N‐terminus of chicken annexin A5 in vesicle aggregation

Annexins are calcium‐dependent phospholipid‐binding proteins involved in calcium signaling and intracellular membrane trafficking among other functions. Vesicle aggregation is a crucial event to make possible the membrane remodeling but this process is energetically unfavorable, and phospholipid membranes do not aggregate and fuse spontaneously. This issue can be circumvented by the presence of different agents such as divalent cations and/or proteins, among them some annexins. Although human annexin A5 lacks the ability to aggregate vesicles, here we demonstrate that its highly similar chicken ortholog induces aggregation of vesicles containing acidic phospholipids even at low protein and/or calcium concentration by establishment of protein dimers. Our experiments show that the ability to aggregate vesicles mainly resides in the N‐terminus as truncation of the N‐terminus of chicken annexin A5 significantly decreases this process and replacement of the N‐terminus of human annexin A5 by that of chicken switches on aggregation; in both cases, there are no changes in the overall protein structure and only minor changes in phospholipid binding. Electrostatic repulsions between negatively charged residues in the concave face of the molecule, mainly in the N‐terminus, seem to be responsible for the impairment of dimer formation in human annexin A5. Taking into account that chicken annexin A5 presents a high sequence and structural similarity with mammalian annexins absent in birds, as annexins A3 and A4, some of the physiological functions exerted by these proteins may be carried out by chicken annexin A5, even those that could require calcium‐dependent membrane aggregation.     

S100-annexin complexes--structural insights

Annexins and S100 proteins represent two large, but distinct, calcium-binding protein families. Annexins are made up of a highly alpha-helical core domain that binds calcium ions, allowing them to interact with phospholipid membranes. Furthermore, some annexins, such as annexins A1 and A2, contain an N-terminal region that is expelled from the core domain on calcium binding. These events allow for the interaction of the annexin N-terminus with target proteins, such as S100. In addition, when an S100 protein binds calcium ions, it undergoes a structural reorientation of its helices, exposing a hydrophobic patch capable of interacting with its targets, including the N-terminal sequences of annexins. Structural studies of the complexes between members of these two families have revealed valuable details regarding the mechanisms of the interactions, including the binding surfaces and conformation of the annexin N-terminus. However, other S100-annexin interactions, such as those between S100A11 and annexin A6, or between dicalcin and annexins A1, A2 and A5, appear to be more complicated, involving the annexin core region, perhaps in concert with the N-terminus. The diversity of these interactions indicates that multiple forms of recognition exist between S100 proteins and annexins. S100-annexin interactions have been suggested to play a role in membrane fusion events by the bridging together of two annexin proteins, bound to phospholipid membranes, by an S100 protein. The structures and differential interactions of S100-annexin complexes may indicate that this process has several possible modes of protein-protein recognition.     

Pathways of lipid vesicle deposition on solid surfaces: a combined QCM-D and AFM study

Supported lipid bilayers (SLBs) are popular models of cell membranes with potential biotechnological applications, yet the mechanism of SLB formation is only partially understood. In this study, the adsorption and subsequent conformational changes of sonicated unilamellar vesicles on silica supports were investigated by quartz crystal microbalance with dissipation monitoring and atomic force microscopy, using mixtures of zwitterionic, negatively charged, and positively charged lipids, both in the presence and in the absence of Ca(2+) ions. Four different pathways of vesicle deposition could be distinguished. Depending on their charge, vesicles i). did not adsorb; ii). formed a stable vesicular layer; or iii). decomposed into an SLB after adsorption at high critical coverage or iv). at low coverage. Calcium was shown to enhance the tendency of SLB formation for negatively charged and zwitterionic vesicles. The role of vesicle-support, interbilayer, and intrabilayer interactions in the formation of SLBs is discussed.     

Characterization of the growth of 2D protein crystals on a lipid monolayer by ellipsometry and rigidity measurements coupled to electron microscopy.

We present here some sensitive optical and mechanical experiments for monitoring the process of formation and growth of two-dimensional (2D) crystals of proteins on a lipid monolayer at an air-water interface. The adsorption of proteins on the lipid monolayer was monitored by ellipsometry measurements. An instrument was developed to measure the shear elastic constant (in plane rigidity) of the monolayer. These experiments have been done using cholera toxin B subunit (CTB) and annexin V as model proteins interacting with a monosialoganglioside (GM1) and dioleoylphosphatidylserine (DOPS), respectively. Electron microscopy observations of the protein-lipid layer transferred to grids were systematically used as a control. We found a good correlation between the measured in-plane rigidity of the monolayer and the presence of large crystalline domains observed by electron microscopy grids. Our interpretation of these data is that the crystallization process of proteins on a lipid monolayer passes through at least three successive stages: 1) molecular recognition between protein and lipid-ligand, i.e., adsorption of the protein on the lipid layer; 2) nucleation and growth of crystalline patches whose percolation is detected by the appearance of a non-zero in-plane rigidity; and 3) annealing of the layer producing a slower increase of the lateral or in-plane rigidity.     

Formation of irreversibly bound annexin A1 protein domains on POPC/POPS solid supported membranes

The specific interaction of annexin A1 with phospholipid bilayers is scrutinized by means of scanning force and fluorescence microscopy, quartz crystal microbalance, ellipsometry, and modeled by dynamic Monte Carlo simulations. It was found that POPC/POPS bilayers exhibit phase separation in POPC- and POPS-enriched domains as a function of Ca2+ concentration. Annexin A1 interacts with POPC/POPS bilayers by forming irreversibly bound protein domains with monolayer thickness on POPS-enriched nanodomains, while the attachment of proteins to the POPC-enriched regions is fully reversible. A thorough kinetic analysis of the process reveals that both, the binding constant of annexin A1 at the POPC-rich areas as well as the irreversible adsorption rate to the POPS-rich domains increases with calcium ion concentration. Based on the thermodynamic and kinetic data, a possible mechanism of the annexin A1 membrane interaction can be proposed.     

Regulation of the chromaffin granule aggregating activity of annexin I by phosphorylation.

Annexin I (lipocortin I) binds to secretory granule membranes and promotes their aggregation in a Ca(2+)-dependent manner [Creutz, C. E., et al. (1987) J. Biol. Chem. 262, 1860-1868; Drust, D. S., & Creutz, C. E. (1988) Nature 331, 88-91]. It is also phosphorylated on serine residues when bovine chromaffin cells are stimulated to secrete [Michener, M. L., et al. (1986) J. Biol. Chem. 261, 6548-6555], suggesting phosphorylation may be involved in modulating the function of annexin I. We report here that phosphorylation of the N-terminal tail by protein kinase C strongly inhibits the ability of annexin I to aggregate chromaffin granules by increasing the calcium requirement 4-fold. This inhibition was readily reversed when the protein was dephosphorylated by protein phosphatase 2A. The inhibition was not due to inability of phosphorylated annexin I to bind to chromaffin granules, since the phosphorylated form bound to the granule membrane at slightly lower levels of calcium than the native form. The phosphorylated annexin I also bound to 20% phosphatidylserine/80% phosphatidylcholine vesicles at lower Ca2+ levels than the native form. The inhibitory effect of phosphorylation on the granule aggregating activity of annexin I was found to be amplified by an unusual mechanism: The phosphorylated form inhibited the activity of the unphosphorylated form. The possible importance of the regulation of annexin I activity by phosphorylation in exocytosis is discussed.     

Activation of annexin 7 by protein kinase C in vitro and in vivo

Annexin 7, a Ca(2+)/GTP-activated membrane fusion protein, is preferentially phosphorylated in intact chromaffin cells, and the levels of annexin 7 phosphorylation increase quantitatively in proportion to the extent of catecholamine secretion. Consistently, various protein kinase C inhibitors proportionately reduce both secretion and phosphorylation of annexin 7 in these cells. In vitro, annexin 7 is quantitatively phosphorylated by protein kinase C to a mole ratio of 2.0, and phosphorylation is extraordinarily sensitive to variables such as pH, calcium, phospholipid, phorbol ester, and annexin 7 concentration. Phosphorylation of annexin 7 by protein kinase C significantly potentiates the ability of the protein to fuse phospholipid vesicles and lowers the half-maximal concentration of calcium needed for this fusion process. Furthermore, other protein kinases, including cAMP-dependent protein kinase, cGMP-dependent protein kinase, and protein-tyrosine kinase pp60(c-)(src), also label annexin 7 with high efficiency but do not have this effect on membrane fusion. In the case of pp60(c-)(src), we note that this kinase, if anything, modestly suppresses the membrane fusion activity of annexin 7. These results thus lead us to hypothesize that annexin 7 may be a positive mediator for protein kinase C action in the exocytotic membrane fusion reaction in chromaffin cells.     

Human annexin V binds to sulfatide: contribution to regulation of blood coagulation

Annexin V is a calcium-dependent phospholipid-binding protein that exhibits anticoagulant activity on binding to phosphatidylserine exposed on the activated surfaces of endothelial cells and platelets, inhibiting activation of factor X and prothrombin in the blood coagulation cascade. Sulfatide (galactosylceramide I(3)-sulfate), one of the glycosphingolipids of the platelet cell membrane, is thought to be involved in blood coagulation systems via activation of factor XII. In this study, we examined whether or not annexin V binds to sulfatide and affects the coagulant activity of sulfatide. Solid phase assaying of annexin V revealed that it binds specifically to sulfatide, i.e. not to galactosylceramide or gangliosides, in the presence of calcium ions. Affinity analysis by means of surface plasmon resonance showed that the K(D) of the interaction between annexin V and sulfatide is 1.2 micro M. Kinetic turbidometric assaying of plasma coagulation initiated by CaCl(2) revealed that the coagulation rate in the presence of sulfatide or phosphatidylserine was decreased by annexin V. These results suggest that annexin V regulates coagulability in the blood stream by binding not only to phosphatidylserine but also to sulfatide.     

Membrane Modulates Affinity for Calcium Ion to Create an Apparent Cooperative Binding Response by Annexin a5

Isothermal titration calorimetry was used to characterize the binding of calcium ion (Ca²⁺) and phospholipid to the peripheral membrane-binding protein annexin a5. The phospholipid was a binary mixture of a neutral and an acidic phospholipid, specifically phosphatidylcholine and phosphatidylserine in the form of large unilamellar vesicles. To stringently define the mode of binding, a global fit of data collected in the presence and absence of membrane concentrations exceeding protein saturation was performed. A partition function defined the contribution of all heat-evolving or heat-absorbing binding states. We find that annexin a5 binds Ca²⁺ in solution according to a simple independent-site model (solution-state affinity). In the presence of phosphatidylserine-containing liposomes, binding of Ca²⁺ differentiates into two classes of sites, both of which have higher affinity compared with the solution-state affinity. As in the solution-state scenario, the sites within each class were described with an independent-site model. Transitioning from a solution state with lower Ca²⁺ affinity to a membrane-associated, higher Ca²⁺ affinity state, results in cooperative binding. We discuss how weak membrane association of annexin a5 prior to Ca²⁺ influx is the basis for the cooperative response of annexin a5 toward Ca²⁺, and the role of membrane organization in this response.     

Annexin A2–dependent actin bundling promotes secretory granule docking to the plasma membrane and exocytosis

Annexin A2, a calcium-, actin-, and lipid-binding protein involved in exocytosis, mediates the formation of lipid microdomains required for the structural and spatial organization of fusion sites at the plasma membrane. To understand how annexin A2 promotes this membrane remodeling, the involvement of cortical actin filaments in lipid domain organization was investigated. 3D electron tomography showed that cortical actin bundled by annexin A2 connected docked secretory granules to the plasma membrane and contributed to the formation of GM1-enriched lipid microdomains at the exocytotic sites in chromaffin cells. When an annexin A2 mutant with impaired actin filament–bundling activity was expressed, the formation of plasma membrane lipid microdomains and the number of exocytotic events were decreased and the fusion kinetics were slower, whereas the pharmacological activation of the intrinsic actin-bundling activity of endogenous annexin A2 had the opposite effects. Thus, annexin A2–induced actin bundling is apparently essential for generating active exocytotic sites.     

Biochemical characterization of annexin B1 from Cysticercus cellulosae

Annexin B1 from Cysticercus cellulosae has recently been identified using immunological screening in an attempt to find novel antigens for vaccine development against cysticercosis. The protein possesses anticoagulant activity and carries significant therapeutic potential due to its thrombus-targeting and thrombolytic properties. We investigated the biochemical properties of annexin B1 using liposome and heparin Sepharose copelleting assays, as well as CD spectroscopy. The calcium-dependent binding to acidic phospholipid membranes is reminiscent of other mammalian annexins with a clear preference for high phosphatidylserine content. A unique property of annexin B1 is its ability to bind to liposomes with high phosphatidylserine content in the absence of calcium, which might be due to the presence of several basic residues on the convex protein surface that harbours the membrane-binding loops. Annexin B1 demonstrates lectin properties and binds to heparin Sepharose in a cooperative, calcium-dependent manner. Although this binding is reversible to a large extent, a small fraction of the protein remains bound to the glycosaminoglycan even in the presence of high concentrations of EDTA. Analogous to annexin A5, we propose a model of heparin wrapped around the protein thereby engaging in calcium-dependent and calcium-independent interactions. Although the calcium-independent heparin-binding sites identified in annexin A5 are not conserved, we hypothesize three possible sites in annexin B1. Results from CD spectroscopy and thermal denaturation indicate that, in solution, the protein binds calcium with a low affinity that leads to a slight increase in folding stability.     

Trimers, dimers of trimers, and trimers of trimers are common building blocks of annexin a5 two-dimensional crystals

Annexin A5 is a member of a family of homologous proteins sharing the ability to bind to negatively charged phospholipid membranes in a Ca(2+)-dependent manner. Annexin A5, as well as other annexins, self-assembles into two-dimensional (2D) ordered arrays upon binding to membranes, a property that has been proposed to have functional implications. Electron microscopy and atomic force microscopy experiments have revealed that annexin A5 forms two types of 2D crystals-with either p6 or p3 symmetry-that are both based on annexin trimers. In this study, we describe three other crystal forms that coexist with the p6 crystals. All crystal forms are made of the same building blocks, namely, dimers of trimers and trimers of trimers. A mechanistic model of the formation of the annexin A5 2D crystals is proposed.     

Interactions of annexins with the mu subunits of the clathrin assembly proteins

A number of biochemical and genetic studies have suggested that certain annexins play important roles in the endocytic pathway, possibly involving the generation, localization, or fusion of endocytic compartments. In a yeast two-hybrid screen for proteins that interact with the N-terminal domain of annexin A2 we identified the mu2 subunit of the clathrin assembly protein complex AP-2. The interaction depended upon two copies of a Yxx phi amino acid sequence motif (Y = tyrosine, x = variable residue, phi = bulky, hydrophobic residue) in the annexin that is also characteristic of the binding site for mu2 on the cytoplasmic domains of transmembrane receptors. The interaction between mu2 and full-length annexin A2 was demonstrated in vitro to be direct, to require calcium, and to be functional in the sense that annexin A2 was able to recruit the mu2 to immobilized lipids. Examination of other annexins and mu subunits demonstrated that annexin A2 also binds the mu1 subunit of the AP-1 complex, that annexin A6 binds mu1 and mu2, and that annexin A1 binds only mu1. We propose that annexins can "masquerade" as transmembrane receptors when they are attached to membranes in the presence of calcium and that they might therefore function to initiate calcium-regulated coated pit formation at the cell surface or on intracellular organelles.