The conserved core domains of annexins A1, A2, A5, and B12 can be divided into two groups with different Ca2+-dependent membrane-binding properties

The hallmark of the annexin super family of proteins is Ca(2+)-dependent binding to phospholipid bilayers, a property that resides in the conserved core domain of these proteins. Despite the structural similarity between the core domains, studies reported herein showed that annexins A1, A2, A5, and B12 could be divided into two groups with distinctively different Ca(2+)-dependent membrane-binding properties. The division correlates with the ability of the annexins to form Ca(2+)-dependent membrane-bound trimers. Site-directed spin-labeling and Forster resonance energy transfer experimental approaches confirmed the well-known ability of annexins A5 and B12 to form trimers, but neither method detected self-association of annexin A1 or A2 on bilayers. Studies of chimeras in which the N-terminal and core domains of annexins A2 and A5 were swapped showed that trimer formation was mediated by the core domain. The trimer-forming annexin A5 and B12 group had the following Ca(2+)-dependent membrane-binding properties: (1) high Ca(2+) stoichiometry for membrane binding ( approximately 12 mol of Ca(2+)/mol of protein); (2) binding to membranes was very exothermic (> -60 kcal/ mol of protein); and (3) binding to bilayers that were in the liquid-crystal phase but not to bilayers in the gel phase. In contrast, the nontrimer-forming annexin A1 and A2 group had the following Ca(2+)-dependent membrane-binding properties: (1) lower Ca(2+) stoichiometry for membrane binding (相似文献   

Modes of annexin-membrane interactions analyzed by employing chimeric annexin proteins

Annexin II is a member of the annexin family of Ca(2+)- and phospholipid-binding proteins which is particularly enriched on early endosomal membranes and has been implicated in participating in endocytic events. In contrast to other endosomal annexins the association of annexin II with its target membrane can occur in the absence of Ca(2+) in a manner depending on the unique N-terminal domain of the protein. However, endosome binding of annexin II does not require formation of a protein complex with the intracellular ligand S100A10 (p11) as an annexin II mutant protein (PM AnxII) incapable of interacting with p11 is still present on endosomal membranes. Fusion of the N-terminal sequence of this PM AnxII (residues 1-27) to the conserved protein core of annexin I transfers the capability of Ca(2+)-independent membrane binding to the otherwise Ca(2+)-sensitive annexin I. These results underscore the importance of the N-terminal sequence of annexin II for the Ca(2+)-independent endosome association and argue for a direct interaction of this sequence with an endosomal membrane receptor.     

X-ray structure of full-length annexin 1 and implications for membrane aggregation

Annexins comprise a multigene family of Ca2+ and phospholipid- binding proteins. They consist of a conserved C-terminal or core domain that confers Ca2+-dependent phospholipid binding and an N-terminal domain that is variable in sequence and length and responsible for the specific properties of each annexin. Crystal structures of various annexin core domains have revealed a high degree of similarity. From these and other studies it is evident that the core domain harbors the calcium-binding sites that interact with the phospholipid headgroups. However, no structure has been reported of an annexin with a complete N-terminal domain. We have now solved the crystal structure of such a full-length annexin, annexin 1. Annexin 1 is active in membrane aggregation and its refined 1.8 A structure shows an alpha-helical N-terminal domain connected to the core domain by a flexible linker. It is surprising that the two alpha-helices present in the N-terminal domain of 41 residues interact intimately with the core domain, with the amphipathic helix 2-12 of the N-terminal domain replacing helix D of repeat III of the core. In turn, helix D is unwound into a flap now partially covering the N-terminal helix. Implications for membrane aggregation will be discussed and a model of aggregation based on the structure will be presented.     

Cholesterol regulates membrane binding and aggregation by annexin 2 at submicromolar Ca(2+) concentration

Annexin 2 is a member of the annexin family which has been implicated in calcium-regulated exocytosis. This contention is largely based on Ca(2+)-dependent binding of the protein to anionic phospholipids. However, annexin 2 was shown to be associated with chromaffin granules in the presence of EGTA. A fraction of this bound annexin 2 was released by methyl-beta-cyclodextrin, a reagent which depletes cholesterol from membranes. Restoration of the cholesterol content of chromaffin granule membranes with cholesterol/methyl-beta-cyclodextrin complexes restored the Ca(2+)-independent binding of annexin 2. The binding of both, monomeric and tetrameric forms of annexin 2 was also tested on liposomes of different composition. In the absence of Ca(2+), annexin 2, especially in its tetrameric form, bound to liposomes containing phosphatidylserine, and the addition of cholesterol to these liposomes increased the binding. Consistent with this observation, liposomes containing phosphatidylserine and cholesterol were aggregated by the tetrameric form of annexin 2 at submicromolar Ca(2+) concentrations. These results indicate that the lipid composition of membranes, and especially their cholesterol content, is important in the control of the subcellular localization of annexin 2 in resting cells, at low Ca(2+) concentration. Annexin 2 might be associated with membrane domains enriched in phosphatidylserine and cholesterol.     

Cholesterol modulates the membrane binding and intracellular distribution of annexin 6

Annexins are Ca(2+)- and phospholipid-binding proteins that are widely expressed in mammalian tissues and that bind to different cellular membranes. In recent years its role in membrane traffic has emerged as one of its predominant functions, but the regulation of its intracellular distribution still remains unclear. We demonstrated that annexin 6 translocates to the late endocytic compartment in low density lipoprotein-loaded CHO cells. This prompted us to investigate whether cholesterol, one of the major constituents of low density lipoprotein, could influence the membrane binding affinity and intracellular distribution of annexin 6. Treatment of crude membranes or early and late endosomal fractions with digitonin, a cholesterol-sequestering agent, displayed a strong reduction in the binding affinity of a novel EDTA-resistant and cholesterol-sensitive pool of annexin 6 proteins. In addition, U18666A-induced accumulation of cholesterol in the late endosomal compartment resulted in a significant increase of annexin 6 in these vesicles in vivo. This translocation/recruitment correlates with an increased membrane binding affinity of GST-annexin 6 to late endosomes of U18666A-treated cells in vitro. In conclusion, the present study shows that changes in the intracellular distribution and concentration of cholesterol in different subcellular compartments participate in the reorganization of intracellular pools of Ca(2+)-dependent and -independent annexin 6.     

Characterization of a Ca(2+)-binding site in human annexin II by site-directed mutagenesis.

Annexin II, a major cytoplasmic substrate of the src tyrosine kinase, is a member of the annexin family of Ca2+/phospholipid-binding proteins. It is composed of a short N-terminal tail (30 residues) followed by four so-called annexin repeats (each 70-80 residues in length) which share sequence homologies and are thought to form (a) new type(s) of Ca(2+)-binding site(s). We have produced wild-type and site specifically mutated annexin II molecules to compare their structure and biochemistry. The recombinant wild-type annexin II displays biochemical and spectroscopical properties resembling those of the authentic protein purified from mammalian cells. In particular, it shows the Ca(2+)-induced blue shift in fluorescence emission which is typical for this annexin. Replacement of the single tryptophan in annexin II (Trp-212) by a phenylalanine abolishes the fluorescence signal and allows the unambiguous assignment of the Ca(2+)-sensitive spectroscopic properties to Trp-212. This residue is located in the third annexin repeat in a highly conserved stretch of 17 amino acids which are also found in the other repeats and known as the endonexin fold. To study the precise architecture of the Ca2+ site which must reside in close proximity to Trp-212, we changed several residues of the endonexin fold in repeat 3 by site-directed mutagenesis. An analysis of these mutants by fluorescence spectroscopy and Ca(2+)-dependent phospholipid binding reveals that Gly-206 and Thr-207 seem indispensible for a correct folding of this Ca(2+)-binding site.     

Annexin A6 is an organizer of membrane microdomains to regulate receptor localization and signalling

Annexin A6 (AnxA6) belongs to the conserved annexin protein family--a group of Ca(2+) -dependent membrane binding proteins. It is the largest of all annexin proteins and upon activation, binds to negatively charged phospholipids in the plasma membrane and endosomes. In addition, AnxA6 associates with cholesterol-rich membrane microdomains termed lipid rafts. Membrane cholesterol triggers Ca(2+) -independent translocation of AnxA6 to membranes and AnxA6 levels determine the number of caveolae, a form of specialized rafts at the cell surface. AnxA6 also has an F-actin binding domain and interacts with cytoskeleton components. Taken together, this suggests that AnxA6 has a scaffold function to link membrane microdomains with the organization of the cytoskeleton. Such a link facilitates AnxA6 to participate in plasma membrane repair and it would also impact on receptor signalling at the cell surface, growth factor, and lipoprotein receptor trafficking, Ca(2+) -channel activity and T cell activation. Hence, the regulation of cell surface receptors by AnxA6 may be facilitated by its unique structure that allows recruitment of interaction partners and simultaneously bridging specialized membrane domains with cortical actin surrounding activated receptors.     

Insight into the location and dynamics of the annexin A2 N-terminal domain during Ca(2+)-induced membrane bridging

Annexin A2 (AnxA2) is a Ca(2+)- and phospholipid-binding protein involved in many cellular regulatory processes. Like other annexins, it is constituted by two domains: a conserved core, containing the Ca(2+) binding sites, and a variable N-terminal segment, containing sites for interactions with other protein partners like S100A10 (p11). A wealth of data exists on the structure and dynamics of the core, but little is known about the N-terminal domain especially in the Ca(2+)-induced membrane-bridging process. To investigate this protein region in the monomeric AnxA2 and in the heterotetramer (AnxA2-p11)(2), the reactive Cys8 residue was specifically labelled with the fluorescent probe acrylodan and the interactions with membranes were studied by steady-state and time-resolved fluorescence. In membrane junctions formed by the (AnxA2-p11)(2) heterotetramer, the flexibility of the N-terminal domain increased as compared to the protein in solution. In "homotypic" membrane junctions formed by monomeric AnxA2, acrylodan moved to a more hydrophobic environment than in the protein in solution and the flexibility of the N-terminal domain also increased. In these junctions, this domain is probably not in close contact with the membrane surface, as suggested by the weak quenching of acrylodan observed with doxyl-PCs, but pairs of N-termini likely interact, as revealed by the excimer-forming probe pyrene-maleimide bound to Cys8. We present a model of monomeric AnxA2 N-terminal domain organization in "homotypic" bridged membranes in the presence of Ca(2+).     

Calcium-dependent interaction of annexin I with annexin II and mapping of the interaction sites

Annexins are multifunctional intracellular proteins with Ca²⁺- and phospholipid-binding properties. Their structures consist of four conserved repeat domains that form the core and a diverse N-terminal tail, from which their functional differences may arise. We searched for cellular proteins that interact with the N-terminal tail plus domain I of annexin I (ANX1) by using the yeast two-hybrid method. Screening of a HeLa cell cDNA library yielded annexin II (ANX2) cDNA. The interaction between ANX1 and ANX2 also occurred in vitro in a Ca²⁺-dependent manner. Mapping of the interaction sites revealed that interaction between domain I of ANX1 and domain IV of ANX2 was stronger than the other combinations.     

The N-terminal domain of annexin 2 serves as a secondary binding site during membrane bridging

Annexin A2 (AnxA2) is a Ca(2+)- and acidic phospholipid-binding protein involved in many cellular processes. It undergoes Ca(2+)-mediated membrane bridging at neutral pH and has been demonstrated to be involved in an H(+)-mediated mechanism leading to a novel AnxA2-membrane complex structure. We used fluorescence techniques to characterize this H(+)-dependent mechanism at the molecular level; in particular, the involvement of the AnxA2 N-terminal domain. This domain was labeled at Cys-8 either with acrylodan or pyrene-maleimide fluorescent probes. Steady-state and time-resolved fluorescence analysis for acrylodan and fluorescence quenching by doxyl-labeled phospholipids revealed direct interaction between the N-terminal domain and the membrane. The absence of pyrene excimer suggested that interactions between N termini are not involved in the H(+)-mediated mechanism. These findings differ from those previously observed for the Ca(2+)-mediated mechanism. Protein titration experiments showed that the protein concentration for half-maximal membrane aggregation was twice for Ca(2+)-mediated compared with H(+)-mediated aggregation, suggesting that AnxA2 was able to bridge membranes either as a dimer or as a monomer, respectively. An N-terminally deleted AnxA2 was 2-3 times less efficient than the wild-type protein for H(+)-mediated membrane aggregation. We propose a model of AnxA2-membrane assemblies, highlighting the different roles of the N-terminal domain in the H(+)- and Ca(2+)-mediated membrane bridging mechanisms.     

Novel organization and properties of annexin 2-membrane complexes

Annexin 2 belongs to the annexin family of proteins that bind to phospholipid membranes in a Ca(2+)-dependent manner. Here we show that, under mild acidic conditions, annexin 2 binds to and aggregates membranes containing anionic phospholipids, a fact that questions the mechanism of its interaction with membranes via Ca(2+) bridges only. The H(+) sensitivity of annexin 2-mediated aggregation is modulated by lipid composition (i.e. cholesterol content). Cryo-electron microscopy of aggregated liposomes revealed that both the monomeric and the tetrameric forms of the protein form bridges between the liposomes at acidic pH. Monomeric annexin 2 induced two different organizations of the membrane junctions. The first resembled that obtained at pH 7 in the presence of Ca(2+). For the tetramer, the arrangement was different. These bridges seemed more flexible than the Ca(2+)-mediated junctions allowing the invagination of membranes. Time-resolved fluorescence analysis at mild acidic pH and the measurement of Stokes radius revealed that the protein undergoes conformational changes similar to those induced by Ca(2+). Labeling with the lipophilic probe 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine indicated that the protein has access to the hydrophobic part of the membrane at both acidic pH in the absence of Ca(2+) and at neutral pH in the presence of Ca(2+). Models for the membrane interactions of annexin 2 at neutral pH in the presence of Ca(2+) and at acidic pH are discussed.     

A ten-residue domain (Y11-A20) in the NH2-terminus modulates membrane association of annexin A7

Annexin A7 (synexin, annexin VII) is postulated to promote membrane fusion during surfactant secretion in alveolar type II cells and catecholamine secretion in adrenal chromaffin cells. Recently, we demonstrated that the 1-29 residues in the NH(2)-terminus could, possibly by interaction with the COOH-terminus, influence the Ca(2+)-dependent membrane binding, aggregation, and fusion properties of annexin A7 (A7). In this study, we further investigated this 29-residue domain by evaluating several deletion and point mutations for membrane-associated functions of A7. In comparison to A7, the mutants lacking 1-29 residues (A7Delta(1-29)) or 1-21 residues (A7Delta(1-21)), but not those lacking 1-10 residues (A7Delta(1-10)) or 21-29 residues (A7Delta(21-29)), showed diminished membrane binding. Segmental deletion of 10-20 residues (A7Delta(10-20)) also decreased the protein binding to membranes. The Ca(2+)-dependent membrane aggregation of PLV with A7Delta(1-29) was maximally diminished but less so with A7Delta(10-20) or A7Delta(1-21) in comparison to that with A7. However, phospholipid vesicle (PVL) aggregation was unaffected with A7Delta(1-10) or A7Delta(21-29). The Ca(2+)-dependent membrane fusion of PLV was also diminished with A7Delta(10-20) and A7Delta(1-29), but not with A7Delta(1-10). Since the mode of annexin A7 association and function with biological membranes could be different, we also evaluated these proteins for functional changes with isolated lung lamellar bodies. In comparison to A7, the binding to lamellar bodies was diminished for A7Delta(1-29) and A7Delta(1-21) but not for A7Delta(1-10). The Ca(2+)-dependent fusion of isolated lamellar bodies with PLV was also diminished with A7Delta(1-29), but not with A7Delta(10-20) or A7Delta(1-21). Taken together, our studies suggest that the 10-residue domain (Y(11)-A(20)) in the NH(2)-terminus modifies the phospholipid binding and aggregation properties of annexin A7. For binding and fusion of biological membranes, the 10-29-residue domain may be required although the annexin A7 properties are primarily modulated through the Y(11)-A(20) domain.     

Allosterism in membrane binding: a common motif of the annexins?

Annexins are a family of proteins generally described as Ca(2+)-dependent for phospholipid binding. Yet, annexins have a wide variety of binding behaviors and conformational states, some of which are lipid-dependent and Ca(2+)-independent. We present a model that captures the cation and phospholipid binding behavior of the highly conserved core of the annexins. Experimental data for annexins A4 and A5, which have short N-termini, were globally modeled to gain an understanding of how the lipid-binding affinity of the conserved protein core is modulated. Analysis of the binding behavior was achieved through use of the lanthanide Tb(3+) as a Ca(2+) analogue. Binding isotherms were determined experimentally from the quenching of the intrinsic fluorescence of annexins A4 and A5 by Tb(3+) in the presence or absence of membranes. In the presence of lipid, the affinity of annexin for cation increases, and the binding isotherms change from hyperbolic to weakly sigmoidal. This behavior was modeled by isotherms derived from microscopic binding partition functions. The change from hyperbolic to sigmoidal binding occurs because of an allosteric transition from the annexin solution state to its membrane-associated state. Protein binding to lipid bilayers renders cation binding by annexins cooperative. The two annexin states denote two affinities of the protein for cation, one in the absence and another in the presence of membrane. In the framework of this model, we discuss membrane binding as well as the influence of the N-terminus in modifying the annexin cation-binding affinity by changing the probability of the protein to undergo the postulated two-state transition.     

Phosphorylation of annexin A1 by TRPM7 kinase: a switch regulating the induction of an α-helix

TRPM7 is an unusual bifunctional protein consisting of an α-kinase domain fused to a TRP ion channel. Previously, we have identified annexin A1 as a substrate for TRPM7 kinase and found that TRPM7 phosphorylates annexin A1 at Ser5 within the N-terminal α-helix. Annexin A1 is a Ca(2+)-dependent membrane binding protein, which has been implicated in membrane trafficking and reorganization. The N-terminal tail of annexin A1 can interact with either membranes or S100A11 protein, and it adopts the conformation of an amphipathic α-helix upon these interactions. Moreover, the existing evidence indicates that the formation of an α-helix is essential for these interactions. Here we show that phosphorylation at Ser5 prevents the N-terminal peptide of annexin A1 from adopting an α-helical conformation in the presence of membrane-mimetic micelles as well as phospholipid vesicles. We also show that phosphorylation at Ser5 dramatically weakens the binding of the peptide to S100A11. Our data suggest that phosphorylation at Ser5 regulates the interaction of annexin A1 with membranes as well as S100A11 protein.     

Regulation of the sarcoplasmic reticulum Ca(2+)-release channel requires intact annexin VI.

Annexin VI has eight highly conserved repeated domains; all other annexins have four. Díaz-Mu?oz et al. (J Biol Chem 265:15894, 1990) reported that annexin VI alters the gating properties of the ryanodine-sensitive Ca(2+)-release channel isolated from sarcoplasmic reticulum. The investigate the domain structure of rat annexin VI (67 kDa calcimedin) required for this channel regulation, various proteolytic digestions were performed. In each case, protease-resistant core polypeptides were produced. Annexin VI was digested with V8 protease and two core polypeptides were purified by Ca(2+)-dependent phospholipid binding followed by HPLC. The purified fragments were shown to be derived from the N- and C-terminal halves of annexin VI, and demonstrated differential immunoreactivity with monoclonal antibodies to rat annexin VI. While both core polypeptides retained their ability to bind phospholipids in a Ca(2+)-dependent manner, they did not regulate the sarcoplasmic reticulum Ca(2+)-dependent manner, they did not regulate the sarcoplasmic reticulum Ca(2+)-release channel as did intact annexin VI.     

The lack of annexin A7 affects functions of primary astrocytes

Annexin A7 is a Ca(2+)- and phospholipid-binding protein, which is thought to function in membrane organization and Ca(2+)-dependent signaling processes. It localizes to different cellular compartments and exists in a 47- and 51-kDa isoform with the large isoform being expressed in brain, skeletal, and heart muscle. In human temporal brain annexin A7 was found exclusively in astroglial cells. As astrocytes are thought to play key roles in several processes of the brain we focused on Ca(2+)-dependent signaling processes and astrocyte proliferation. Primary astrocytes from an anxA7(-/-) mouse exhibited an increased velocity of mechanically induced astrocytic Ca(2+) waves as compared to wild type. We also observed a remarkably increased proliferation rate in cultured mutant astrocytes. A search for annexin A7 binding partners with advanced biochemical methods confirmed sorcin as the major binding protein. However, in vivo GFP-tagged annexin A7 and sorcin appeared to redistribute mainly independently from each other in wild type and in mutant astrocytes. Our results favor an involvement of annexin A7 in Ca(2+)-dependent signaling or Ca(2+) homeostasis in astrocytes.     

Identification of a domain that mediates vesicle aggregation reveals functional diversity of annexin repeats.

Annexins are structurally related proteins that bind phospholipids in a Ca2(+)-dependent manner and possess at least four conserved 70-amino acid repeat domains. The ability of certain annexins to promote contact between vesicle membranes in vitro has prompted the suggestion that these proteins regulate membrane traffic in exocytosis. We have previously found that annexins I and II promote contact between vesicles whereas annexin V does not. In order to understand the mechanism of annexin I-mediated vesicle-vesicle contact, we prepared a monoclonal antibody that specifically inhibits annexin I-mediated vesicle aggregation. We identified the domain of annexin I recognized by this monoclonal antibody by using it to screen an expression library containing random fragments of annexin I cDNA. The antibody identified a fragment encoding amino acids 41-118 (the first repeat plus 8 residues of the amino-terminal tail). We constructed a chimeric protein containing these amino acids of annexin I fused to the second, third, and fourth repeats of annexin V. Transfer of this domain conferred the ability to promote vesicle aggregation, confirming that this domain participates directly in mediating contact between vesicle membranes.     

Annexin I-mediated vesicular aggregation: mechanism and role in human neutrophils.

Whole cytosol isolated from human neutrophils was found to accelerate the Ca(2+)-dependent fusion of phospholipid vesicles with neutrophil plasma membranes as measured by several fluorescence resonance energy transfer lipid dilution assays or by the fate of an encapsulated aqueous soluble fluorophore. The Ca2+ (threshold of 2-10 microM) and protein concentration dependencies for fusion mediated by purified human neutrophil annexin I (lipocortin I), recombinant annexin I and des(1-9)annexin I showed behavior similar to that of whole cytosol. A monoclonal antibody against the N-terminal region of annexin I strongly inhibited the action of isolated annexins as well as whole cytosol, indicating that annexin I is the major activity of this type in whole neutrophil cytosol and that it functions even in this complex mixture of proteins. Residual Ca(2+)-dependent fusion activity in the absence of cytosol or annexin I was not inhibited by several antibodies against annexin I, implicating an as yet unknown protein. Kinetic analysis of liposomal fusion showed that annexin I, as in the case of synexin, accelerates aggregation of vesicles but not the actual fusion event per se. The disposition of annexin I in liposomal aggregates was studied by monitoring binding of the protein with a pyrene-phospholipid and by simultaneously monitoring vesicular aggregation by turbidity. An antibody to the N-terminus of annexin I inhibited vesicular aggregation but not binding, suggesting that initial binding of annexin I is similar to that of annexin V. A relatively small proportion of the bound annexin was involved in intervesicular linkage, and no exchange of bound annexin to subsequently added vesicles was observed. The lack of extensive contact between lipids of aggregated vesicles was supported by a lack of energy transfer between phospholipid probes on separate aggregating vesicles. Covalent linkage of maleimidyl or photoaffinity phospholipid derivatives with annexin I in vesicular aggregates did not allow complete disaggregation of vesicles by EDTA, suggesting that monomers of annexin I can contact two membranes simultaneously at the point of intervesicular linkage. These data are discussed in terms of possible models for the structure of this site.     

Annexins as intracellular calcium sensors

The annexins are a multigene family of Ca(2+)- and charged phospholipid-binding proteins. Although they have been ascribed with diverse functions, there is no consensus about the role played by this family as a whole. We have mapped the Ca(2+)-induced translocations of four members of the annexin family and of two truncated annexins in live cells, and demonstrated that these proteins interact with the plasma membrane as well as with internal membrane systems in a highly coordinated manner. Annexin 2 was the most Ca(2+) sensitive of the studied proteins, followed by annexins 6, 4 and 1. The calcium sensitivity of annexin 2 increased further following co-expression with S100A10. Upon elevation of [Ca(2+)](i), annexins 2 and 6 translocated to the plasma membrane, whereas annexins 4 and 1 also became associated with intracellular membranes and the nuclear envelope. The NH(2)-terminus had a modulatory effect on plasma membrane binding: its truncation increased the Ca(2+) sensitivity of annexin 1, and decreased that of annexin 2. Given the fact that several annexins are present within any one cell, it is likely that they form a sophisticated [Ca(2+)] sensing system, with a regulatory influence on other signaling pathways.