Mutation of a critical arginine in the GTP-binding site of transglutaminase 2 disinhibits intracellular cross-linking activity

Transglutaminase type 2 (TG2; also known as G(h)) is a multifunctional protein involved in diverse cellular processes. It has two well characterized enzyme activities: receptor-stimulated signaling that requires GTP binding and calcium-activated transamidation or cross-linking that is inhibited by GTP. In addition to the GDP binding residues identified from the human TG2 crystal structure (Liu, S., Cerione, R. A., and Clardy, J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 2743-2747), we have previously implicated Ser171 in GTP binding, as binding is lost with glutamate substitution (Iismaa, S. E., Wu, M.-J., Nanda, N., Church, W. B., and Graham, R. M. (2000) J. Biol. Chem. 275, 18259-18265). Here, we have shown that alanine substitution of homologous residues in rat TG2 (Phe174 in the core domain or Arg476, Arg478, or Arg579 in barrel 1) does not affect TG activity but reduces or abolishes GTP binding and GTPgammaS inhibition of TG activity in vitro, indicating that these residues are important in GTP binding. Alanine substitution of Ser171 does not impair GTP binding, indicating this residue does not interact directly with GTP. Arg579 is particularly important for GTP binding, as isothermal titration calorimetry demonstrated a 100-fold reduction in GTP binding affinity by the R579A mutant. Unlike wild-type TG2 or its S171E or F174A mutants, which are sensitive to both trypsin and mu-calpain digestion, R579A is inherently more resistant to mu-calpain, but not trypsin, digestion, indicating reduced accessibility and/or flexibility of this mutant in the region of the calpain cleavage site(s). Basal TG activity of intact R579A stable SH-SY5Y neuroblastoma cell transfectants was slightly increased relative to wild-type transfectants and, in contrast to the TG activity of the latter, was further stimulated by muscarinic receptor-activated calcium mobilization. Thus, loss of GTP binding sensitizes TG2 to intracellular calcium concentrations. These findings are consistent with the notion that intracellularly, under physiological conditions, TG2 is maintained largely as a latent enzyme, its calcium-activated cross-linking activity being suppressed allosterically by guanine nucleotide binding.     

The annexin A3-membrane interaction is modulated by an N-terminal tryptophan

The crystal structure of annexin A3 (human annexin III) solved recently revealed a well-ordered folding of its N-terminus with the side chain of tryptophan 5 interacting with residues at the extremity of the central pore. Since the pore of annexins has been suggested as the ion pathway involved in membrane permeabilization by these proteins, we investigated the effect of the N-terminal tryptophan on the channel activity of annexin A3 by a comparative study of the wild-type and the W5A mutant in structural and functional aspects. Calcium influx and patch-clamp recordings revealed that the mutant exhibited an enhanced membrane permeabilization activity as compared to the wild-type protein. Analysis of the phospholipid binding behavior of wild-type and mutant protein was carried out by cosedimentation with lipids and inhibition of PLA(2) activity. Both methods reveal a much stronger binding of the mutant to phospholipids. The structure is very similar for the wild-type and the mutant protein. The exchange of the tryptophan for an alanine results in a disordered N-terminal segment. Urea-induced denaturation of the wild-type and mutant monitored by intrinsic fluorescence indicates a separate unfolding of the N-terminal region which occurs at lower urea concentrations than unfolding of the protein core. We therefore conclude that the N-terminal domain of annexin A3, and especially tryptophan 5, is involved in the modulation of membrane binding and permeabilization by annexin A3.     

Human annexin A6 interacts with influenza a virus protein M2 and negatively modulates infection

The influenza A virus M2 ion channel protein has the longest cytoplasmic tail (CT) among the three viral envelope proteins and is well conserved between different viral strains. It is accessible to the host cellular machinery after fusion with the endosomal membrane and during the trafficking, assembly, and budding processes. We hypothesized that identification of host cellular interactants of M2 CT could help us to better understand the molecular mechanisms regulating the M2-dependent stages of the virus life cycle. Using yeast two-hybrid screening with M2 CT as bait, a novel interaction with the human annexin A6 (AnxA6) protein was identified, and their physical interaction was confirmed by coimmunoprecipitation assay and a colocalization study of virus-infected human cells. We found that small interfering RNA (siRNA)-mediated knockdown of AnxA6 expression significantly increased virus production, while its overexpression could reduce the titer of virus progeny, suggesting a negative regulatory role for AnxA6 during influenza A virus infection. Further characterization revealed that AnxA6 depletion or overexpression had no effect on the early stages of the virus life cycle or on viral RNA replication but impaired the release of progeny virus, as suggested by delayed or defective budding events observed at the plasma membrane of virus-infected cells by transmission electron microscopy. Collectively, this work identifies AnxA6 as a novel cellular regulator that targets and impairs the virus budding and release stages of the influenza A virus life cycle.     

Cooperative Binding of Annexin A2 to Cholesterol- and Phosphatidylinositol-4,5-Bisphosphate-Containing Bilayers

Biological membranes are organized into dynamic microdomains that serve as sites for signal transduction and membrane trafficking. The formation and expansion of these microdomains are driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Annexin A2 (AnxA2) is a peripherally associated membrane protein that can support microdomain formation in a Ca²⁺-dependent manner and has been implicated in membrane transport processes. Here, we performed a quantitative analysis of the binding of AnxA2 to solid supported membranes containing the annexin binding lipids phosphatidylinositol-4,5-bisphosphate and phosphatidylserine in different compositions. We show that the binding is of high specificity and affinity with dissociation constants ranging between 22.1 and 32.2 nM. We also analyzed binding parameters of a heterotetrameric complex of AnxA2 with its S100A10 protein ligand and show that this complex has a higher affinity for the same membranes with K_d values of 12 to 16.4 nM. Interestingly, binding of the monomeric AnxA2 and the AnxA2-S100A10 complex are characterized by positive cooperativity. This cooperative binding is mediated by the conserved C-terminal annexin core domain of the protein and requires the presence of cholesterol. Together our results reveal for the first time, to our knowledge, that AnxA2 and its derivatives bind cooperatively to membranes containing cholesterol, phosphatidylserine, and/or phosphatidylinositol-4,5-bisphosphate, thus providing a mechanistic model for the lipid clustering activity of AnxA2.     

Annexin A6-regulator of the EGFR/Ras signalling pathway and cholesterol homeostasis

Annexin A6 (AnxA6) belongs to the highly conserved annexin protein family. Like other annexins, the function of AnxA6 is linked to its ability to bind phospholipids in a Ca²⁺-dependent manner, thereby interacting with cellular membranes in a dynamic, reversible and regulated fashion. Upon cell activation, AnxA6 is recruited to the plasma membrane, endosomes and caveolae/membrane rafts to interact with signalling proteins, the endocytic machinery and actin cytoskeleton to inhibit epidermal growth factor receptor and Ras signalling. In addition, AnxA6 associates with late endosomes to regulate cholesterol export leading to reduced cytoplasmic phospholipase A2 activity and caveolae formation. Accordingly, AnxA6 may function as an organizer of membrane domains (i) to create a scaffold for the formation of multifactorial signalling complexes, (ii) to regulate transient membrane–actin interactions during endocytic transport, and (iii) to modulate intracellular cholesterol homeostasis. Altogether, this will regulate critical physiological processes including proliferation, differentiation, inflammation and cell migration.     

Cooperative Binding of Annexin A2 to Cholesterol- and Phosphatidylinositol-4,5-Bisphosphate-Containing Bilayers

Biological membranes are organized into dynamic microdomains that serve as sites for signal transduction and membrane trafficking. The formation and expansion of these microdomains are driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Annexin A2 (AnxA2) is a peripherally associated membrane protein that can support microdomain formation in a Ca²⁺-dependent manner and has been implicated in membrane transport processes. Here, we performed a quantitative analysis of the binding of AnxA2 to solid supported membranes containing the annexin binding lipids phosphatidylinositol-4,5-bisphosphate and phosphatidylserine in different compositions. We show that the binding is of high specificity and affinity with dissociation constants ranging between 22.1 and 32.2 nM. We also analyzed binding parameters of a heterotetrameric complex of AnxA2 with its S100A10 protein ligand and show that this complex has a higher affinity for the same membranes with K_d values of 12 to 16.4 nM. Interestingly, binding of the monomeric AnxA2 and the AnxA2-S100A10 complex are characterized by positive cooperativity. This cooperative binding is mediated by the conserved C-terminal annexin core domain of the protein and requires the presence of cholesterol. Together our results reveal for the first time, to our knowledge, that AnxA2 and its derivatives bind cooperatively to membranes containing cholesterol, phosphatidylserine, and/or phosphatidylinositol-4,5-bisphosphate, thus providing a mechanistic model for the lipid clustering activity of AnxA2.     

Annexin XII E105K crystal structure: identification of a pH-dependent switch for mutant hexamerization

Annexins are a family of calcium- and phospholipid-binding proteins involved with numerous cellular processes including membrane fusion, ion channel activity, and heterocomplex formation with other proteins. The annexin XII (ANXB12) crystal structure presented evidence that calcium mediates the formation of a hexamer through a novel intermolecular calcium-binding site [Luecke et al. (1995) Nature 378, 512-515]. In an attempt to disrupt hexamerization, we mutated a conserved key ligand in the intermolecular calcium-binding site, Glu105, to lysine. Despite its occurrence in a new spacegroup, the 1.93 A resolution structure reveals a hexamer with the Lys105 epsilon-amino group nearly superimposable with the original intermolecular calcium position. Our analysis shows that the mutation is directly involved in stabilizing the hexamer. The local residues are reoriented to retain affinity between the two trimers via a pH-dependent switch residue, Glu76, which is now protonated, allowing it to form tandem hydrogen bonds with the backbone carbonyl and nitrogen atoms of Thr103 located across the trimer interface. The loss of the intermolecular calcium-binding site is recuperated by extensive hydrogen bonding favoring hexamer stabilization. The presence of this mutant structure provides further evidence for hexameric annexin XII, and possible in vivo roles are discussed.     

Annexin A6 is recruited into lipid rafts of Niemann–Pick type C disease fibroblasts in a Ca-dependent manner

Niemann–Pick type C (NPC) disease is characterized by excessive accumulation of cholesterol in the late endosome/lysosome compartment. Some members of the annexin family of proteins such as annexin A2 (AnxA2) and annexin A6 (AnxA6) follow the same route as cholesterol during the endocytic pathway and are found, as AnxA6, attached to the membranes of the cholesterol storage compartment in NPC disease fibroblasts. Therefore, the purpose of this work was to test the hypothesis that AnxA6 participates in the NPC-induced changes in the organization of membrane microdomains resistant to solubilization by a nonionic detergent, Triton X-100, i.e., detergent-resistant microdomains (DRMs). Using cellular fractionation, fluorescence microscopy and specific antibodies we observed that in the absence of calcium AnxA6 was found in the DRM-depleted membrane fractions isolated from NPC and control fibroblasts. In the presence of calcium, AnxA6 re-located to the fractions enriched in DRMs only in the NPC cells, suggestive of AnxA6 participation in organization of these microdomains.     

Annexin A6 is recruited into lipid rafts of Niemann-Pick type C disease fibroblasts in a Ca2+-dependent manner

Niemann-Pick type C (NPC) disease is characterized by excessive accumulation of cholesterol in the late endosome/lysosome compartment. Some members of the annexin family of proteins such as annexin A2 (AnxA2) and annexin A6 (AnxA6) follow the same route as cholesterol during the endocytic pathway and are found, as AnxA6, attached to the membranes of the cholesterol storage compartment in NPC disease fibroblasts. Therefore, the purpose of this work was to test the hypothesis that AnxA6 participates in the NPC-induced changes in the organization of membrane microdomains resistant to solubilization by a nonionic detergent, Triton X-100, i.e., detergent-resistant microdomains (DRMs). Using cellular fractionation, fluorescence microscopy and specific antibodies we observed that in the absence of calcium AnxA6 was found in the DRM-depleted membrane fractions isolated from NPC and control fibroblasts. In the presence of calcium, AnxA6 re-located to the fractions enriched in DRMs only in the NPC cells, suggestive of AnxA6 participation in organization of these microdomains.     

The activity of one soluble component of the cell-free NADPH:O2 oxidoreductase of human neutrophils depends on guanosine 5'-O-(3-thio)triphosphate

Neutrophil NADPH:O2 oxidoreductase activity, essential in the killing of bacteria by neutrophils, can be elicited in a cell-free system that requires plasma membranes, cytosol and sodium dodecyl sulfate. In addition, GTP or its nonhydrolyzable analog guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) enhances NADPH oxidase activity. We investigated the mechanism of this effect of GTP gamma S in the cell-free system. Cytosol from human neutrophils was separated in three different soluble oxidase components (SOC I, SOC II, and SOC III). Previously we (Bolscher, B. G. J. M., Van Zwieten, R., Kramer, I. J. M., Weening, R. S., Verhoeven, A. J., and Roos, D. (1989) J. Clin. Invest. 83, 757-763) reported that the cytosol contains two components which act synergistically. We now report that one component (previously labeled SOC II) contains two different components that can be separated by ion exchange chromatography. Immunoblotting with antiserum B-1 (Volpp, B. D., Nauseef, W. M., and Clark, R. A. (1988) Science 242, 1295-1297), directed against a cytosolic complex capable of activating latent membranes in the cell-free system, showed a 47-kDa protein in SOC II and a 67-kDa protein in SOC III. SOC II also contains the 47-kDa phosphoprotein, which indicates that this phosphoprotein and the protein recognized by the antiserum are identical. Low rates of NADPH-dependent O2 consumption can be elicited by SOC II and SOC III in the absence of SOC I. This activity is independent of GTP gamma S. Addition of SOC I increases this activity 3-4-fold, only when GTP gamma S is present. Plasma membranes, incubated with SOC I plus GTP gamma S and re-isolated, showed a similar 3-4-fold enhanced O2 consumption with SOC II and SOC III. The GTP gamma S effect is exerted primarily at the level of the plasma membrane. The concentration of GTP gamma S that causes a half-maximal stimulation was 0.4 mu M. It is concluded that SOC I is a functional component of the NADPH oxidase.     

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.     

Annexin A2 Mediates Apical Trafficking of Renal Na+-K+-2Cl? Cotransporter

The furosemide-sensitive Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2) is responsible for urine concentration and helps maintain systemic salt homeostasis. Its activity depends on trafficking to, and insertion into, the apical membrane, as well as on phosphorylation of conserved N-terminal serine and threonine residues. Vasopressin (AVP) signaling via PKA and other kinases activates NKCC2. Association of NKCC2 with lipid rafts facilitates its AVP-induced apical translocation and activation at the surface. Lipid raft microdomains typically serve as platforms for membrane proteins to facilitate their interactions with other proteins, but little is known about partners that interact with NKCC2. Yeast two-hybrid screening identified an interaction between NKCC2 and the cytosolic protein, annexin A2 (AnxA2). Annexins mediate lipid raft-dependent trafficking of transmembrane proteins, including the AVP-regulated water channel, aquaporin 2. Here, we demonstrate that AnxA2, which binds to phospholipids in a Ca²⁺-dependent manner and may organize microdomains, is codistributed with NKCC2 to promote its apical translocation in response to AVP stimulation and low chloride hypotonic stress. NKCC2 and AnxA2 interact in a phosphorylation-dependent manner. Phosphomimetic AnxA2 carrying a mutant phosphoacceptor (AnxA2-Y24D-GFP) enhanced surface expression and raft association of NKCC2 by 5-fold upon low chloride hypotonic stimulation, whereas AnxA2-Y24A-GFP and PKC-dependent AnxA2-S26D-GFP did not. As the AnxA2 effect involved only nonphosphorylated NKCC2, it appears to affect NKCC2 trafficking. Overexpression or knockdown experiments further supported the role of AnxA2 in the apical translocation and surface expression of NKCC2. In summary, this study identifies AnxA2 as a lipid raft-associated trafficking factor for NKCC2 and provides mechanistic insight into the regulation of this essential cotransporter.     

Annexin A2 phosphorylation mediates cell scattering and branching morphogenesis via cofilin Activation

Dynamic remodeling of the actin cytoskeleton is required for cell spreading, motility, and migration and can be regulated by tyrosine kinase activity. Phosphotyrosine proteomic screening revealed phosphorylation of the lipid-, calcium-, and actin-binding protein annexin A2 (AnxA2) at Tyr23 as a major event preceding ts-v-Src kinase-induced cell scattering. Expression of the phospho-mimicking mutant Y23E-AnxA2 itself was sufficient to induce actin reorganization and cell scattering in MDCK cells. While Y23E-AnxA2, but not Y23A-AnxA2, enhanced Src- or hepatocyte growth factor (HGF)-induced cell scattering, short hairpin RNA-mediated knockdown of AnxA2 inhibited both v-Src- and HGF-induced cell scattering. Three-dimensional branching morphogenesis was induced in wild-type-AnxA2-expressing cells only in the presence of HGF, while Y23E-AnxA2 induced HGF-independent branching morphogenesis. Knockdown of AnxA2 prevented lumen formation during cystogenesis. The Y23E-AnxA2-induced scattering was associated with dephosphorylation/activation of the actin-severing protein cofilin. Likewise, inactive S3E-cofilin and constitutively active LIM kinase, a direct upstream kinase of cofilin, inhibited Y23E-AnxA2-induced scattering. Together, our studies indicate an essential role for AnxA2 phosphorylation in regulating cofilin-dependent actin cytoskeletal dynamics in the context of cell scattering and branching morphogenesis.     

Evidence for a GTP-dependent increase in membrane permeability for calcium in NG108-15 microsomes

The effect of GTP on Ca2+ uptake and release was studied in a microsomal fraction isolated from neuroblastoma x glioma hybrid NG108-15 cells. GTP did not alter the ATP-dependent initial uptake of Ca2+ but markedly enhanced the efflux of Ca2+ from microsomes. GTP-dependent Ca2+ release requires the presence of millimolar concentration of Mg2+. The effect of GTP was not mimicked by other nucleotides and was competitively blocked by the thiophosphate analogue of GTP, GTP gamma S but not by the non-hydrolyzable nucleotide GMP-PNP. Addition of an inhibiting concentration of GTP gamma S after completion of GTP-induced calcium release did not result in a re-uptake of Ca2+, showing the irreversibility of the releasing effect of GTP. Our data are consistent with the hypothesis of Ca2+-dependent GTP-induced opening of a channel responsible for vectorial transport of Ca2+ ions from one intracellular compartment to another. A model is proposed suggesting that the GTP-binding protein is a GTP-specific diacylglycerol kinase.     

Effect of Annexin A5 on CFTR: regulated traffic or scaffolding?

Previous studies have implicated annexins in regulating ion channels and in particular annexin A5 (AnxA5) in the traffic of the cystic fibrosis transmembrane conductance regulator (CFTR). In the present study, we further investigated the role of AnxA5 in regulating CFTR function and intracellular trafficking in both Xenopus oocytes and mammalian cells. Although we could confirm the previously reported CFTR/AnnxA5 interaction, we found that in oocytes AnxA5 inhibits CFTR-mediated whole-cell membrane conductance presumably by a mechanism independent of PDZ-binding domain at the C-terminus of CFTR but protein kinase C (PKC)-dependent and results from either endocytosis activation and/or exocytosis block. In contrast, in human cells, co-expression of AnxA5 augmented CFTR whole-cell currents, an effect that was independent of CFTR PDZ-binding domain. We conclude that annexin A5 has multiple effects on CFTR, so that the net effect observed is cell system-dependent. Nevertheless, both effects observed here are consistent with the described role of annexins forming scaffolding platforms at cell membranes, thus contributing to a decrease in their dynamics. Finally, we could not confirm that AnxA5 overexpression rescues traffic/function of the most frequent disease-causing mutant F508del-CFTR, thus concluding that AnxA5 is not a promising tool for correction of the F508del-CFTR defect.     

Annexin A2 binding to endosomes and functions in endosomal transport are regulated by tyrosine 23 phosphorylation

The phospholipid-binding annexin A2 (AnxA2) is known to play a role in the regulation of membrane and actin dynamics, in particular in the endocytic pathway. The protein is present on early endosomes, where it regulates membrane traffic, including the biogenesis of multivesicular transport intermediates destined for late endosomes. AnxA2 membrane association depends on the protein N terminus and membrane cholesterol but does not involve the AnxA2 ligand p11/S100A10. However, the precise mechanisms that control AnxA2 membrane association and function are not clear. In the present study, we have investigated the role of AnxA2 N-terminal phosphorylation in controlling association to endosomal membranes and functions. We found that endosomal AnxA2 was partially tyrosine-phosphorylated and that mutation of Tyr-23 to Ala (AnxA2Y23A), but not of Ser-25 to Ala, impaired AnxA2 endosome association. We then found that the AnxA2Y23A mutant was unable to bind endosomes in vivo, whereas a phospho-mimicking AnxA2 mutant (Y23D) showed efficient endosome binding capacity. Similarly, we found that AnxA2Y23D interacted more efficiently with liposomes in vitro when compared with AnxA2Y23A. To investigate the role of Tyr-23 in vivo, AnxA2 was knocked down with small interfering RNAs, and then cells were recomplemented with RNA interference-resistant forms of the protein. Using this strategy, we could show that AnxA2Y23D, but not AnxA2Y23A, could restore early-to-late endosome transport after AnxA2 depletion. We conclude that phosphorylation of Tyr-23 is essential for proper endosomal association and function of AnxA2, perhaps because it stabilizes membrane-associated protein via a conformational change.     

Chemical rescue of histidine selectivity filter mutants of the M2 ion channel of influenza A virus

The influenza virus M2 proton-selective ion channel activity facilitates virus uncoating, a process that occurs in the acidic environment of the endosome. The M2 channel causes acidification of the interior of the virus particle, which results in viral protein-protein dissociation. The M2 protein is a homotetramer that contains in its aqueous pore a histidine residue (His-37) that acts as a selectivity filter and a tryptophan residue (Trp-41) that acts as a channel gate. Substitution of His-37 modifies M2 ion channel properties drastically. However, the results of such experiments are difficult to interpret because substitution of His-37 could cause gross structural changes to the channel pore. We described here experiments in which partial or, in some cases, full rescue of specific M2 ion channel properties of His-37 substitution mutants was achieved by addition of imidazole to the bathing medium. Chemical rescue was demonstrated for three histidine substitution mutant ion channels (M2-H37G, M2-H37S, and M2-H37T) and for two double mutants in which the Trp-41 channel gate was also mutated (H37G/W41Y and H37G/W41A). Currents of the M2-H37G mutant ion channel were inhibited by Cu(II), which has been shown to coordinate with His-37 in the wild-type channel. Chemical rescue was very specific for imidazole. Buffer molecules that were neutral when protonated (4-morpholineethanesulfonic acid and 3-morpholino-2-hydroxypropanesulfonic acid) did not rescue ion channel activity of the M2-H37G mutant ion channel, but 1-methylimidazole did provide partial rescue of function. These results were consistent with a model for proton transport through the pore of the wild-type channel in which the imidazole side chain of His-37 acted as an intermediate proton acceptor/donor group.     

The frontotemporal dementia mutation R406W blocks tau's interaction with the membrane in an annexin A2-dependent manner

Changes of the microtubule-associated protein tau are central in Alzheimer's disease (AD) and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). However, the functional consequence of the FTDP-17 tau mutation R406W, which causes a tauopathy clinically resembling AD, is not well understood. We find that the R406W mutation does not affect microtubule interaction but abolishes tau's membrane binding. Loss of binding is associated with decreased trapping at the tip of neurites and increased length fluctuations during process growth. Tandem affinity purification tag purification and mass spectrometry identify the calcium-regulated plasma membrane-binding protein annexin A2 (AnxA2) as a potential interaction partner of tau. Consistently, wild-type tau but not R406W tau interacts with AnxA2 in a heterologous yeast expression system. Sequestration of Ca(2+) or knockdown of AnxA2 abolishes the differential trapping of wild-type and R406W tau. We suggest that the pathological effect of the R406W mutation is caused by impaired membrane binding, which involves a functional interaction with AnxA2 as a membrane-cytoskeleton linker.