Multiple C-terminal motifs of the 46-kDa mannose 6-phosphate receptor tail contribute to efficient binding of medium chains of AP-2 and AP-3

The interaction of adaptor protein (AP) complexes with signal structures in the cytoplasmic domains of membrane proteins is required for intracellular sorting. Tyrosine- or dileucine-based motifs have been reported to bind to medium chain subunits (mu) of AP-1, AP-2, or AP-3. In the present study, we have examined the interaction of the entire 67-amino acid cytoplasmic domain of the 46-kDa mannose 6-phosphate receptor (MPR46-CT) containing tyrosine- as well as dileucine-based motifs with mu2 and mu3A chains using the yeast two-hybrid system. Both mu2 and mu3A bind specifically to the MPR46-CT. In contrast, mu3A fails to bind to the cytoplasmic domain of the 300-kDa mannose 6-phosphate receptor. Mutational analysis of the MPR46-CT revealed that the tyrosine-based motif and distal sequences rich in acidic amino acid residues are sufficient for effective binding to mu2. However, the dileucine motif was found to be one part of a consecutive complex C-terminal structure comprising tyrosine and dileucine motifs as well as clusters of acidic residues necessary for efficient binding of mu3A. Alanine substitution of 2 or 4 acidic amino acid residues of this cluster reduces the binding to mu3A much more than to mu2. The data suggest that the MPR46 is capable of interacting with different AP complexes using multiple partially overlapping sorting signals, which might depend on posttranslational modifications or subcellular localization of the receptor.     

Functional binding interaction identified between the axonal CAM L1 and members of the ERM family

A yeast two-hybrid library was screened using the cytoplasmic domain of the axonal cell adhesion molecule L1 to identify binding partners that may be involved in the regulation of L1 function. The intracellular domain of L1 bound to ezrin, a member of the ezrin, radixin, and moesin (ERM) family of membrane-cytoskeleton linking proteins, at a site overlapping that for AP2, a clathrin adaptor. Binding of bacterial fusion proteins confirmed this interaction. To determine whether ERM proteins interact with L1 in vivo, extracellular antibodies to L1 were used to force cluster the protein on cultured hippocampal neurons and PC12 cells, which were then immunolabeled for ERM proteins. Confocal analysis revealed a precise pattern of codistribution between ERMs and L1 clusters in axons and PC12 neurites, whereas ERMs in dendrites and spectrin labeling remained evenly distributed. Transfection of hippocampal neurons grown on an L1 substrate with a dominant negative ERM construct resulted in extensive and abnormal elaboration of membrane protrusions and an increase in axon branching, highlighting the importance of the ERM-actin interaction in axon development. Together, our data indicate that L1 binds directly to members of the ERM family and suggest this association may coordinate aspects of axonal morphogenesis.     

Recruitment of Dbl by Ezrin and Dystroglycan Drives Membrane Proximal Cdc42 Activation and Filopodia Formation

Dystroglycan is an essential laminin binding cell adhesion molecule which is also an adaptor for several SH2 domain-containing signalling molecules and as a scaffold for the ERK-MAP kinase cascade. Loss of dystroglycan function is implicated in muscular dystrophies and the aetiology of epithelial cancers. We have previously demonstrated a role for dystroglycan and ezrin in the formation of filopodia structures. Here we demonstrate the existence of a dystroglycan:ezrin:Dbl complex that is targeted to the membrane by dystroglycan where it drives local Cdc42 activation and the formation of filopodial. Deletion of an ezrin binding site in dystroglycan prevented the association with ezrin and Dbl and the formation of filopodia. Furthermore, expression of the dystroglycan cytoplasmic domain alone had a dominant-negative effect on filopodia formation and Cdc42 activation by sequestering ezrin and Dbl away from the membrane. Depletion of dystroglycan inhibited Cdc42-induced filopodia formation. For the first time we also demonstrate co-localisation of Cdc42 and dystroglycan at the tips of dynamic filopodia.     

Ca2+-dependent binding and activation of dormant ezrin by dimeric S100P

S100 proteins are EF hand type Ca2+ binding proteins thought to function in stimulus-response coupling by binding to and thereby regulating cellular targets in a Ca2+-dependent manner. To isolate such target(s) of the S100P protein we devised an affinity chromatography approach that selects for S100 protein ligands requiring the biologically active S100 dimer for interaction. Hereby we identify ezrin, a membrane/F-actin cross-linking protein, as a dimer-specific S100P ligand. S100P-ezrin complex formation is Ca2+ dependent and most likely occurs within cells because both proteins colocalize at the plasma membrane after growth factor or Ca2+ ionophore stimulation. The S100P binding site is located in the N-terminal domain of ezrin and is accessible for interaction in dormant ezrin, in which binding sites for F-actin and transmembrane proteins are masked through an association between the N- and C-terminal domains. Interestingly, S100P binding unmasks the F-actin binding site, thereby at least partially activating the ezrin molecule. This identifies S100P as a novel activator of ezrin and indicates that activation of ezrin's cross-linking function can occur directly in response to Ca2+ transients.     

Endocytic activity of HIV‐1 Vpu: Phosphoserine‐dependent interactions with clathrin adaptors

HIV‐1 Vpu modulates cellular transmembrane proteins to optimize viral replication and provide immune‐evasion, triggering ubiquitin‐mediated degradation of some targets but also modulating endosomal trafficking to deplete them from the plasma membrane. Interactions between Vpu and the heterotetrameric clathrin adaptor protein (AP) complexes AP‐1 and AP‐2 have been described, yet the molecular basis and functional roles of such interactions are incompletely defined. To investigate the trafficking signals encoded by Vpu, we fused the cytoplasmic domain (CD) of Vpu to the extracellular and transmembrane domains of the CD8 α‐chain. CD8‐VpuCD was rapidly endocytosed in a clathrin‐ and AP‐2‐dependent manner. Multiple determinants within the Vpu CD contributed to endocytic activity, including phosphoserines of the β‐TrCP binding site and a leucine‐based ExxxLV motif. Using recombinant proteins, we confirmed ExxxLV‐dependent binding of the Vpu CD to the α/σ2 subunit hemicomplex of AP‐2 and showed that this is enhanced by serine‐phosphorylation. Remarkably, the Vpu CD also bound directly to the medium (μ) subunits of AP‐2 and AP‐1; this interaction was dependent on serine‐phosphorylation of Vpu and on basic residues in the μ subunits. We propose that the flexibility with which Vpu binds AP complexes broadens the range of cellular targets that it can misdirect to the virus' advantage.      

Mutagenesis of the phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding site in the NH(2)-terminal domain of ezrin correlates with its altered cellular distribution

The cytoskeleton-membrane linker protein ezrin has been shown to associate with phosphatidyl-inositol 4,5-bisphosphate (PIP(2))-containing liposomes via its NH(2)-terminal domain. Using internal deletions and COOH-terminal truncations, determinants of PIP(2) binding were located to amino acids 12-115 and 233-310. Both regions contain a KK(X)(n)K/RK motif conserved in the ezrin/radixin/moesin family. K/N mutations of residues 253 and 254 or 262 and 263 did not affect cosedimentation of ezrin 1-333 with PIP(2)-containing liposomes, but their combination almost completely abolished the capacity for interaction. Similarly, double mutation of Lys 63, 64 to Asn only partially reduced lipid interaction, but combined with the double mutation K253N, K254N, the interaction of PIP(2) with ezrin 1-333 was strongly inhibited. Similar data were obtained with full-length ezrin. When residues 253, 254, 262, and 263 were mutated in full-length ezrin, the in vitro interaction with the cytoplasmic tail of CD44 was not impaired but was no longer PIP(2) dependent. This construct was also expressed in COS1 and A431 cells. Unlike wild-type ezrin, it was not any more localized to dorsal actin-rich structures, but redistributed to the cytoplasm without strongly affecting the actin-rich structures. We have thus identified determinants of the PIP(2) binding site in ezrin whose mutagenesis correlates with an altered cellular localization.     

Clathrin adaptor AP2 and NSF interact with overlapping sites of GluR2 and play distinct roles in AMPA receptor trafficking and hippocampal LTD

Proteins that bind to the cytoplasmic tails of AMPA receptors control receptor trafficking and thus the strength of postsynaptic responses. Here we show that AP2, a clathrin adaptor complex important for endocytosis, associates with a region of GluR2 that overlaps the NSF binding site. Peptides used previously to interfere with NSF binding also antagonize GluR2-AP2 interaction. Using GluR2 mutants and peptide variants that dissociate NSF and AP2 interaction, we find that AP2 is involved specifically in NMDA receptor-induced (but not ligand-dependent) internalization of AMPA receptors, and is essential for hippocampal long-term depression (LTD). NSF function, on the other hand, is needed to maintain synaptic AMPA receptor responses, but is not directly required for NMDA receptor-mediated internalization and LTD.     

A novel serine-rich motif in the intercellular adhesion molecule 3 is critical for its ezrin/radixin/moesin-directed subcellular targeting.

Intercellular adhesion molecule 3 (ICAM-3) is a leukocyte-specific receptor involved in primary immune responses. We have investigated the interaction between ICAM-3 and ezrin/radixin/moesin (ERM) proteins and its role in LFA-1-induced cell-cell interactions and membrane positioning of ICAM-3 in polarized migrating lymphocytes. Protein-protein binding assays demonstrated a phosphatidylinositol 4,5-bisphosphate-induced association between ICAM-3 and the amino-terminal domain of ERM proteins. This interaction was not essential for the binding of ICAM-3 to LFA-1. Dynamic fluorescence videomicroscopy studies of cells demonstrated that moesin and ICAM-3 coordinately redistribute on the plasma membrane during lymphocyte migration. Furthermore, overexpression of the amino-terminal domain of moesin, which lacks the consensus moesin actin-binding site, caused the subcellular mislocalization of ICAM-3. A CD4 chimerical protein containing the cytoplasmic tail of ICAM-3 was targeted to the trailing edge. Point mutation of Ser(487), Ser(489), and Ser(496) to alanine in the juxtamembrane region of ICAM-3 significantly impaired both ERM binding and polarization of ICAM-3. ERM-directed polarization of ICAM-3 was also impaired by phosphorylation-like mutation of Ser(487) and Ser(489), but not of Ser(496). Our results underscore the key role of specific serine residues within the cytoplasmic region of ICAM-3 for its ERM-directed positioning at the trailing edge of motile lymphocytes.     

Ezrin has a COOH-terminal actin-binding site that is conserved in the ezrin protein family

Ezrin, previously also known as cytovillin, p81, and 80K, is a cytoplasmic protein enriched in microvilli and other cell surface structures. Ezrin is postulated to have a membrane-cytoskeleton linker role. Recent findings have also revealed that the NH2-terminal domain of ezrin is associated with the plasma membrane and the COOH-terminal domain with the cytoskeleton (Algrain, M., O. Turunen, A. Vaheri, D. Louvard, and M. Arpin. 1993. J. Cell Biol. 120: 129-139). Using bacterially expressed fragments of ezrin we now demonstrate that ezrin has an actin-binding capability. We used glutathione-S-transferase fusion proteins of truncated ezrin in affinity chromatography to bind actin from the cell extract or purified rabbit muscle actin. We detected a binding site for filamentous actin that was localized to the COOH-terminal 34 amino acids of ezrin. No binding of monomeric actin was detected in the assay. The region corresponding to the COOH- terminal actin-binding site in ezrin is highly conserved in moesin, actin-capping protein radixin and EM10 protein of E. multilocularis, but not in merlin/schwannomin. Consequently, this site is a potential actin-binding site also in the other members of the protein family. Furthermore, the actin-binding site in ezrin shows sequence homology to the actin-binding site in the COOH terminus of the beta subunit of the actin-capping protein CapZ and one of the potential actin-binding sites in myosin heavy chain. The actin-binding capability of ezrin supports its proposed role as a membrane-cytoskeleton linker.     

Dynamic interaction between soluble tubulin and C-terminal domains of N-methyl-D-aspartate receptor subunits

The cytoplasmic C-terminal domains (CTs) of the NR1 and NR2 subunits of the NMDA receptor have been implicated in its anchoring to the subsynaptic cytoskeleton. Here, we used affinity chromatography with glutathione S-transferase-NR1-CT and -NR2B-CT fusion proteins to identify novel binding partner(s) of these NMDA receptor subunits. Upon incubation with rat brain cytosolic protein fraction, both NR1-CT and NR2B-CT, but not glutathione S-transferase, specifically bound tubulin. The respective fusion proteins also bound tubulin purified from brain, suggesting a direct interaction between the two binding partners. In tubulin polymerization assays, NR1-CT and NR2B-CT significantly decreased the rate of microtubule formation without destabilizing preformed microtubules. Moreover, only minor fractions of either fusion protein coprecipitated with the newly formed microtubules. Consistent with these findings, ultrastructural analysis of the newly formed microtubules revealed a limited association only with the CTs of the NR1 and NR2B. These data suggest a direct interaction of the NMDA receptor channel subunit CTs and tubulin dimers or soluble forms of tubulin. The efficient modulation of microtubule dynamics by the NR1 and NR2 cytoplasmic domains suggests a functional interaction of the receptor and the subsynaptic cytoskeletal network that may play a role during morphological adaptations, as observed during synaptogenesis and in adult CNS plasticity.     

Ezrin directly interacts with the alpha1b-adrenergic receptor and plays a role in receptor recycling

Using the yeast two-hybrid system, we identified ezrin as a protein interacting with the C-tail of the alpha1b-adrenergic receptor (AR). The interaction was shown to occur in vitro between the receptor C-tail and the N-terminal portion of ezrin, or Four-point-one ERM (FERM) domain. The alpha1b-AR/ezrin interaction occurred inside the cells as shown by the finding that the transfected alpha1b-AR and FERM domain or ezrin could be coimmunoprecipitated from human embryonic kidney 293 cell extracts. Mutational analysis of the alpha1b-AR revealed that the binding site for ezrin involves a stretch of at least four arginines on the receptor C-tail. The results from both receptor biotinylation and immunofluorescence experiments indicated that the FERM domain impaired alpha1b-AR recycling to the plasma membrane without affecting receptor internalization. The dominant negative effect of the FERM domain, which relies on its ability to mask the ezrin binding site for actin, was mimicked by treatment of cells with cytochalasin D, an actin depolymerizing agent. A receptor mutant (DeltaR8) lacking its binding site in the C-tail for ezrin displayed delayed receptor recycling. These findings identify ezrin as a new protein directly interacting with a G protein-coupled receptor and demonstrate the direct implication of ezrin in GPCR trafficking via an actin-dependent mechanism.     

Ezrin controls the macromolecular complexes formed between an adapter protein Na+/H+ exchanger regulatory factor and the cystic fibrosis transmembrane conductance regulator

Na(+)/H(+) exchanger regulatory factor (NHERF) is an adapter protein that is responsible for organizing a number of cell receptors and channels. NHERF contains two amino-terminal PDZ (postsynaptic density 95/disk-large/zonula occluden-1) domains that bind to the cytoplasmic domains of a number of membrane channels or receptors. The carboxyl terminus of NHERF interacts with the FERM domain (a domain shared by protein 4.1, ezrin, radixin, and moesin) of a family of actin-binding proteins, ezrin-radixin-moesin. NHERF was shown previously to be capable of enhancing the channel activities of cystic fibrosis transmembrane conductance regulator (CFTR). Here we show that binding of the FERM domain of ezrin to NHERF regulates the cooperative binding of NHERF to bring two cytoplasmic tails of CFTR into spatial proximity to each other. We find that ezrin binding activates the second PDZ domain of NHERF to interact with the cytoplasmic tails of CFTR (C-CFTR), so as to form a specific 2:1:1 (C-CFTR)(2).NHERF.ezrin ternary complex. Without ezrin binding, the cytoplasmic tail of CFTR only interacts strongly with the first amino-terminal PDZ domain to form a 1:1 C-CFTR.NHERF complex. Immunoprecipitation and immunoblotting confirm the specific interactions of NHERF with the full-length CFTR and with ezrin in vivo. Because of the concentrated distribution of ezrin and NHERF in the apical membrane regions of epithelial cells and the diverse binding partners for the NHERF PDZ domains, the regulation of NHERF by ezrin may be employed as a general mechanism to assemble channels and receptors in the membrane cytoskeleton.     

DCC regulates cell adhesion in human colon cancer derived HT-29 cells and associates with ezrin

The deleted in colorectal cancer (DCC) gene encodes a 170- to 190-kDa protein of the Immunoglobulin superfamily. Firstly identified as a tumor suppressor gene in human colorectal carcinomas, the main function for DCC has been described in the nervous system as part of a receptor complex for netrin-1. Moreover, roles in mucosecretory cell differentiation and as inducer of apoptosis have also been reported. DCC knockout mice supported a crucial role for this gene in axonal migration, yet questioned its implication in tumor suppression and mucosecretory differentiation. The work presented here demonstrates that a DCC-transfected HT-29 colonic human cell line (HT-29/DCC) displays an increase in cell-cell adhesion to the detriment of cell-matrix interactions: HT-29/DCC cells exhibit more and better-structured desmosomes while focal adhesions and hemidesmosomes are disrupted. HT-29/DCC cells show no changes in adherent junctions but upon treatment with TPA, HT-29/DCC cells show resistance to scattering, and maintain E-cadherin in the membrane. In addition, the actin cytoskeleton is affected in HT-29/DCC cells: stress fibers are disrupted while cortical actin remains intact. We identified a putative ERM-M (ezrin/radixin/moesin and merlin) binding domain in the juxtamembrane region of the DCC protein. In vitro pull-down assays demonstrate the interaction of the DCC cytoplasmic domain with the N-terminal region of ezrin and merlin, and co-immunoprecipitation assays in transiently DCC-transfected COS-1 cells showed that the interaction between DCC and ezrin also takes place in vivo. Altogether, our results suggest that DCC could regulate cell adhesion and migration through its association with ERM-M proteins.     

Mechanism of interaction between leucine-based sorting signals from the invariant chain and clathrin-associated adaptor protein complexes AP1 and AP2

The cytoplasmic tail of the invariant chain contains two leucine-based sorting signals, and each of those seems sufficient to route the invariant chain to its intracellular destination in either normal or polarized cells. It is believed that the intracellular routing of the invariant chain is mediated by its interactions with the clathrin-associated adaptor protein complexes AP1 and AP2. We () have previously demonstrated the in vitro interactions between the cytoplasmic tail of the invariant chain and AP1/AP2 complexes. These interactions were specific and depended on the critical leucine residues in the invariant chain's sorting signals. In the present study, we decided to investigate the molecular mechanism of these interactions. To this end, we constructed a set of glutathione S-transferase fusion proteins that contained the intact cytoplasmic tail of the invariant chain and its various mutants to define residues important for its interactions with AP1 and AP-2. Our results demonstrated the importance of several residues other than the critical leucine residues for such interactions. A strong correlation between in vitro binding of AP2 to the invariant chain and in vivo internalization of the invariant chain was observed, confirming the primary role of AP2 in recognition of endocytic signals. In addition, we demonstrated different requirements for AP1 and AP2 binding to cytoplasmic tail of the invariant chain, which may reflect that the different sorting pathways mediated by AP1 and AP2 involve their recognition of the primary structure of the sorting signal.     

RNA aptamers selectively modulate protein recruitment to the cytoplasmic domain of beta-secretase BACE1 in vitro

The beta-amyloid peptide (Abeta) is a major component of the Alzheimer's disease (AD)-associated senile plaques and is generated by sequential cleavage of the beta-amyloid precursor protein (APP) by beta-secretase and gamma-secretase. Since BACE1 initiates Abeta generation it represents a valuable target to interfere with Abeta production and treatment of AD. While the enzymatic activity of BACE1 resides in the extracellular domain, the protein also contains a short cytoplasmic tail (B1-CT). This domain serves as a binding site for at least two proteins, the copper chaperone for superoxide dismutase-1 (CCS), and the Golgi-localized, gamma-ear-containing, ADP ribosylation factor-binding (GGA1) protein, and contains a single phosphorylation site. However, the precise role of the B1-CT for the overall biological function of this protein is largely unknown. Functional studies focusing on the activity of this domain would strongly benefit from the availability of domain-specific inhibitors. Here we describe the isolation and characterization of RNA aptamers that selectively target the B1-CT. We show that these RNAs bind to authentic BACE1 and provide evidence that the binding site is restricted to the membrane-proximal half of the C terminus. Aptamer-binding specifically interferes with the recruitment of CCS, but still permits GGA1 association and casein kinase-dependent phosphorylation, consistent with selective binding site targeting within this short peptide. Because phosphorylation and GGA1 binding to B1-CT regulate BACE1 transport, these RNA inhibitors could be applied to investigate B1-CT activity without affecting the subcellular localization of BACE1.     

Ezrin interacts with focal adhesion kinase and induces its activation independently of cell-matrix adhesion

Ezrin, a membrane-cytoskeleton linker, is required for cell morphogenesis, motility, and survival through molecular mechanisms that remain to be elucidated. Using the N-terminal domain of ezrin as a bait, we found that p125 focal adhesion kinase (FAK) interacts with ezrin. We show that the two proteins coimmunoprecipitate from cultured cell lysates. However, FAK does not interact with full-length ezrin in vitro, indicating that the FAK binding site on ezrin is cryptic. Mapping experiments showed that the entire N-terminal domain of FAK (amino acids 1-376) is required for optimal ezrin binding. While investigating the role of the ezrin-FAK interaction, we observed that, in suspended kidney-derived epithelial LLC-PK1 cells, overproduction of ezrin promoted phosphorylation of FAK Tyr-397, the major autophosphorylation site, creating a docking site for FAK signaling partners. Treatment of the cells with a Src family kinase inhibitor reduced the phosphorylation of Tyr-577 but not that of Tyr-397, indicating that ezrin-mediated FAK activation does not require the activity of Src kinases. Altogether, these observations indicate that ezrin is able to trigger FAK activation in signaling events that are not elicited by cell-matrix adhesion.     

Properties of an Ezrin Mutant Defective in F-actin Binding

Ezrin, radixin and moesin are a family of proteins that provide a link between the plasma membrane and the cortical actin cytoskeleton. The regulated targeting of ezrin to the plasma membrane and its association with cortical F-actin are more than likely functions necessary for a number of cellular processes, such as cell adhesion, motility, morphogenesis and cell signalling. The interaction with F-actin was originally mapped to the last 34 residues of ezrin, which correspond to the last three helices (αB, αC and αD) of the C-terminal tail. We set out to identify and mutate the ezrin/F-actin binding site in order to pinpoint the role of F-actin interaction in morphological processes as well as signal transduction. We report here the generation of an ezrin mutant defective in F-actin binding. We identified four actin-binding residues, T576, K577, R579 and I580, that form a contiguous patch on the surface of the last helix, αD. Interestingly, mutagenesis of R579 also eliminated the interaction of band four-point one, ezrin, radixin, moesin homology domains (FERM) and the C-terminal tail domain, identifying a hotspot of the FERM/tail interaction. In vivo expression of the ezrin mutant defective in F-actin binding and FERM/tail interaction (R579A) altered the normal cell surface structure dramatically and inhibited cell migration. Further, we showed that ezrin/F-actin binding is required for the receptor tyrosine kinase signal transfer to the Ras/MAP kinase signalling pathway. Taken together, these observations highlight the importance of ezrin/F-actin function in the development of dynamic membrane/actin structures critical for cell shape and motility, as well as signal transduction.     

Transforming growth factor-beta receptors interact with AP2 by direct binding to beta2 subunit

Transforming growth factor-beta (TGF-beta) superfamily members regulate a wide range of biological processes by binding to two transmembrane serine/threonine kinase receptors, type I and type II. We have previously shown that the internalization of these receptors is inhibited by K(+) depletion, cytosol acidification, or hypertonic medium, suggesting the involvement of clathrin-coated pits. However, the involvement of the clathrin-associated adaptor complex AP2 and the identity of the AP2 subunit that binds the receptors were not known. Herein, we have studied these issues by combining studies on intact cells with in vitro assays. Using fluorescence photobleaching recovery to measure the lateral mobility of the receptors on live cells (untreated or treated to alter their coated pit structure), we demonstrated that their mobility is restricted by interactions with coated pits. These interactions were transient and mediated through the receptors' cytoplasmic tails. To measure direct binding of the receptors to specific AP2 subunits, we used yeast two-hybrid screens and in vitro biochemical assays. In contrast to most other plasma membrane receptors that bind to AP2 via the mu2 subunit, AP2/TGF-beta receptor binding was mediated by a direct interaction between the beta2-adaptin N-terminal trunk domain and the cytoplasmic tails of the receptors; no binding was observed to the mu2, alpha, or sigma2 subunits of AP2 or to mu1 of AP1. The data uniquely demonstrate both in vivo and in vitro the ability of beta2-adaptin to directly couple TGF-beta receptors to AP2 and to clathrin-coated pits, providing the first in vivo evidence for interactions of a transmembrane receptor with beta2-adaptin.     

Quantitative analysis of the binding of ezrin to large unilamellar vesicles containing phosphatidylinositol 4,5 bisphosphate

The plasma membrane-cytoskeleton interface is a dynamic structure participating in a variety of cellular events. Among the proteins involved in the direct linkage between the cytoskeleton and the plasma membrane is the ezrin/radixin/moesin (ERM) family. The FERM (4.1 ezrin/radixin/moesin) domain in their N-terminus contains a phosphatidylinositol 4,5 bisphosphate (PIP₂) (membrane) binding site whereas their C-terminus binds actin. In this work, our aim was to quantify the interaction of ezrin with large unilamellar vesicles (LUVs) containing PIP₂. For this purpose, we produced human recombinant ezrin bearing a cysteine residue at its C-terminus for subsequent labeling with Alexa488 maleimide. The functionality of labeled ezrin was checked by comparison with that of wild-type ezrin. The affinity constant between ezrin and LUVs was determined by cosedimentation assays and fluorescence correlation spectroscopy. The affinity was found to be ∼5 μM for PIP₂-LUVs and 20-to 70-fold lower for phosphatidylserine-LUVs. These results demonstrate, as well, that the interaction between ezrin and PIP₂-LUVs is not cooperative. Finally, we found that ezrin FERM domain (area of ∼30 nm²) binding to a single PIP₂ can block access to neighboring PIP₂ molecules and thus contributes to lower the accessible PIP₂ concentration. In addition, no evidence exists for a clustering of PIP₂ induced by ezrin addition.