EWI-2 association with α-actinin regulates T cell immune synapses and HIV viral infection.

EWI motif-containing protein 2 (EWI-2) is a member of the Ig superfamily that links tetraspanin-enriched microdomains to the actin cytoskeleton. We found that EWI-2 colocalizes with CD3 and CD81 at the central supramolecular activation cluster of the T cell immune synapse. Silencing of the endogenous expression or overexpression of a cytoplasmic truncated mutant of EWI-2 in T cells increases IL-2 secretion upon Ag stimulation. Mass spectrometry experiments of pull-downs with the C-term intracellular domain of EWI-2 revealed the specific association of EWI-2 with the actin-binding protein α-actinin; this association was regulated by PIP2. α-Actinin regulates the immune synapse formation and is required for efficient T cell activation. We extended these observations to virological synapses induced by HIV and found that silencing of either EWI-2 or α-actinin-4 increased cell infectivity. Our data suggest that the EWI-2-α-actinin complex is involved in the regulation of the actin cytoskeleton at T cell immune and virological synapses, providing a link between membrane microdomains and the formation of polarized membrane structures involved in T cell recognition.

The C-terminal tail of tetraspanin protein CD9 contributes to its function and molecular organization.

Tetraspanin protein CD9 supports sperm-egg fusion, and regulates cell adhesion, motility, metastasis, proliferation and signaling. The large extracellular loop and transmembrane domains of CD9 engage in functionally important interactions with partner proteins. However, neither functional nor biochemical roles have been shown for the CD9 C-terminal tail, despite it being highly conserved throughout vertebrate species. To gain new insight into the CD9 tail, three C-terminal amino acids (Glu-Met-Val) were replaced with residues corresponding to C-terminal amino acids from tetraspanin protein CD82 (Pro-Lys-Tyr). Wild-type and mutant CD9 were then stably expressed in MOLT-4, K562, U937, RD and HT1080 cells. Whereas wild-type CD9 inhibited cell adhesion and spreading on fibronectin, mutant CD9 did not. Wild-type CD9 also promoted homotypic cell-cell aggregation and microvilli formation, whereas mutant CD9 did not. Protein interactions of wild-type and mutant CD9 were compared quantitatively using stable isotope labeling with amino acids in cell culture (SILAC) in conjunction with liquid-chromatography-tandem mass spectrometry (LC-MS/MS) technology. SILAC results showed that, despite wild-type and mutant CD9 having identical expression levels, mutant CD9 and its major transmembrane interacting partners were recovered in substantially reduced amounts from 1% Brij 96 lysates. Immunoprecipitation experiments confirmed that mutant CD9 recovery was decreased in Brij 96, but not in more stringent Triton X-100 detergent. Additionally, compared with wild-type CD9 complexes, mutant CD9 complexes were larger and more oligomerized in Brij 96 detergent, consistent with decreased Brij 96 solubility, perhaps due to more membrane domains packing more tightly together. In conclusion, multiple CD9 functions depend on its C-terminal tail, which affects the molecular organization of CD9 complexes, as manifested by their altered solubilization in Brij 96 and organization on the cell surface.

EWI-2 regulates alpha3beta1 integrin-dependent cell functions on laminin-5.

EWI-2, a cell surface immunoglobulin SF protein of unknown function, associates with tetraspanins CD9 and CD81 with high stoichiometry. Overexpression of EWI-2 in A431 epidermoid carcinoma cells did not alter cell adhesion or spreading on laminin-5, and had no effect on reaggregation of cells plated on collagen I (alpha2beta1 integrin ligand). However, on laminin-5 (alpha3beta1 integrin ligand), A431 cell reaggregation and motility functions were markedly impaired. Immunodepletion and reexpression experiments revealed that tetraspanins CD9 and CD81 physically link EWI-2 to alpha3beta1 integrin, but not to other integrins. CD81 also controlled EWI-2 maturation and cell surface localization. EWI-2 overexpression not only suppressed cell migration, but also redirected CD81 to cell filopodia and enhanced alpha3beta1-CD81 complex formation. In contrast, an EWI-2 chimeric mutant failed to suppress cell migration, redirect CD81 to filopodia, or enhance alpha3beta1-CD81 complex formation. These results show how laterally associated EWI-2 might regulate alpha3beta1 function in disease and development, and demonstrate how tetraspanin proteins can assemble multiple nontetraspanin proteins into functional complexes.

Functional domains in tetraspanin proteins.

Exciting new findings have emerged about the structure, function and biochemistry of tetraspanin proteins. Five distinct tetraspanin regions have now been delineated linking structural features to specific functions. Within the large extracellular loop of tetraspanins, there is a variable region that mediates specific interactions with other proteins, as well as a more highly conserved region that has been suggested to mediate homodimerization. Within the transmembrane region, the four tetraspanin transmembrane domains are probable sites of both intra- and inter-molecular interactions that are crucial during biosynthesis and assembly of the network of tetraspanin-linked membrane proteins known as the 'tetraspanin web'. In the intracellular juxtamembrane region, palmitoylation of cysteine residues also contributes to tetraspanin web assembly, and the C-terminal cytoplasmic tail region could provide specific functional links to cytoskeletal or signaling proteins.

Evidence for specific tetraspanin homodimers: inhibition of palmitoylation makes cysteine residues available for cross-linking.

It is a well-established fact that tetraspanin proteins, a large family of integral membrane proteins involved in cell motility, fusion and signalling, associate extensively with one another and with other transmembrane and membrane-proximal proteins. In this study, we present results strongly suggesting that tetraspanin homodimers are fundamental units within larger tetraspanin complexes. Evidence for constitutive CD9 homodimers was obtained using several cell lines, utilizing the following four methods: (1) spontaneous cross-linking via intermolecular disulphide bonds, (2) use of a cysteine-reactive covalent cross-linking agent, (3) use of an amino-reactive covalent cross-linking agent, and (4) covalent cross-linking via direct intermolecular disulphide bridging between unpalmitoylated membrane-proximal cysteine residues. In the last case, incubation of cells with the palmitoylation inhibitor 2-bromopalmitate exposed membrane-proximal cysteine residues, thus effectively promoting 'zero-length' cross-linking to stabilize homodimers. Similar to CD9, other tetraspanins (CD81 and CD151) also showed a tendency to homodimerize. Tetraspanin homodimers were assembled from newly synthesized proteins in the Golgi, as evidenced by cycloheximide and Brefeldin A inhibition studies. Importantly, tetraspanin homodimers appeared on the cell surface and participated in typical 'tetraspanin web' interactions with other proteins. Whereas homodimers were the predominant cross-linked species, we also observed some higher-order complexes (trimers, tetramers or higher) and a much lower level of cross-linking between different tetraspanins (CD81-CD9, CD9-CD151, CD81-CD151). In conclusion, our results strongly suggest that tetraspanin homodimers, formed in the Golgi and present at the cell surface, serve as building blocks for the assembly of larger, multicomponent tetraspanin protein complexes.

Glioblastoma inhibition by cell surface immunoglobulin protein EWI-2, in vitro and in vivo.

EWI-2, a cell surface IgSF protein, is highly expressed in normal human brain but is considerably diminished in glioblastoma tumors and cell lines. Moreover, loss of EWI-2 expression correlated with a shorter survival time in human glioma patients, suggesting that EWI-2 might be a natural inhibitor of glioblastoma. In support of this idea, EWI-2 expression significantly impaired both ectopic and orthotopic tumor growth in nude mice in vivo. In vitro assays provided clues regarding EWI-2 functions. Expression of EWI-2 in T98G and/or U87-MG malignant glioblastoma cell lines failed to alter two-dimensional cell proliferation but inhibited glioblastoma colony formation in soft agar and caused diminished cell motility and invasion. At the biochemical level, EWI-2 markedly affects the organization of four molecules (tetraspanin proteins CD9 and CD81 and matrix metalloproteinases MMP-2 and MT1-MMP), which play key roles in the biology of astrocytes and gliomas. EWI-2 causes CD9 and CD81 to become more associated with each other, whereas CD81 and other tetraspanins become less associated with MMP-2 and MT1-MMP. We propose that EWI-2 inhibition of glioblastoma growth in vivo is at least partly explained by the capability of EWI-2 to inhibit growth and/or invasion in vitro. Underlying these functional effects, EWI-2 causes a substantial molecular reorganization of multiple molecules (CD81, CD9, MMP-2, and MT1-MMP) known to affect proliferation and/or invasion of astrocytes and/or glioblastomas.

Contrasting effects of EWI proteins, integrins, and protein palmitoylation on cell surface CD9 organization.

CD9, a tetraspanin protein, makes crucial contributions to sperm egg fusion, other cellular fusions, epidermal growth factor receptor signaling, cell motility, and tumor suppression. Here we characterize a low affinity anti-CD9 antibody, C9BB, which binds preferentially to homoclustered CD9. Using mAb C9BB as a tool, we show that cell surface CD9 homoclustering is promoted by expression of alpha3beta1 and alpha6beta4 integrins and by palmitoylation of the CD9 and beta4 proteins. Conversely, CD9 is shifted toward heteroclusters upon expression of CD9 partner proteins (EWI-2 and EWI-F) or other tetraspanins, or upon ablation of CD9 palmitoylation. Furthermore, unpalmitoylated CD9 showed enhanced EWI-2 association, thereby demonstrating a previously unappreciated role for tetraspanin palmitoylation, and underscoring how depalmitoylation and EWI-2 association may collaborate to shift CD9 from homo- to heteroclusters. In conclusion, we have used a novel molecular probe (mAb C9BB) to demonstrate the existence of multiple types of CD9 complex on the cell surface. A shift from homo- to heteroclustered CD9 may be functionally significant because the latter was especially obvious on malignant epithelial tumor cells. Hence, because of its specialized properties, C9BB may be more useful than other anti-CD9 antibodies for monitoring CD9 during tumor progression.

EWI-2 modulates lymphocyte integrin alpha4beta1 functions.

The most prominent cell-surface integrin alpha4beta1 partner, a 70-kDa protein, was isolated from MOLT-4 T leukemia cells, using anti-alpha4beta1 integrin antibody-coated beads. By mass spectrometry, this protein was identified as EWI-2, a previously described cell-surface partner for tetraspanin proteins CD9 and CD81. Wild-type EWI-2 overexpression had no effect on MOLT-4 cell tethering and adhesion strengthening on the alpha4beta1 ligand, vascular cell adhesion molecule-1 (VCAM-1), in shear flow assays. However, EWI-2 markedly impaired spreading and ruffling on VCAM-1. In contrast, a mutant EWI-2 molecule, with a different cytoplasmic tail, neither impaired cell spreading nor associated with alpha4beta1 and CD81. The endogenous wild-type EWI-2-CD81-alpha4beta1 complex was fully soluble, and highly specific as seen by the absence of other MOLT-4 cell-surface proteins. Also, it was relatively small in size (0.5 x 10(6) Da to 4 x 10(6) Da), as estimated by size exclusion chromatography. Overexpression of EWI-2 in MOLT-4 cells caused reorganization of cell-surface CD81, increased the extent of CD81-CD81, CD81-alpha4beta1, and alpha4beta1-alpha4beta1 associations, and increased the apparent size of CD81-alpha4beta1 complexes. We suggest that EWI-2-dependent reorganization of alpha4beta1-CD81 complexes on the cell surface is responsible for EWI-2 effects on integrin-dependent morphology and motility functions.