Tetraspanin CD151 regulates glycosylation of (alpha)3(beta)1 integrin

The tetraspanin CD151 forms a stoichiometric complex with integrin alpha3beta1 and regulates its endocytosis. We observed that down-regulation of CD151 in various epithelial cell lines changed glycosylation of alpha3beta1. In contrast, glycosylation of other transmembrane proteins, including those associated with CD151 (e.g. alpha6beta1, CD82, CD63, and emmprin/CD147) was not affected. The detailed analysis has shown that depletion of CD151 resulted in the reduction of Fucalpha1-2Gal and bisecting GlcNAc-beta(1-->4) linkage on N-glycans of the alpha3 integrin subunit. The modulatory activity of CD151 toward alpha3beta1 was specific, because stable knockdown of three other tetraspanins (i.e. CD9, CD63, and CD81) did not affect glycosylation of the integrin. Analysis of alpha3 glycosylation in CD151-depleted breast cancer cells with reconstituted expression of various CD151 mutants has shown that a direct contact with integrin is required but not sufficient for the modulatory activity of the tetraspanin toward alpha3beta1. We also found that glycosylation of CD151 is also critical; Asn(159) --> Gln mutation in the large extracellular loop did not affect interactions of CD151 with other tetraspanins or alpha3beta1 but negated its modulatory function. Changes in the glycosylation pattern of alpha3beta1 observed in CD151-depleted cells correlated with a dramatic decrease in cell migration toward laminin-332. Migration toward fibronectin or static adhesion of cells to extracellular matrix ligands was not affected. Importantly, reconstituted expression of the wild-type CD151 but not glycosylation-deficient mutant restored the migratory potential of the cells. These results demonstrate that CD151 plays an important role in post-translation modification of alpha3beta1 integrin and strongly suggest that changes in integrin glycosylation are critical for the promigratory activity of this tetraspanin.     

Interacting regions of CD81 and two of its partners, EWI-2 and EWI-2wint, and their effect on hepatitis C virus infection

CD81 is a tetraspanin protein that is involved in several essential cellular functions, as well as in the hepatitis C virus (HCV) infection. CD81 interacts with a high stoichiometry with its partner proteins EWI-2, EWI-2wint, and EWI-F. These latter proteins modify the functions of CD81 and can thereby potentially inhibit infection or modulate cell migration. Here, we characterized the cleavage of EWI-2 leading to the production of EWI-2wint, which has been shown to inhibit HCV infection. We determined the regions of EWI-2/EWI-2wint and CD81 that are important for their interaction and their functionality. More precisely, we identified a glycine zipper motif in the transmembrane domain of EWI-2/EWI-2wint that is essential for the interaction with CD81. In addition, we found that palmitoylation on two juxtamembranous cysteines in the cytosolic tail of EWI-2/EWI-2wint is required for their interaction with CD81 as well as with CD9, another tetraspanin. Thus, we have shown that palmitoylation of a tetraspanin partner protein can influence the interaction with a tetraspanin. We therefore propose that palmitoylation not only of tetraspanins, but also of their partner proteins is important in regulating the composition of complexes in tetraspanin networks. Finally, we identified the regions in CD81 that are necessary for its functionality in HCV entry and we demonstrated that EWI-2wint needs to interact with CD81 to exert its inhibitory effect on HCV infection.     

Beta-1 and beta-4 integrins, and laminin 67 kDa receptors in interaction between A431 cells and laminin isoforms

Laminins, as basal membrane glycoproteins, are able to stimulate cell adhesion and migration, and to influence gene expression. The laminin molecule has a set of bioactive sites that interact with different integrin and nonintegrin receptors, and, as a result, the reaction of the same cell type to different laminin isoforms may be different. The aim of this study was to determine the contributions of both integrins with beta 1 and beta 4 chains and 67 kDa laminin receptor in the interaction of A431 cells with two laminin isoforms: laminin-1 and laminin-2/4. The obtained data show that integrin alpha 6 beta 4 is more specific for interaction with laminin-2/4 than with laminin-1 and takes part in the stage of attachment of A431 cells to laminin. 67 kDa receptor promotes cell spreading on laminin-2/4 and inhibits cell spreading on laminin-1. An assumption was made about the complex action of receptors for interaction of A431 cells with laminins ("integrin alpha 6 beta 4--67 kDa receptors" complex).     

Characterization of integrin-tetraspanin adhesion complexes: role of tetraspanins in integrin signaling.

Tetraspanins (or proteins from the transmembrane 4 superfamily, TM4SF) form membrane complexes with integrin receptors and are implicated in integrin-mediated cell migration. Here we characterized cellular localization, structural composition, and signaling properties of alpha3beta1-TM4SF adhesion complexes. Double-immunofluorescence staining showed that various TM4SF proteins, including CD9, CD63, CD81, CD82, and CD151 are colocalized within dot-like structures that are particularly abundant at the cell periphery. Differential extraction in conjunction with chemical cross-linking indicated that the cell surface fraction of alpha3beta1-TM4SF protein complexes may not be directly linked to the cytoskeleton. However, in cells treated with cytochalasin B alpha3beta1-TM4SF protein complexes are relocated into intracellular vesicles suggesting that actin cytoskeleton plays an important role in the distribution of tetraspanins into adhesion structures. Talin and MARCKS are partially codistributed with TM4SF proteins, whereas vinculin is not detected within the tetraspanin-containing adhesion structures. Attachment of serum-starved cells to the immobilized anti-TM4SF mAbs induced dephosphorylation of focal adhesion kinase (FAK). On the other hand, clustering of tetraspanins in cells attached to collagen enhanced tyrosine phosphorylation of FAK. Furthermore, ectopic expression of CD9 in fibrosarcoma cells affected adhesion-induced tyrosine phosphorylation of FAK, that correlated with the reorganization of the cortical actin cytoskeleton. These results show that tetraspanins can modulate integrin signaling, and point to a mechanism by which TM4SF proteins regulate cell motility.     

Expression of the palmitoylation-deficient CD151 weakens the association of alpha 3 beta 1 integrin with the tetraspanin-enriched microdomains and affects integrin-dependent signaling

Transmembrane proteins of the tetraspanin superfamily are assembled in multimeric complexes on the cell surface. Spatial orientation of tetraspanins within these complexes may affect signaling functions of the associated transmembrane receptors (e.g. integrins, receptor-type tyrosine kinases). The structural determinants that control assembly of the tetraspanin complexes are unknown. We have found that various tetraspanins and the alpha(3) integrin subunit are palmitoylated. The stability and molecular composition of the palmitoylated alpha(3)beta(1)-tetraspanin complexes are not affected by adhesion. To assess the significance of palmitoylation in the function of the alpha(3)beta(1)-tetraspanin complexes we mapped the sites of palmitoylation for CD151. Mutation of six cysteines, Cys(11), Cys(15), Cys(79), Cys(80), Cys(242), and Cys(243) was necessary to completely abolish palmitoylation of CD151. The association of the palmitoylation-deficient mutant of CD151 (CD151Cys8) with CD81 and CD63 was markedly decreased, but the interaction of the alpha(3)beta(1)-CD151Cys8 complex with phosphatidylinositol 4-kinase was not affected. Ectopic expression of CD151Cys8 in Rat-1 cells impaired the interactions of the endogenous CD63 and CD81 with the alpha(3)beta(1) integrin. Although the expression of the palmitoylation-deficient CD151 does not change cell spreading on the extracellular matrix, the number of focal adhesions increased. Adhesion-induced phosphorylation of PKB/c-Akt is markedly increased in cells expressing a palmitoylation-deficient mutant, thereby providing direct evidence for the role of the tetraspanin microdomains in regulation of the integrin-dependent phosphatidylinositol 3-kinase signaling pathway. In contrast, activation of FAK and ERK1/2 were not affected by the expression of CD151Cys8. Our results demonstrate that palmitoylation of tetraspanins is critical not only for the organization of the integrin-tetraspanin microdomains but also has a specific role in modulation of adhesion-dependent signaling.     

The tetraspanin CD151 regulates cell morphology and intracellular signaling on laminin-511

The tetraspanin CD151 forms a stable complex with integrin alpha3beta1, a widely expressed laminin receptor, and is implicated in the regulation of integrin alpha3beta1-mediated cellular responses, including cell attachment, spreading and migration. However, the molecular mechanism by which CD151 regulates integrin alpha3beta1 functions remains unclear. To address this issue, we knocked down CD151 expression in A549 human lung adenocarcinoma cells by RNA interference. When plated on laminin-511 (laminin-10), the CD151-knocked-down cells showed aberrant membrane protrusions and exhibited reductions in the tyrosine phosphorylation of focal adhesion kinase, Src, p130Cas and paxillin. The formation of membrane protrusions was attenuated when the cells were either plated on surfaces coated with higher concentrations of laminin-511 or treated with the integrin beta1-activating mAb TS2/16; however, neither treatment could rescue the reduced tyrosine phosphorylation. These results indicate that CD151 knockdown weakens the integrin alpha3beta1-mediated adhesion to laminin-511 and thereby provokes an aberrant morphology, but this reduced adhesive activity is not involved in the decline of signaling events in CD151-knocked-down cells. Thus, our results suggest that CD151 regulates integrin alpha3beta1 functions in two independent aspects: potentiation of integrin alpha3beta1-mediated cell adhesion and promotion of integrin alpha3beta1-stimulated signaling events involving tyrosine phosphorylation.     

Structure-function analysis of tetraspanin CD151 reveals distinct requirements for tumor cell behaviors mediated by α3β1 versus α6β4 integrin

The basement membrane protein laminin-332 (laminin-5) mediates both stable cell adhesion and rapid cell migration and thus has the potential to either restrain or promote tumor cell metastasis. The major cellular receptors for laminin-332 are integrin α3β1, which mediates rapid tumor cell migration, and integrin α6β4, which often mediates stable cell attachment. Tetraspanin protein CD151 interacts directly with both α3β1 and α6β4 integrins and with other tetraspanins, thereby promoting α3β1 and α6β4 association with tetraspanin-enriched microdomains on the cell surface. To explore the possibility of selectively modulating tumor cell responses to laminin-332, we re-expressed a series of CD151 mutants in epidermoid carcinoma cells with near total, RNAi-mediated silencing of endogenous CD151. The interactions of CD151 with its integrin partners or its interactions with other tetraspanins were selectively disrupted by specific mutations in the CD151 large extracellular loop (EC2 domain) or in intracellular CD151 palmitoylation sites, respectively. CD151-integrin association and CD151-tetraspanin association were both important for α3β1 integrin-dependent initial adhesion and rapid migration on laminin-332. Remarkably, however, only CD151-integrin association was required for stable, α6β4 integrin-dependent cell attachment on laminin-332. In addition, we found that a QRD amino acid motif in the CD151 EC2 domain, which had been thought to be crucial for CD151-integrin interaction, is not essential for CD151-integrin association or for the ability of CD151 to promote several different integrin functions. These new data suggest potential strategies for selectively modulating migratory cell responses to laminin-332, while leaving stable cell attachment on laminin-332 intact.     

A critical role for tetraspanin CD151 in alpha3beta1 and alpha6beta4 integrin-dependent tumor cell functions on laminin-5

The basement membrane protein laminin-5 supports tumor cell adhesion and motility and is implicated at multiple steps of the metastatic cascade. Tetraspanin CD151 engages in lateral, cell surface complexes with both of the major laminin-5 receptors, integrins alpha3beta1 and alpha6beta4. To determine the role of CD151 in tumor cell responses to laminin-5, we used retroviral RNA interference to efficiently silence CD151 expression in epidermal carcinoma cells. Near total loss of CD151 had no effect on steady state cell surface expression of alpha3beta1, alpha6beta4, or other integrins with which CD151 associates. However, CD151-silenced carcinoma cells displayed markedly impaired motility on laminin-5, accompanied by unusually persistent lateral and trailing edge adhesive contacts. CD151 silencing disrupted alpha3beta1 integrin association with tetraspanin-enriched microdomains, reduced the bulk detergent extractability of alpha3beta1, and impaired alpha3beta1 internalization in cells migrating on laminin-5. Both alpha3beta1- and alpha6beta4-dependent cell adhesion to laminin-5 were also impaired in CD151-silenced cells. Reexpressing CD151 in CD151-silenced cells reversed the adhesion and motility defects. Finally, loss of CD151 also impaired migration but not adhesion on substrates other than laminin-5. These data show that CD151 plays a critical role in tumor cell responses to laminin-5 and reveal promotion of integrin recycling as a novel potential mechanism whereby CD151 regulates tumor cell migration.     

The CD9/CD81 Tetraspanin Complex and Tetraspanin CD151 Regulate α3β1 Integrin-Dependent Tumor Cell Behaviors by Overlapping but Distinct Mechanisms

Integrin α3β1 potently promotes cell motility on its ligands, laminin-332 and laminin-511, and this may help to explain why α3β1 has repeatedly been linked to breast carcinoma progression and metastasis. The pro-migratory functions of α3β1 depend strongly on lateral interactions with cell surface tetraspanin proteins. Tetraspanin CD151 interacts directly with the α3 integrin subunit and links α3β1 integrin to other tetraspanins, including CD9 and CD81. Loss of CD151 disrupts α3β1 association with other tetraspanins and impairs α3β1-dependent motility. However, the extent to which tetraspanins other than CD151 are required for specific α3β1 functions is unclear. To begin to clarify which aspects of α3β1 function require which tetraspanins, we created breast carcinoma cells depleted of both CD9 and CD81 by RNA interference. Silencing both of these closely related tetraspanins was required to uncover their contributions to α3β1 function. We then directly compared our CD9/CD81-silenced cells to CD151-silenced cells. Both CD9/CD81-silenced cells and CD151-silenced cells showed delayed α3β1-dependent cell spreading on laminin-332. Surprisingly, however, once fully spread, CD9/CD81-silenced cells, but not CD151-silenced cells, displayed impaired α3β1-dependent directed motility and altered front-rear cell morphology. Also unexpectedly, the CD9/CD81 complex, but not CD151, was required to promote α3β1 association with PKCα in breast carcinoma cells, and a PKC inhibitor mimicked aspects of the CD9/CD81-silenced cell motility defect. Our data reveal overlapping, but surprisingly distinct contributions of specific tetraspanins to α3β1 integrin function. Importantly, some of CD9/CD81''s α3β1 regulatory functions may not require CD9/CD81 to be physically linked to α3β1 by CD151.     

EWI-2 negatively regulates TGF-β signaling leading to altered melanoma growth and metastasis

In normal melanocytes, TGF-β signaling has a cytostatic effect. However, in primary melanoma cells, TGF-β-induced cytostasis is diminished, thus allowing melanoma growth. Later, a second phase of TGF-β signaling supports melanoma EMT-like changes, invasion and metastasis. In parallel with these “present-absent-present” TGF-β signaling phases, cell surface protein EWI motif-containing protein 2 (EWI-2 or IgSF8) is “absent-present-absent” in melanocytes, primary melanoma, and metastatic melanoma, respectively, suggesting that EWI-2 may serve as a negative regulator of TGF-β signaling. Using melanoma cell lines and melanoma short-term cultures, we performed RNAi and overexpression experiments and found that EWI-2 negatively regulates TGF-β signaling and its downstream events including cytostasis (in vitro and in vivo), EMT-like changes, cell migration, CD271-dependent invasion, and lung metastasis (in vivo). When EWI-2 is present, it associates with cell surface tetraspanin proteins CD9 and CD81 — molecules not previously linked to TGF-β signaling. Indeed, when associated with EWI-2, CD9 and CD81 are sequestered and have no impact on TβR2-TβR1 association or TGF-β signaling. However, when EWI-2 is knocked down, CD9 and CD81 become available to provide critical support for TβR2-TβR1 association, thus markedly elevating TGF-β signaling. Consequently, all of those TGF-β-dependent functions specifically arising due to EWI-2 depletion are reversed by blocking or depleting cell surface tetraspanin proteins CD9 or CD81. These results provide new insights into regulation of TGF-β signaling in melanoma, uncover new roles for tetraspanins CD9 and CD81, and strongly suggest that EWI-2 could serve as a favorable prognosis indicator for melanoma patients.     

Protein kinase C-dependent mobilization of the alpha6beta4 integrin from hemidesmosomes and its association with actin-rich cell protrusions drive the chemotactic migration of carcinoma cells.

We explored the hypothesis that the chemotactic migration of carcinoma cells that assemble hemidesmosomes involves the activation of a signaling pathway that releases the alpha6beta4 integrin from these stable adhesion complexes and promotes its association with F-actin in cell protrusions enabling it to function in migration. Squamous carcinoma-derived A431 cells were used because they express alpha6beta4 and migrate in response to EGF stimulation. Using function-blocking antibodies, we show that the alpha6beta4 integrin participates in EGF-stimulated chemotaxis and is required for lamellae formation on laminin-1. At concentrations of EGF that stimulate A431 chemotaxis ( approximately 1 ng/ml), the alpha6beta4 integrin is mobilized from hemidesmosomes as evidenced by indirect immunofluorescence microscopy using mAbs specific for this integrin and hemidesmosomal components and its loss from a cytokeratin fraction obtained by detergent extraction. EGF stimulation also increased the formation of lamellipodia and membrane ruffles that contained alpha6beta4 in association with F-actin. Importantly, we demonstrate that this mobilization of alpha6beta4 from hemidesmosomes and its redistribution to cell protrusions occurs by a mechanism that involves activation of protein kinase C-alpha and that it is associated with the phosphorylation of the beta4 integrin subunit on serine residues. Thus, the chemotactic migration of A431 cells on laminin-1 requires not only the formation of F-actin-rich cell protrusions that mediate alpha6beta4-dependent cell movement but also the disruption of alpha6beta4-containing hemidesmosomes by protein kinase C.     

The ADAM-integrin-tetraspanin complex in fetal and postnatal testicular cords

New insights have emerged about the expression, during testicular cord formation, of the ADAM (a disintegrin and metalloprotease) domain family of proteins that combines both cell surface adhesion and proteolytic activity; this family includes integrins alpha3beta1 and alpha6beta1 and tetraspanins, a distinct family of proteins containing four transmembrane domains, a small and a large extracellular loop, and short cytoplasmic tails. ADAM3 (cyritestin), ADAM5, ADAM6, and ADAM15 are expressed in fetal rat testes. In contrast, the expression of the ADAM1/ADAM2 pair (fertilin alpha/fertilin beta, respectively) is not detected in fetal testis. Yet the expression of ADAM1 starts immediately after birth, and is followed within 24 hr by the expression of ADAM2. Therefore, the ADAM1/ADAM2 heterodimer is visualized far in advance of the meiotic and spermiogenic phase of spermatogenesis. A similar expression pattern was observed for integrin subunits alpha3, alpha6, and beta1, as well as for tetraspanins CD9, CD81, and CD98; the latter is a single-pass integrin subunit beta1-binding protein. ADAM2, integrin subunits alpha3, alpha6, and beta1, and tetraspanin CD9 and CD81 immunoreactive sites are observed in prespermatogonia (also known as primordial germ cells or gonocytes). A model is proposed in which the ADAM-integrin-tetraspanin complex, known to constitute a network of membrane microdomains called the tetraspanin web, may be involved in the migration of prespermatogonia from the center to the periphery of the testicular cords and in the reinitiation of mitotic activity during the initial wave of spermatogenesis. A complementary model consists in the rearrangement of the tetraspanin web in prespermatogonia/spermatogonia undergoing spontaneous or Fas-induced apoptosis upon coculturing with Sertoli cells. In this model, the cellular site involved in the formation of preapoptotic bodies is devoid of tetraspanin-integrin clusters, in contrast with nonapoptotic cells, which display a diffuse circumferential distribution. In apoptotic prespermatogonia, immunoreactive clusters are restricted to sites where the attachment of prespermatogonia/spermatogonia to Sertoli cell surfaces is still preserved.     

The Ig Domain Protein CD9P-1 Down-regulates CD81 Ability to Support Plasmodium yoelii Infection

Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a malaria natural infection. The molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that CD81 is required on hepatocytes for infection by Plasmodium falciparum and Plasmodium yoelii sporozoites. CD81 belongs to the tetraspanin superfamily of transmembrane proteins. By interacting with each other and with other transmembrane proteins, tetraspanins may play a role in the lateral organization of membrane proteins. In this study, we investigated the role of the two major molecular partners of CD81 in hepatocytic cells, CD9P-1/EWI-F and EWI-2, two transmembrane proteins belonging to a novel subfamily of immunoglobulin proteins. We show that CD9P-1 silencing increases the host cell susceptibility to P. yoelii sporozoite infection, whereas EWI-2 knock-down has no effect. Conversely, overexpression of CD9P-1 but not EWI-2 partially inhibits infection. Using CD81 and CD9P-1 chimeric molecules, we demonstrate the role of transmembrane regions in CD81-CD9P-1 interactions. Importantly, a CD9P-1 chimera that no longer associates with CD81 does not affect infection. Based on these data, we conclude that CD9P-1 acts as a negative regulator of P. yoelii infection by interacting with CD81 and regulating its function.     

The interplay between integrins alphaMbeta2 and alpha5beta1 during cell migration to fibronectin

A directed migration of leukocytes through the extracellular matrix requires the regulated engagement of integrin cell adhesion receptors. The integrin alpha(M)beta(2) (CD11b/CD18, Mac-1) is progressively upregulated to high levels on migrating phagocytic leukocytes in response to inflammatory stimuli and is able to bind numerous ligands in the interstitial matrix. The role of alpha(M)beta(2) in migration of leukocytes through the extracellular matrix and its cooperation with other leukocyte integrins during migration are not understood. Using a model system consisting of cells that express different levels of alpha(M)beta(2) and an invariable level of endogenous integrin alpha(5)beta(1), we have explored a situation relevant to migrating neutrophils when alpha(M)beta(2) and alpha(5)beta(1) engage the same ligand, fibronectin. We show that fibronectin is a ligand for alpha(M)beta(2) and that both alpha(M)beta(2) and alpha(5)beta(1) on the alpha(M)beta(2)-expressing cells contribute to adhesion to fibronectin. However, migration of these cells to fibronectin is mediated by alpha(5)beta(1), whereas alpha(M)beta(2) retards migration. The decrease in migration correlates directly with the increased alpha(M)beta(2) density. Ligation of alpha(M)beta(2) with function-blocking antibodies can reverse this effect. The restorative effects of antibodies are caused by the removal of restraint imposed by the excess of alpha(M)beta(2)-fibronectin adhesive bonds. These findings indicate that alpha(M)beta(2) can increase general cell adhesiveness which results in braking of cell migration mediated by integrin alpha(5)beta(1). Because alpha(M)beta(2) binds numerous proteins in the extracellular matrix with a specificity overlapping that of the beta(1) integrins, the results suggest that alpha(M)beta(2) can affect the beta(1) integrin-mediated cell migration.     

Multiple levels of interactions within the tetraspanin web

The tetraspanin web refers to a network of molecular interactions involving tetraspanins and other molecules. Inside the tetraspanin web, small primary complexes containing only one tetraspanin and one specific partner molecule such as CD151/alpha3beta1 integrin and CD9/CD9P-1 (FPRP) can be observed under particular conditions. Here we demonstrate that when cells are lysed with Brij97, the tetraspanins CD151 and CD9 allow and/or stabilize the interaction of their partner molecules with other tetraspanins and that their two partners associate under conditions maintaining tetraspanin/tetraspanin interactions. The tetraspanins were also found to partition into a detergent-resistant membrane environment to which the integrin alpha3beta1 was relocalized upon expression of CD151.     

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.     

Effect of ganglioside and tetraspanins in microdomains on interaction of integrins with fibroblast growth factor receptor

The functional interaction ("cross-talk") of integrins with growth factor receptors has become increasingly clear as a basic mechanism in cell biology, defining cell growth, adhesion, and motility. However, no studies have addressed the microdomains in which such interaction takes place nor the effect of gangliosides and tetraspanins (TSPs) on such interaction. Growth of human embryonal WI38 fibroblasts is highly dependent on fibroblast growth factor (FGF) and its receptor (FGFR), stably associated with ganglioside GM3 and TSPs CD9 and CD81 in the ganglioside-enriched microdomain. Adhesion and motility of these cells are mediated by laminin-5 ((LN5) and fibronectin (FN) through alpha3beta1 and alpha5beta1 integrin receptors, respectively. When WI38 cells or its transformant VA13 cells were adhered to LN5 or FN, alpha3beta1 or alpha5beta1 were stimulated, giving rise to signaling to activate FGFR through tyrosine phosphorylation and inducing cell proliferation under serum-free conditions without FGF addition. Types and intensity of signaling during the time course differed significantly depending on the type of integrin stimulated (alpha3beta1 versus alpha5beta1), and on cell type (WI38 versus VA13). Such effect of cross-talk between integrins and FGFR was influenced strongly by the change of GM3 and TSPs. (i) GM3 depletion by P4 caused enhanced tyrosine phosphorylation of FGFR and Akt followed by MAPK activation, without significant change of ceramide level. GM3 depletion also caused enhanced co-immunoprecipitation of FGFR with alpha3/alpha5/beta1 and of these integrins with CD9/CD81. (ii) LN5- or FN-dependent proliferation of both WI38 and VA13 was strongly enhanced by GM3 depletion and by CD9/CD81 knockdown by siRNA. Thus, integrin-FGFR cross-talk is strongly influenced by GM3 and/or TSPs within the ganglioside-enriched microdomain.     

Analysis of the CD151-alpha3beta1 integrin and CD151-tetraspanin interactions by mutagenesis

Transmembrane proteins of the tetraspanin superfamily are associated with various integrins and modulate their function. We performed mutagenesis analysis to establish structural requirements for the interaction of CD151 with the alpha3beta1 integrin and with other tetraspanins. Using a panel of CD151/CD9 chimeras and CD151 deletion mutants we show that the minimal region, which confers stable (e.g. Triton X-100-resistant) association of the tetraspanin with alpha3beta1, maps within the large extracellular loop (LECL) of CD151 (the amino acid sequence between residues Leu(149) and Glu(213)). Furthermore, the substitution of 11 amino acids (residues 195-205) from this region for a corresponding sequence from CD9 LECL or point mutations of cysteines in the conserved CCG and PXXCC motifs abolish the interaction. The removal of the LECL CD151 does not affect the association of the protein with other tetraspanins (e.g. CD9, CD81, CD63, and wild-type CD151). On the other hand, the mutation of the CCG motif selectively prevents the homotypic CD151-CD151 interaction but does not influence the association of the mutagenized CD151 with other tetraspanins. These results demonstrate the differences in structural requirements for the heterotypic and homotypic tetraspanin-tetraspanin interactions. Various deletions involving the small extracellular loop and the first three transmembrane domains prevent surface expression of the CD151 mutants but do not affect the CD151-alpha3beta1 interaction. The CD151 deletion mutants are accumulated in the endoplasmic reticulum and redirected to the lysosomes. The assembly of the CD151-alpha3beta1 complex occurs early during the integrin biosynthesis and precedes the interaction of CD151 with other tetraspanins. Collectively, these data show that the incorporation of CD151 into the "tetraspanin web" can be controlled at various levels by different regions of the protein.