Transmembrane-4 superfamily proteins associate with activated protein kinase C (PKC) and link PKC to specific beta(1) integrins

Translocation of conventional protein kinases C (PKCs) to the plasma membrane leads to their specific association with transmembrane-4 superfamily (TM4SF; tetraspanin) proteins (CD9, CD53, CD81, CD82, and CD151), as demonstrated by reciprocal co-immunoprecipitation and covalent cross-linking experiments. Although formation and maintenance of TM4SF-PKC complexes are not dependent on integrins, TM4SF proteins can act as linker molecules, recruiting PKC into proximity with specific integrins. Previous studies showed that the extracellular large loop of TM4SF proteins determines integrin associations. In contrast, specificity for PKC association probably resides within cytoplasmic tails or the first two transmembrane domains of TM4SF proteins, as seen from studies with chimeric CD9 molecules. Consistent with a TM4SF linker function, only those integrins (alpha(3)beta(1), alpha(6)beta(1), and a chimeric "X3TC5" alpha(3) mutant) that associated strongly with tetraspanins were found in association with PKC. We propose that PKC-TM4SF-integrin structures represent a novel type of signaling complex. The simultaneous binding of TM4SF proteins to the extracellular domains of the integrin alpha(3) subunit and to intracellular PKC helps to explain why the integrin alpha3 extracellular domain is needed for both intracellular PKC recruitment and PKC-dependent phosphorylation of the alpha(3) integrin cytoplasmic tail.     

An extracellular site on tetraspanin CD151 determines alpha 3 and alpha 6 integrin-dependent cellular morphology

The alpha 3 beta 1 integrin shows strong, stoichiometric, direct lateral association with the tetraspanin CD151. As shown here, an extracellular CD151 site (QRD(194-196)) is required for strong (i.e., Triton X-100-resistant) alpha 3 beta 1 association and for maintenance of a key CD151 epitope (defined by monoclonal antibody TS151r) that is blocked upon alpha 3 integrin association. Strong CD151 association with integrin alpha 6 beta 1 also required the QRD(194-196) site and masked the TS151r epitope. For both alpha 3 and alpha 6 integrins, strong QRD/TS151r-dependent CD151 association occurred early in biosynthesis and involved alpha subunit precursor forms. In contrast, weaker associations of CD151 with itself, integrins, or other tetraspanins (Triton X-100-sensitive but Brij 96-resistant) were independent of the QRD/TS151r site, occurred late in biosynthesis, and involved mature integrin subunits. Presence of the CD151-QRD(194-196)-->INF mutant disrupted alpha 3 and alpha 6 integrin-dependent formation of a network of cellular cables by Cos7 or NIH3T3 cells on basement membrane Matrigel and markedly altered cell spreading. These results provide definitive evidence that strong lateral CD151-integrin association is functionally important, identify CD151 as a key player during alpha 3 and alpha 6 integrin-dependent matrix remodeling and cell spreading, and support a model of CD151 as a transmembrane linker between extracellular integrin domains and intracellular cytoskeleton/signaling molecules.     

Characterization of novel complexes on the cell surface between integrins and proteins with 4 transmembrane domains (TM4 proteins).

Here we identified several new integrin/TM4 protein complexes on the cell surface. By immunoprecipitation using nonstringent conditions, and by reciprocal immunoprecipitation, we found that alpha 3 beta 1 and alpha 6 beta 1 integrins but not alpha 2 beta 1, alpha 5 beta 1, or alpha 6 beta 4 integrins associated with CD9 and CD81 in alpha 3 beta 1/CD81, alpha 3 beta 1/CD9, alpha 6 beta 1/CD81, and alpha 6 beta 1/CD9 complexes. Also, cross-linking experiments established that alpha 3 beta 1/CD81, alpha 3 beta 1/CD9, and alpha 3 beta 1/CD63 associations occur on the surface of intact cells and suggested that a critical interaction site is located within extracellular domains. Cross-linking in conjunction with reimmunoprecipitation indicated that larger multi-component alpha 3 beta 1/TM4/TM4 complexes (alpha 3 beta 1/CD9/CD63, alpha 3 beta 1/CD81/CD63, and alpha 3 beta 1/CD9/CD81) also could be detected on the cell surface. Immunofluorescent staining showed redistribution of alpha 3 beta 1/TM4 complexes toward the periphery of cells plated on various extracellular matrix substrates and also showed that these complexes were localized in cell footprints. Staining of human tissues yielded additional results consistent with co-localization of alpha 3 beta 1 and CD9, CD63, and CD81 proteins. In conclusion we suggest that the prevalence of integrin/TM4 complexes in diverse cellular environments is indicative of their general physiological importance.     

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.     

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.     

Evaluation of prototype transmembrane 4 superfamily protein complexes and their relation to lipid rafts

Recent literature suggests that tetraspanin proteins (transmembrane 4 superfamily; TM4SF proteins) may associate with each other and with many other transmembrane proteins to form large complexes that sometimes may be found in lipid rafts. Here we show that prototype complexes of CD9 or CD81 (TM4SF proteins) with alpha(3)beta(1) (an integrin) and complexes of CD63 (a TM4SF protein) with phosphatidylinositol 4-kinase (PtdIns 4-K) may indeed localize within lipid raft-like microdomains, as seen by three different criteria. First, these complexes localize to low density light membrane fractions in sucrose gradients. Second, CD9 and alpha(3) integrin colocalized with ganglioside GM1 as seen by double staining of fixed cells. Third, CD9-alpha3beta1 and CD81-alpha3beta1 complexes were shifted to a higher density upon cholesterol depletion from intact cells or cell lysate. However, CD9-alpha3beta1, CD81-alpha3beta1, and CD63-PtdIns 4-K complex formation itself was not dependent on localization into raftlike lipid microdomains. These complexes did not require cholesterol for stabilization, were maintained within well solubilized dense fractions from sucrose gradients, were stable at 37 degrees C, and were small enough to be included within CL6B gel filtration columns. In summary, prototype TM4SF protein complexes (CD9-alpha3beta1, CD81-alpha3beta1, and CD63-PtdIns 4-K) can be solubilized as discrete units, independent of lipid microdomains, although they do associate with microdomains resembling lipid rafts.     

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.     

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.     

FPRP, a major, highly stoichiometric, highly specific CD81- and CD9-associated protein

CD81 and CD9, members of the transmembrane-4 superfamily (TM4SF; tetraspanins), form extensive complexes with other TM4SF proteins, integrins, and other proteins, especially in mild detergents. In moderately stringent Brij 96 lysis conditions, CD81 and CD9 complexes are virtually identical to each other, but clearly distinct from other TM4SF complexes. One of the most prominent proteins within CD81 and CD9 complexes is identified here as FPRP, the 133-kDa prostaglandin F(2alpha) receptor regulatory protein. FPRP, a cell-surface Ig superfamily protein, associates specifically with CD81 or with CD81 and CD9, but not with integrins or other TM4SF proteins. In contrast to other CD81- and CD9-associating proteins, FPRP associates at very high stoichiometry, with essentially 100% of cell-surface FPRP on 293 cells being CD81- and CD9-associated. Also, CD81.CD9.FPRP complexes have a discrete size (<4 x 10(6) Da) as measured by gel permeation chromatography and remain intact after disruption of cholesterol-rich membrane microdomains by methyl-beta-cyclodextrin. Although CD81 associated with both alpha(3) integrin and FPRP in 293 cells, the alpha(3)beta(1).CD81 and CD81.CD9.FPRP complexes were distinct, as determined by immunoprecipitation and immunodepletion experiments. In conclusion, our data affirm the existence of distinct TM4SF complexes with unique compositions and specifically characterize FPRP as the most robust, highly stoichiometric CD81- and/or CD9-associated protein yet described.     

The tetraspan molecule CD151, a novel constituent of hemidesmosomes, associates with the integrin alpha6beta4 and may regulate the spatial organization of hemidesmosomes

CD151 is a cell surface protein that belongs to the tetraspan superfamily. It associates with other tetraspan molecules and certain integrins to form large complexes at the cell surface. CD151 is expressed by a variety of epithelia and mesenchymal cells. We demonstrate here that in human skin CD151 is codistributed with alpha3beta1 and alpha6beta4 at the basolateral surface of basal keratinocytes. Immunoelectron microscopy showed that CD151 is concentrated in hemidesmosomes. By immunoprecipitation from transfected K562 cells, we established that CD151 associates with alpha3beta1 and alpha6beta4. In beta4-deficient pyloric atresia associated with junctional epidermolysis bullosa (PA-JEB) keratinocytes, CD151 and alpha3beta1 are clustered together at the basal cell surface in association with patches of laminin-5. Focal adhesions are present at the periphery of these clusters, connected with actin filaments, and they contain both CD151 and alpha3beta1. Transient transfection studies of PA-JEB cells with beta4 revealed that the integrin alpha6beta4 becomes incorporated into the alpha3beta1-CD151 clusters where it induces the formation of hemidesmosomes. As a result, the amount of alpha3beta1 in the clusters diminishes and the protein becomes restricted to the peripheral focal adhesions. Furthermore, CD151 becomes predominantly associated with alpha6beta4 in hemidesmosomes, whereas its codistribution with alpha3beta1 in focal adhesions becomes partial. The localization of alpha6beta4 in the pre-hemidesmosomal clusters is accompanied by a strong upregulation of CD151, which is at least partly due to increased cell surface expression. Using beta4 chimeras containing the extracellular and transmembrane domain of the IL-2 receptor and the cytoplasmic domain of beta4, we found that for recruitment of CD151 into hemidesmosomes, the beta4 subunit must be associated with alpha6, confirming that integrins associate with tetraspans via their alpha subunits. CD151 is the only tetraspan identified in hemidesmosomal structures. Others, such as CD9 and CD81, remain diffusely distributed at the cell surface.In conclusion, we show that CD151 is a major component of (pre)-hemidesmosomal structures and that its recruitment into hemidesmosomes is regulated by the integrin alpha6beta4. We suggest that CD151 plays a role in the formation and stability of hemidesmosomes by providing a framework for the spatial organization of the different hemidesmosomal components.     

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.     

Insights into the substrate specificity of plant peptide deformylase, an essential enzyme with potential for the development of novel biotechnology applications in agriculture

CD151, a member of the tetraspanin family of proteins, forms a stable complex with integrin alpha 3 beta 1 and regulates integrin-mediated cell-substrate adhesion. However, the molecular basis of the stable association of CD151 with integrin alpha 3 beta 1 remains poorly understood. In the present study, we show that a panel of anti-human CD151 mAbs (monoclonal antibodies) could be divided into three groups on the basis of their abilities to co-immunoprecipitate integrin alpha 3: Group-1 mAbs were devoid of sufficient activities to co-precipitate integrin alpha 3 under both low- and high-stringency detergent conditions; Group-2 mAbs co-precipitated integrin alpha 3 under low-stringency conditions; and Group-3 mAbs exhibited strong co-precipitating activities under both conditions. Group-1 mAbs in particular exhibited increased reactivity toward integrin alpha 3 beta 1-unbound CD151, indicating that the binding sites for Group-1 mAbs are partly blocked by bound integrin alpha 3 beta 1. Epitope mapping using a series of CD151 mutants with substitutions at amino acid residues that are not conserved between human and mouse CD151 revealed that Gly(176)/Gly(177), Leu(191) and Gln(194) comprise epitopes characteristic of Group-1 mAbs. Replacement of short peptide segments, each containing one of these epitopes, with those of other tetraspanins lacking stable interactions with integrin alpha 3 beta 1 demonstrated that the segment from Cys(185) to Cys(192), including Leu(191), was involved in the stable association of CD151 with integrin alpha 3 beta 1, as was the Gln(194)-containing QRD peptide. Taken together these results indicate that two consecutive segments including two Group-1 epitopes, Leu(191) and Gln(194), comprise an interface between CD151 and integrin alpha 3 beta 1, and, along with the epitope including Gly(176)/Gly(177), are concealed by bound integrin.     

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.     

Correlations between Transmembrane 4 L6 Family Member 5 (TM4SF5), CD151, and CD63 in Liver Fibrotic Phenotypes and Hepatic Migration and Invasive Capacities

Transmembrane 4 L6 family member 5 (TM4SF5) is overexpressed during CCl₄-mediated murine liver fibrosis and in human hepatocellular carcinomas. The tetraspanins form tetraspanin-enriched microdomains (TEMs) consisting of large membrane protein complexes on the cell surface. Thus, TM4SF5 may be involved in the signal coordination that controls liver malignancy. We investigated the relationship between TM4SF5-positive TEMs with liver fibrosis and tumorigenesis, using normal Chang hepatocytes that lack TM4SF5 expression and chronically TGFβ1-treated Chang cells that express TM4SF5. TM4SF5 expression is positively correlated with tumorigenic CD151 expression, but is negatively correlated with tumor-suppressive CD63 expression in mouse fibrotic and human hepatic carcinoma tissues, indicating cooperative roles of the tetraspanins in liver malignancies. Although CD151 did not control the expression of TM4SF5, TM4SF5 appeared to control the expression levels of CD151 and CD63. TM4SF5 interacted with CD151, and caused the internalization of CD63 from the cell surface into late lysosomal membranes, presumably leading to terminating the tumor-suppressive functions of CD63. TM4SF5 could overcome the tumorigenic effects of CD151, especially cell migration and extracellular matrix (ECM)-degradation. Taken together, TM4SF5 appears to play a role in liver malignancy by controlling the levels of tetraspanins on the cell surface, and could provide a promising therapeutic target for the treatment of liver malignancies.     

Analysis of the alpha4beta1 integrin-osteopontin interaction

The integrin alpha4beta1 is involved in mediating exfiltration of leukocytes from the vasculature. It interacts with a number of proteins up-regulated during the inflammatory response including VCAM-1 and the CS-1 alternatively spliced region of fibronectin. In addition it binds the multifunctional protein osteopontin (OPN), which can act as both a cytokine and an extracellular matrix molecule. Here we map the region of human OPN that supports cell adhesion via alpha4beta1 using GST fusion proteins. We show that alpha4beta1 expressed in J6 cells interacts with intact OPN when the integrin is in a high activation state, and by deletion mapping that the alpha4beta1 binding region in OPN lies between amino acid residues 125 and 168 (aa125-168). This region contains the central RGD motif of OPN, which also interacts with integrins alphavbeta3, alphavbeta5, alphavbeta1, alpha8beta1, and alpha5beta1. Mutating the RGD motif to RAD had no effect on the interaction with alpha4beta1. To define the binding site the region incorporating aa125-168 was divided into 5 overlapping peptides expressed as GST fusion proteins. Two peptides supported adhesion via alpha4beta1, aa132-146, and aa153-168; of these only a synthetic peptide, SVVYGLR (aa162-168), derived from aa153-168 was able to inhibit alpha4beta1 binding to CS-1. These data identify the motif SVVYGLR as a novel peptide inhibitor of alpha4beta1, and the primary alpha4beta1 binding site within OPN.     

Function of the Tetraspanin CD151–α6β1 Integrin Complex during Cellular Morphogenesis

Upon plating on basement membrane Matrigel, NIH3T3 cells formed an anastomosing network of cord-like structures, inhibitable by anti-alpha6beta1 integrin antibodies. For NIH3T3 cells transfected with human CD151 protein, the formation of a cord-like network was also inhibitable by anti-CD151 antibodies. Furthermore, CD151 and alpha6beta1 were physically associated within NIH3T3 cells. On removal of the short 8-amino acid C-terminal CD151 tail (by deletion or exchange), exogenous CD151 exerted a dominant negative effect, as it almost completely suppressed alpha6beta1-dependent cell network formation and NIH3T3 cell spreading on laminin-1 (an alpha6beta1 ligand). Importantly, mutant CD151 retained alpha6beta1 association and did not alter alpha6beta1-mediated cell adhesion to Matrigel. In conclusion, the CD151-alpha6beta1 integrin complex acts as a functional unit that markedly influences cellular morphogenesis, with the CD151 tail being of particular importance in determining the "outside-in" functions of alpha6beta1-integrin that follow ligand engagement. Also, antibodies to alpha6beta1 and CD151 inhibited formation of endothelial cell cord-like networks, thus pointing to possible relevance of CD151-alpha6beta1 complexes during angiogenesis.     

Transmembrane signal transduction of the alpha(IIb)beta(3) integrin

Integrins are composed of noncovalently bound dimers of an alpha- and a beta-subunit. They play an important role in cell-matrix adhesion and signal transduction through the cell membrane. Signal transduction can be initiated by the binding of intracellular proteins to the integrin. Binding leads to a major conformational change. The change is passed on to the extracellular domain through the membrane. The affinity of the extracellular domain to certain ligands increases; thus at least two states exist, a low-affinity and a high-affinity state. The conformations and conformational changes of the transmembrane (TM) domain are the focus of our interest. We show by a global search of helix-helix interactions that the TM section of the family of integrins are capable of adopting a structure similar to the structure of the homodimeric TM protein Glycophorin A. For the alpha(IIb)beta(3) integrin, this structural motif represents the high-affinity state. A second conformation of the TM domain of alpha(IIb)beta(3) is identified as the low-affinity state by known mutational and nuclear magnetic resonance (NMR) studies. A transition between these two states was determined by molecular dynamics (MD) calculations. On the basis of these calculations, we propose a three-state mechanism.     

Transmembrane domain helix packing stabilizes integrin alphaIIbbeta3 in the low affinity state

Regulated changes in the affinity of integrin adhesion receptors ("activation") play an important role in numerous biological functions including hemostasis, the immune response, and cell migration. Physiological integrin activation is the result of conformational changes in the extracellular domain initiated by the binding of cytoplasmic proteins to integrin cytoplasmic domains. The conformational changes in the extracellular domain are likely caused by disruption of intersubunit interactions between the alpha and beta transmembrane (TM) and cytoplasmic domains. Here, we reasoned that mutation of residues contributing to alpha/beta interactions that stabilize the low affinity state should lead to integrin activation. Thus, we subjected the entire intracellular domain of the beta3 integrin subunit to unbiased random mutagenesis and selected it for activated mutants. 25 unique activating mutations were identified in the TM and membrane-proximal cytoplasmic domain. In contrast, no activating mutations were identified in the more distal cytoplasmic tail, suggesting that this region is dispensable for the maintenance of the inactive state. Among the 13 novel TM domain mutations that lead to integrin activation were several informative point mutations that, in combination with computational modeling, suggested the existence of a specific TM helix-helix packing interface that maintains the low affinity state. The interactions predicted by the model were used to identify additional activating mutations in both the alpha and beta TM domains. Therefore, we propose that helical packing of the alpha and beta TM domains forms a clasp that regulates integrin activation.     

Focal adhesion and actin organization by a cross-talk of TM4SF5 with integrin alpha2 are regulated by serum treatment

The biological functions of transmembrane 4 L6 family member 5 (TM4SF5) homologues to a tumor-associated antigen L6 are unknown, although it is over-expressed in certain forms of cancer. In the present study, the ectopic expression of TM4SF5 in Cos7 cells reduced integrin signaling under serum-containing conditions, but increased integrin signaling upon serum-free replating on substrates. TM4SF5 regulated actin organization and focal contact dynamics via the serum treatment-dependent differential regulation of FAK Tyr925 and paxillin Tyr118 phosphorylations and their localizations on peripheral cell boundaries. Y925F FAK mutation abolished the TM4SF5 effects. TM4SF5 associated with integrin alpha2 subunit, and this association was abolished by serum treatment. Furthermore, functional blocking anti-integrin alpha2 antibody abolished TM4SF5-enhanced signaling activity and caused membrane blebbing with abnormal actin organization. TM4SF5 increased chemotactic but decreased haptotactic migration. Altogether, this study reveals the functions of TM4SF5 collaborative with integrin signaling to alter focal contact dynamics, actin reorganization, and migration. Furthermore, this study suggests a mechanism of cross-talk between TM4SF5 and integrin which is further regulated by growth factor signaling.     

Tetraspanin CD151 promotes cell migration by regulating integrin trafficking

Regulation of cell migration is an important feature of tetraspanin CD151. Although it is well established that CD151 physically associates with integrins, the mechanism by which CD151 regulates integrin-dependent cell migration is basically unknown. Given the fact that CD151 is localized in both the plasma membrane and intracellular vesicles, we found that CD151 and its associated alpha3beta1, alpha5beta1, and alpha6beta1 integrins undergo endocytosis and accumulate in the same intracellular vesicular compartments. CD151 contains a YRSL sequence, a YXXvarphi type of endocytosis/sorting motif, in its C-terminal cytoplasmic domain. Mutation of this motif markedly attenuated CD151 internalization. The loss of CD151 trafficking completely abrogated CD151-promoted cell migration on extracellular matrices such as laminin and diminished the internalization of its associated integrins, indicating a critical role for integrin trafficking in regulating cell motility. In conclusion, the YXXvarphi motif-mediated internalization of CD151 promotes integrin-dependent cell migration by modulating the endocytosis and/or vesicular trafficking of its associated integrins.