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.     

DHHC2 affects palmitoylation, stability, and functions of tetraspanins CD9 and CD151

Although palmitoylation markedly affects tetraspanin protein biochemistry and functions, relevant palmitoylating enzymes were not known. There are 23 mammalian "DHHC" (Asp-His-His-Cys) proteins, which presumably palmitoylate different sets of protein substrates. Among DHHC proteins tested, DHHC2 best stimulated palmitoylation of tetraspanins CD9 and CD151, whereas inactive DHHC2 (containing DH-->AA or C-->S mutations within the DHHC motif) failed to promote palmitoylation. Furthermore, DHHC2 associated with CD9 and CD151, but not other cell surface proteins, and DHHC2 knockdown diminished CD9 and CD151 palmitoylation. Knockdown of six other Golgi-resident DHHC proteins (DHHC3, -4, -8, -17, -18, and -21) had no effect on CD9 or CD151. DHHC2 selectively affected tetraspanin palmitoylation, but not the palmitoylations of integrin beta4 subunit and bulk proteins visible in [(3)H]palmitate-labeled whole cell lysates. DHHC2-dependent palmitoylation also had multiple functional effects. First, it promoted physical associations between CD9 and CD151, and between alpha3 integrin and other proteins. Second, it protected CD151 and CD9 from lysosomal degradation. Third, the presence of DHHC2, but not other DHHC proteins, shifted cells away from a dispersed state and toward increased cell-cell contacts.     

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.     

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.     

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.     

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.     

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.     

Identification of Tspan9 as a novel platelet tetraspanin and the collagen receptor GPVI as a component of tetraspanin microdomains

Platelets are essential for wound healing and inflammatory processes, but can also play a deleterious role by causing heart attack and stroke. Normal platelet activation is dependent on tetraspanins, a superfamily of glycoproteins that function as 'organisers' of cell membranes by recruiting other receptors and signalling proteins into tetraspanin-enriched microdomains. However, our understanding of how tetraspanin microdomains regulate platelets is hindered by the fact that only four of the 33 mammalian tetraspanins have been identified in platelets. This is because of a lack of antibodies to most tetraspanins and difficulties in measuring mRNA, due to low levels in this anucleate cell. To identify potentially platelet-expressed tetraspanins, mRNA was measured in their nucleated progenitor cell, the megakaryocyte, using serial analysis of gene expression and DNA microarrays. Amongst 19 tetraspanins identified in megakaryocytes, Tspan9, a previously uncharacterized tetraspanin, was relatively specific to these cells. Through generating the first Tspan9 antibodies, Tspan9 expression was found to be tightly regulated in platelets. The relative levels of CD9, CD151, Tspan9 and CD63 were 100, 14, 6 and 2 respectively. Since CD9 was expressed at 49000 cell surface copies per platelet, this suggested a copy number of 2800 Tspan9 molecules. Finally, Tspan9 was shown to be a component of tetraspanin microdomains that included the collagen receptor GPVI (glycoprotein VI) and integrin alpha6beta1, but not the von Willebrand receptor GPIbalpha or the integrins alphaIIbbeta3 or alpha2beta1. These findings suggest a role for Tspan9 in regulating platelet function in concert with other platelet tetraspanins and their associated proteins.     

Palmitoylation of tetraspanin proteins: modulation of CD151 lateral interactions, subcellular distribution, and integrin-dependent cell morphology

Here we demonstrate that multiple tetraspanin (transmembrane 4 superfamily) proteins are palmitoylated, in either the Golgi or a post-Golgi compartment. Using CD151 as a model tetraspanin, we identified and mutated intracellular N-terminal and C-terminal cysteine palmitoylation sites. Simultaneous mutations of C11, C15, C242, and C243 (each to serine) eliminated >90% of CD151 palmitoylation. Notably, palmitoylation had minimal influence on the density of tetraspanin protein complexes, did not promote tetraspanin localization into detergent-resistant microdomains, and was not required for CD151-alpha 3 beta 1 integrin association. However, the CD151 tetra mutant showed markedly diminished associations with other cell surface proteins, including other transmembrane 4 superfamily proteins (CD9, CD63). Thus, palmitoylation may be critical for assembly of the large network of cell surface tetraspanin-protein interactions, sometimes called the "tetraspanin web." Also, compared with wild-type CD151, the tetra mutant was much more diffusely distributed and showed markedly diminished stability during biosynthesis. Finally, expression of the tetra-CD151 mutant profoundly altered alpha 3 integrin-deficient kidney epithelial cells, such that they converted from a dispersed, elongated morphology to an epithelium-like cobblestone clustering. These results point to novel biochemical and biological functions for tetraspanin palmitoylation.     

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.     

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.     

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.     

Tetraspanins in the humoral immune response

The tetraspanins represent a large superfamily of four-transmembrane proteins that are expressed on all nucleated cells. Tetraspanins play a prominent role in the organization of the plasma membrane by co-ordinating the spatial localization of transmembrane proteins and signalling molecules into 'tetraspanin microdomains'. In immune cells, tetraspanins interact with key leucocyte receptors [including MHC molecules, integrins, CD4/CD8 and the BCR (B-cell receptor) complex] and as such can modulate leucocyte receptor activation and downstream signalling pathways. There is now ample evidence that tetraspanins on B-lymphocytes are important in controlling antibody production. The tetraspanin CD81 interacts with the BCR complex and is critical for CD19 expression and IgG production, whereas the tetraspanin CD37 inhibits IgA production and is important for IgG production. By contrast, the tetraspanins CD9, Tssc6 and CD151 appear dispensable for humoral immune responses. Thus individual tetraspanin family members have specific functions in B-cell biology, which is evidenced by recent studies in tetraspanin-deficient mice and humans. The present review focuses on tetraspanins expressed by B-lymphocytes and discusses novel insights into the function of tetraspanins in the humoral immune response.     

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.     

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.     

Tetraspanin CD82 attenuates cellular morphogenesis through down-regulating integrin alpha6-mediated cell adhesion

Tetraspanin CD82 has been implicated in integrin-mediated functions such as cell motility and invasiveness. Although tetraspanins associate with integrins, it is unknown if and how CD82 regulates the functionality of integrins. In this study, we found that Du145 prostate cancer cells underwent morphogenesis on the reconstituted basement membrane Matrigel to form an anastomosing network of multicellular structures. This process entirely depends on integrin alpha6, a receptor for laminin. After CD82 is expressed in Du145 cells, this cellular morphogenesis was abolished, indicating a functional cross-talk between CD82 and alpha6 integrins. Interestingly, antibodies against other tetraspanins expressed in Du145 cells such as CD9, CD81, and CD151 did not block this integrin alpha6-dependent morphogenesis. We further found that CD82 significantly inhibited cell adhesion on laminin 1. Notably, the level of alpha6 integrins on the cell surface was down-regulated upon CD82 expression, although total cellular alpha6 protein levels remained unchanged in CD82-expressing cells. This down-regulation indicates that the diminished cell adhesiveness of CD82-expressing Du145 cells on laminin likely resulted from less cell surface expression of alpha6 integrins. As expected, CD82 physically associated with the integrin alpha6 in Du145-CD82 transfectant cells, suggesting that the formation of the CD82-integrin alpha6 complex reduces alpha6 integrin cell surface expression. Finally, the internalization of cell surface integrin alpha6 is significantly enhanced upon CD82 expression. In conclusion, our results indicate that 1) CD82 attenuates integrin alpha6 signaling during a cellular morphogenic process; 2) the decreased surface expression of alpha6 integrins in CD82-expressing cells is likely responsible for the diminished adhesiveness on laminin and, subsequently, results in the attenuation of alpha6 integrin-mediated cellular morphogenesis; and 3) the accelerated internalization of integrin alpha6 upon CD82 expression correlates with the down-regulation of cell surface integrin alpha6.     

Characterization of mice lacking the tetraspanin superfamily member CD151

The tetraspanin membrane protein CD151 is a broadly expressed molecule noted for its strong molecular associations with integrins, especially alpha3beta1, alpha6beta1, alpha7beta1, and alpha6beta4. In vitro functional studies have pointed to a role for CD151 in cell-cell adhesion, cell migration, platelet aggregation, and angiogenesis. It has also been implicated in epithelial tumor progression and metastasis. Here we describe the generation and initial characterization of CD151-null mice. The mice are viable, healthy, and fertile and show normal Mendelian inheritance. They have essentially normal blood and bone marrow cell counts and grossly normal tissue morphology, including hemidesmosomes in skin, and expression of alpha3 and alpha6 integrins. However, the CD151-null mice do show phenotypes in several different tissue types. An absence of CD151 leads to a minor abnormality in hemostasis, with CD151-null mice showing longer average bleeding times, greater average blood loss, and an increased incidence of rebleeding occurrences. CD151-null keratinocytes migrate poorly in skin explant cultures. Finally, CD151-null T lymphocytes are hyperproliferative in response to in vitro mitogenic stimulation.     

BAP31 and its caspase cleavage product regulate cell surface expression of tetraspanins and integrin-mediated cell survival

BAP31, a resident integral protein of the endoplasmic reticulum membrane, regulates the export of other integral membrane proteins to the downstream secretory pathway. Here we show that cell surface expression of the tetraspanins CD9 and CD81 is compromised in mouse cells from which the Bap31 gene has been deleted. CD9 and CD81 facilitate the function of multiprotein complexes at the plasma membrane, including integrins. Of note, BAP31 does not appear to influence the egress of alpha5beta1 or alpha(v)beta3 integrins to the cell surface, but in Bap31-null mouse cells, these integrins are not able to maintain cellular adhesion to the extracellular matrix in the presence of reduced serum. Consequently, Bap31-null cells are sensitive to serum starvation-induced apoptosis. Reconstitution of wild-type BAP31 into these Bap31-null cells restores integrin-mediated cell attachment and cell survival after serum stress, whereas interference with the functions of CD9, alpha5beta1, or alpha(v)beta3 by antagonizing antibodies makes BAP31 cells act similar to Bap31-null cells in these respects. Finally, in human KB epithelial cells protected from apoptosis by BCL-2, the caspase-8 cleavage product, p20 BAP31, inhibits egress of tetraspanin and integrin-mediated cell attachment. Thus, p20 BAP31 can operate upstream of BCL-2 in living cells to influence cell surface properties due to its effects on protein egress from the endoplasmic reticulum.     

Dissociation of the complex between CD151 and laminin-binding integrins permits migration of epithelial cells

Laminin-binding integrins form a complex with CD151, a member of the tetraspanin family suggested to be involved in the regulation of cell migration. In the epidermis, CD151 is localized with alpha3beta1 and alpha6beta4 integrins at cell-cell and cell-matrix contacts, respectively, characteristic structures of non-migrating cells. Taking advantage of a monoclonal antibody against CD151, TS151r, which epitope overlaps with the tetraspanin integrin-binding site, we have investigated the role of CD151 in epithelial cell migration. Under standard culture conditions, the migratory capacity of epithelial HaCaT cells on laminins is low, apparently due to endogenous laminin 5. However, challenging HaCaT cells with TS151r allows a re-arrangement of the actin cytoskeleton, dismantling of cell-cell and beta4 integrin-mediated cell-matrix contacts and cell migration. In vivo, free CD151 is absent in resting epithelial cells of interfollicular epidermis, and all CD151 is bound to integrins in intercellular and cell-matrix contacts. By contrast, free CD151 is present at intercellular contacts in the epithelial sheet lining the deeper region of anagen hair follicles, which is considered to contain migrating cells. Together, these results strongly suggest that dissociation of the CD151-integrin complex permits remodeling of epithelial cell interactions with the extracellular matrix and cell migration.