(1/110) PETA-3/CD151, a member of the transmembrane 4 superfamily, is localised to the plasma membrane and endocytic system of endothelial cells, associates with multiple integrins and modulates cell function.
The Transmembrane 4 Superfamily member, PETA-3/CD151, is ubiquitously expressed by endothelial cells in vivo. In cultured human umbilical vein endothelial cells PETA-3 is present on the plasma membrane and predominantly localises to regions of cell-cell contact. Additionally, this protein is abundant within an intracellular compartment which accounts for up to 66% of the total PETA-3 expressed. Intracellular PETA-3 showed colocalisation with transferrin receptor and CD63 suggesting an endosomal/lysosomal localisation which was supported by immuno-electronmicroscopy studies. Co-immunoprecipitation experiments investigating possible interactions of PETA-3 with other molecules demonstrated associations with several integrin chains including beta1, beta3, beta4, (alpha)2, (alpha)3, (alpha)5, (alpha)6 and provide the first report of Transmembrane 4 Superfamily association with the (alpha)6beta4 integrin. Using 2-colour confocal microscopy, we demonstrated similar localisation of PETA-3 and integrin chains within cytoplasmic vesicles and endothelial cell junctions. In order to assess the functional implications of PETA-3/integrin associations, the effect of anti-PETA-3 antibodies on endothelial function was examined. Anti-PETA-3 mAb inhibited endothelial cell migration and modulated in vitro angiogenesis, but had no detectable effect on neutrophil transendothelial migration. The broad range of integrin associations and the presence of PETA-3 with integrins both on the plasma membrane and within intracellular vesicles, suggests a primary role for PETA-3 in regulating integrin trafficking and/or function. (+info)
(2/110) Selective tetraspan-integrin complexes (CD81/alpha4beta1, CD151/alpha3beta1, CD151/alpha6beta1) under conditions disrupting tetraspan interactions.
The tetraspans are molecules with four transmembrane domains which are engaged in multimolecular complexes (the tetraspan web) containing a subset of beta1 integrins (in particular alpha3beta1, alpha4beta1 and alpha6beta1), MHC antigens and several unidentified molecules. The molecules associated with tetraspans are readily detected after immunoprecipitation performed in mild detergents such as Brij 97 or CHAPS. In this study we show that another classical mild detergent, digitonin, dissociated most of these associated molecules, including integrins, from the tetraspans CD9, CD37, CD53, CD63, CD82, Co-029, Talla-1 and NAG-2. In contrast, reciprocal immunoprecipitations from various cell lines demonstrated that two other tetraspans, CD81 and CD151, formed complexes with integrins not disrupted by digitonin. These complexes were CD81/alpha4beta1, CD151/alpha3beta1 and CD151/alpha6beta1. Furthermore, a new anti-CD151 monoclonal antibody (mAb), TS151r, was shown to have a restricted pattern of expression, inversely related to the sum of the levels of expression of alpha6beta1 and alpha3beta1. This mAb was unable to co-precipitate integrins in digitonin, suggesting that its epitope is blocked by the association with integrins. Indeed, the binding of TS151r to the cell surface was quantitatively diminished following alpha3beta1 overexpression. Altogether, these data suggest that, among tetraspans, CD81 interacts directly with the integrin alpha4beta1, and CD151 interacts directly with integrins alpha3beta1 and alpha6beta1. Because all tetraspan-tetraspan associations are disrupted by digitonin, it is likely that the other tetraspans interact indirectly with integrins, through interactions with CD81 or CD151. (+info)
(3/110) Eukaryotic expression cloning with an antimetastatic monoclonal antibody identifies a tetraspanin (PETA-3/CD151) as an effector of human tumor cell migration and metastasis.
A monoclonal antibody (mAb), 50-6, generated by subtractive immunization, was found to specifically inhibit in vivo metastasis of a human epidermoid carcinoma cell line, HEp-3. The cDNA of the cognate antigen of mAb 50-6 was isolated by a modified eukaryotic expression cloning protocol from a HEp-3 library. Sequence analysis identified the antigen as PETA-3/CD151, a recently described member of the tetraspanin family of proteins. The cloned antigen was also recognized by a previously described antimetastatic antibody, mAb 1A5. Inhibition of HEp-3 metastasis by the mAbs could not be attributed to any effect of the antibodies on tumor cell growth in vitro or in vivo. Rather, the antibodies appeared to inhibit an early step in the formation of metastatic foci. In a chemotaxis assay, HEp-3 migration was blocked by both antibodies. HeLa cells transfected with and overexpressing PETA-3/CD151 were more migratory than control transfectants expressing little CD151. The increase in HeLa migration was inhibitable by both mAb 50-6 and mAb 1A5. PETA-3 appears not to be involved in cell attachment because adhesion did not correlate with levels of PETA-3 expression and was unaffected by mAb 50-6 or mAb 1A5. The ability of PETA-3 to mediate cell migration suggests a mechanism by which this protein may influence metastasis. These data identify PETA-3/CD151 as the first member of the tetraspanin family to be linked as a positive effector of metastasis. (+info)
(4/110) Direct extracellular contact between integrin alpha(3)beta(1) and TM4SF protein CD151.
Previously we established that the alpha(3)beta(1) integrin shows stable, specific, and stoichiometric association with the TM4SF (tetraspannin) protein CD151. Here we used a membrane impermeable cross-linking agent to show a direct association between extracellular domains of alpha(3)beta(1) and CD151. The alpha(3)beta(1)-CD151 association site was then mapped using chimeric alpha(6)/alpha(3) integrins and CD151/NAG2 TM4SF proteins. Complex formation required an extracellular alpha(3) site (amino acids (aa) 570-705) not previously known to be involved in specific integrin contacts with other proteins and a region (aa 186-217) within the large extracellular loop of CD151. Notably, the anti-CD151 monoclonal antibody TS151r binding epitope, previously implicated in alpha(3) integrin association, was mapped to the same region of CD151 (aa 186-217). Finally, we demonstrated that both NH(2)- and COOH-terminal domains of CD151 are located on the inside of the plasma membrane, thus confirming a long suspected model of TM4SF protein topology. (+info)
(5/110) Transmembrane-4-superfamily proteins CD151 and CD81 associate with alpha 3 beta 1 integrin, and selectively contribute to alpha 3 beta 1-dependent neurite outgrowth.
Proteins in the transmembrane-4-superfamily (TM4SF) form many different complexes with proteins in the integrin family, but the functional utility of these complexes has not yet been demonstrated. Here we show that TM4SF proteins CD151, CD81, and CD63 co-distribute with alpha3beta1 integrin on neurites and growth cones of human NT2N cells. Also, stable CD151-alpha3beta1 and CD81-alpha3beta1 complexes were recovered in NT2N detergent lysates. Total NT2N neurite outgrowth on laminin-5 (a ligand for alpha3beta1 integrin) was strongly inhibited by anti-CD151 and -CD81 antibodies either together ( approximately 85% inhibition) or alone ( approximately 45% inhibition). Notably, these antibodies had no inhibitory effect on NT2N neurites formed on laminin-1 or fibronectin, when alpha3beta1integrin was not engaged. Neurite number, length, and rate of extension were all affected by anti-TM4SF antibodies. In summary: (1) these substrate-dependent inhibition results strongly suggest that CD151 and CD81 associations with alpha3beta1 are functionally relevant, (2) TM4SF proteins CD151 and CD81 make a strong positive contribution toward neurite number, length, and rate of outgrowth, and (3) NT2N cells, a well-established model of immature central nervous system neurons, can be a powerful system for studies of integrin function in neurite outgrowth and growth cone motility. (+info)
(6/110) 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. (+info)
(7/110) Tetraspanins are localized at motility-related structures and involved in normal human keratinocyte wound healing migration.
We have described previously that beta1 integrins, which mediate keratinocyte cell adhesion and migration, are in ligand-occupied conformation at the basal surface but not at the lateral and apical surfaces of keratinocytes. This led us to study the cellular localization and function of tetraspanin molecules, which have been postulated to modulate integrin activity. We found that CD9 and CD81 are highly expressed by keratinocytes clearly delineating filopodia at lateral and apical surfaces. CD63 and CD151 are largely expressed in the intracellular compartment, although some membrane expression is observed. We found accumulation of CD9, CD81, and CD151 together with alpha3 and beta1 integrins at intercellular junctions. In low calcium medium, this intercellular space is crossed by a zipper of filopodia enriched in alpha3beta1 and tetraspanin proteins. Interestingly, the expression of CD9, CD81, and beta1 and alpha3 integrins was detected in the footprints and rippings of motile keratinocytes, suggesting their role in both adhesion to extracellular matrix and keratinocyte motility. beta1 integrins were only partially activated in the rips, whereas cytoskeleton-linking proteins such as talin were completely absent. On the other hand, antitetraspanin antibodies did not stain focal adhesions, which contain talin. The involvement of tetraspanins in keratinocyte motility was assessed in a wound healing migration assay. Inhibition of cell migration was observed with antibodies to CD9, CD81, beta1, and alpha3, and, to a lesser extent, to CD151. Together these results indicate that tetraspanin-integrin complexes might be involved in transient adhesion and integrin recycling during keratinocyte migration, as well as in intercellular recognition. (+info)
(8/110) 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. (+info)