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 (␣ 3 ␤ 1 , ␣ 6 ␤ 1 , and a chimeric "X3TC5" ␣ 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 ␣ 3 subunit and to intracellular PKC helps to explain why the integrin ␣3 extracellular domain is needed for both intracellular PKC recruitment and PKC-dependent phosphorylation of the ␣ 3 integrin cytoplasmic tail.

Upon plating on basement membrane Matrigel, NIH3T3 cells formed an anastomosing network of cord-like structures, inhibitable by anti-␣6␤1 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 ␣6␤1 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 ␣6␤1-dependent cell network formation and NIH3T3 cell spreading on laminin-1 (an ␣6␤1 ligand). Importantly, mutant CD151 retained ␣6␤1 association and did not alter ␣6␤1-mediated cell adhesion to Matrigel. In conclusion, the CD151-␣6␤1 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 ␣6␤1-integrin that follow ligand engagement. Also, antibodies to ␣6␤1 and CD151 inhibited formation of endothelial cell cord-like networks, thus pointing to possible relevance of CD151-␣6␤1 complexes during angiogenesis. INTRODUCTIONStudies of integrin-dependent adhesion, migration, and signaling have focused largely on integrin ligand binding sites (Plow et al., 2000) and on cytoplasmic domains (Liu et al., 2000). Cytoplasmic domain perturbations alter ligand binding ("inside-out" signaling) and ligand binding triggers long-range alterations in cytoplasmic domain interactions ("outside-in" signaling). Integrin functions are modulated also by lateral associations with other transmembrane proteins (Hemler, 1998;Woods and Couchman, 2000). As shown here, outside-in signaling through ␣6␤1 integrin is markedly influenced by its lateral association with CD151, a transmembrane-4 superfamily (TM4SF, tetraspanin) protein.Tetraspanin proteins contain two extracellular loops, four hydrophobic transmembrane domains, and two short cytoplasmic tails. Tetraspanins regulate membrane fusion, trafficking, cell motility, and tumor metastasis (Wright and Tomlinson, 1994;. Tetraspanins form multimolecular complexes with many other transmembrane proteins, including integrins (Hemler et al., 1996;Hemler, 1998). Despite several reports of tetraspanin-protein complexes, only a few have documented functional relevance. For example, results from CD81-null mice support the relevance of CD81-CD19 association Miyazaki et al., 1997;Tsitsikov et al., 1997), CD9 influences the activity of associated HB-EGF (Iwamoto et al., 1994), and antibodies to CD81 and CD151 inhibit the functions of associated integrins (Domanico et al., 1997;Yánez-Mó et al., 1998;Yauch et al., 1998;Stipp and Hemler, 2000). Notably, anti-CD81 and anti-CD151 antibodies inhibited neurite outgrowth only when associated ␣3␤1 integrin was engaged with ligand (Stipp and Hemler, 2000).Among tetraspanin complexes, the CD151-␣3␤1 integrin complex has unusually high stoichiometry, proximity, and stability. A specific site in CD151'...

Integrin alpha 3A cytoplasmic tail phosphorylation was mapped to amino acid S1042, as determined by mass spectrometry, and confirmed by mutagenesis. This residue occurs within a "QPSXXE" motif conserved in multiple alpha chains (alpha 3A, alpha 6A, alpha 7A), from multiple species. Phosphorylation of alpha 3A and alpha 6A did not appear to be directly mediated by protein kinase C (PKC) alpha, beta, gamma, delta, epsilon, zeta, or mu, or by any of several other known serine kinases, although PKC has an indirect role in promoting phosphorylation. A S1042A mutation did not affect alpha 3-Chinese hamster ovary (CHO) cell adhesion to laminin-5, but did alter 1) alpha 3-dependent tyrosine phosphorylation of focal adhesion kinase and paxillin (in the presence or absence of phorbol 12-myristate 13 acetate stimulation), and p130(CAS) (in the absence of phorbol 12-myristate 13 acetate stimulation), 2) the shape of cells spread on laminin-5, and 3) alpha 3-dependent random CHO cell migration on laminin-5. In addition, S1042A mutation altered the PKC-dependent, ligand-dependent subcellular distribution of alpha 3 and F-actin in CHO cells. Together, the results demonstrate clearly that alpha 3A phosphorylation is functionally relevant. In addition, the results strongly suggest that alpha 3 phosphorylation may regulate alpha 3 integrin interaction with the cytoskeleton.