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

Abstract

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'...

Tetraspanin CD151 regulates α6β1 integrin adhesion strengthening

“…To observe cellular cable formation, cells were plated on a thick layer of Matrigel in 5% FBS͞DMEM at 5 ϫ 10 4 cells per well in a 24-well plate, analyzed by using a Zeiss Axiovert 135 microscope, and photographed after 18 h as described (20). To measure static cell adhesion, cells were incubated on laminin-1 (coated at 20 g͞ml) for varying times, washed, and attached cells were quantitated by using the Cytofluor 2300 measurement system (Millipore) as described (33).…”

“…In this regard, only the laminin-binding integrins (␣3␤1, ␣6␤1, ␣6␤4, and ␣7␤1) show strong lateral association with CD151, a transmembrane protein in the tetraspanin family (13)(14)(15)(16). mAb perturbation studies indicate that CD151 modulates integrin-dependent migration, neurite outgrowth, and cell morphology on Matrigel (17)(18)(19)(20). The short C-terminal cytoplasmic domain of CD151 was particularly important for ␣6␤1 integrin-mediated spreading, migration, and cellular cable formation on Matrigel (20).…”

“…mAb perturbation studies indicate that CD151 modulates integrin-dependent migration, neurite outgrowth, and cell morphology on Matrigel (17)(18)(19)(20). The short C-terminal cytoplasmic domain of CD151 was particularly important for ␣6␤1 integrin-mediated spreading, migration, and cellular cable formation on Matrigel (20). Besides CD151, several other members of the tetraspanin protein family (such as CD9, CD81, and CD63) also regulate integrin-dependent cell migration.…”

“…Because CD151 strongly influences ␣6␤1 integrin-dependent cellular cable formation on Matrigel (20), we hypothesized that CD151 could be regulating strength of adhesion mediated through the integrin. To test this hypothesis directly, we engaged ␣6␤1 integrin with laminin-coated beads, exerted a defined mechanical force on the beads, and analyzed bead responses in terms of bead detachment.…”

Les tétraspanines dans la physiopathologie de la peau

“…Dans la décennie qui suivit, la fonction dans la régu-lation de ces processus cellulaires d'autres tétraspa-nines, en interaction avec les intégrines, a été étudiée. Ainsi, par des expériences d'immunofluorescence, il a été montré que les protéines CD9, CD81 et CD151 sont fortement exprimées dans les kératinocytes en culture α3β1, α6β1 et α4β1 [5][6][7] pour réguler leurs fonctions, comme l'attestent des expériences de mutagenèse dirigée visant à introduire des mutations dans les domaines fonctionnels de CD151 [8][9][10]. Les tétraspanines possèdent une structure commune comprenant quatre régions hydrophobes hautement conservées, transmembranaires, flanquées de courtes séquences intracytoplasmiques N-et C-terminales et de deux boucles extracellulaires de tailles inégales (les domaines EC1 et EC2) (Figure 1).…”