FUNCTIONAL-ROLE OF THE CYTOPLASMIC DOMAIN OF THE INTEGRIN-ALPHA-5 SUBUNIT

The purpose of this study was to explore the functional role of the cytoplasmic domain of the alpha subunit of the alpha5/beta1 integrin, a fibronectin receptor. Mutant CHO cells that express very low levels of endogenous hamster alpha5 subunit (CHO clone B2) were transfected with an expression vector containing full-length or truncated human alpha5 cDNAs to form chimeric human alpha5/hamster beta1 integrins. Three transfectants were examined: B2a27 expresses a full-length human alpha5 subunit with 27 amino acids in the cytoplasmic domain; B2a10 expresses an alpha5 with a 17-amino acid cytoplasmic truncation; B2a1 expresses an alpha5 with a 26-amino acid truncation. Levels of alpha5/beta1 surface expression in B2a27 and B2a10 cells were similar to that in wild type CHO cells. The expression of alpha5/beta1 in B2a1 cells was less, amounting to 15-20% of WT levels, despite message levels that were three to five times greater than those of B2a27. The transfectants were used to examine the role of the alpha5 cytoplasmic domain in cell adhesion, cell motility, cytoskeletal organization, and integrin-mediated tyrosine phosphorylation. The adhesion characteristics of B2a27 and B2a10 cells on fibronectin substrata were similar to each other and to wild type CHO cells. B2a1 cells displayed slight reductions in the strength and rate of adhesion to fibronectin. Cell motility in the presence of fibronectin was similar for B2a27, B2a10, and wild type CHO cells, while the B2a1 cells were substantially less motile. Comparable degrees of cell spreading and extensive organization of actin filaments were observed for B2a27, B2a10, and wild type CHO cells on fibronectin substrata. The B2a1 cells spread to a lesser degree, and some organization of actin was observed; the untransfected B2 cells remained round on fibronectin substrata and showed no actin reorganization. Since the reduced motility and cell spreading observed in the B2a1 cells might be due either to reduced surface expression of alpha5/beta1 or to the truncation in the alpha5 cytoplasmic domain, we used flow cytometric cell sorting to select populations of B2a1 and B2a27 cells expressing similar levels of cell surface alpha5. The deficits in spreading and motility were present in B2a1 cells expressing high levels of alpha5. Thus the region of the alpha5 cytoplasmic domain adjacent to the membrane seems to play an important role in cytoskeletal organization and cell motility. We also examined whether alpha subunit truncation would affect integrin-mediated tyrosine phosphorylation. When B2a27 cells interacted with fibronectin substrata, increased tyrosine phosphorylation was observed in proteins of approximately 125 kD. A similar pattern of phosphorylation was observed in wild type CHO, B2a10, and B2a1 cells, but not in B2 cells. Thus, the alpha5 cytoplasmic domain does not seem to be essential for integrin-mediated tyrosine phosphorylation of intracellular proteins.