THE 2 PHENYLALANINES IN THE GFFKR MOTIF OF THE INTEGRIN ALPHA-6A SUBUNIT ARE ESSENTIAL FOR HETERODIMERIZATION

The membrane-proximal domain of the integrin alpha subunit contains a conserved motif of five amino acid residues, GFFKR. We deleted this mo tif from the human alpha 6A subunit and found that in COS-7 cells this mutant cannot associate with the beta 1 subunit and is retained in th e endoplasmic reticulum. Point mutations in the GFFKR motif of the gly cine residue or the two highly charged amino acids, or deletion of the lysine and arginine residues, had no effect on the ability of alpha 6 to interact with beta 1 and to be expressed at the cell surface. In c ontrast, by replacing either of the two phenylalanines with alanine, o r by deletion of both of these residues, alpha 6 was incapable of asso ciating with beta 1. The alpha 6 point mutants that associated with be ta 1 were expressed in K562 cells and their responsiveness to integrin -activating factors was determined. None of these transfectants bound spontaneously to laminin-1, but binding could be induced by either PMA or the stimulating anti-beta 1 antibody TS2/16 to the same extent as that of the wild-type transfectant. The ability of these mutants to in itiate focal-contact formation in CHO cells plated on laminin-1 substr ates also appeared to be unaltered. Thus the behaviour of alpha 6 muta nts involving the glycine, lysine or arginine residues was indistingui shable from that of wild-type alpha 6 both in inside-out and outside-i n signalling. In contrast, deletion of the cytoplasmic domain of alpha 6 C-terminal of the GFFKR motif resulted in a loss of responsiveness of alpha 6 beta 1 to PMA stimulation and formation of focal contacts o n laminin-1. However, this mutant was targeted to focal contacts forme d by other integrins, even when they had not bound ligand. Together, t hese results suggest that the two phenylalanine residues of the GFFKR motif provide a site for interaction of the alpha 6A subunit with beta 1, whereas the cytoplasmic domain C-terminal of this motif is involve d in the regulation of bidirectional signalling via alpha 6A beta 1.