Abstract
The membrane-proximal domain of the integrin alpha subunit contains a conserved motif of five amino acid residues, GFFKR. We deleted this motif from the human alpha6A subunit and found that in COS-7 cells this mutant cannot associate with the beta1 subunit and is retained in the endoplasmic reticulum. Point mutations in the GFFKR motif of the glycine residue or the two highly charged amino acids, or deletion of the lysine and arginine residues, had no effect on the ability of alpha6 to interact with beta1 and to be expressed at the cell surface. In contrast, by replacing either of the two phenylalanines with alanine, or by deletion of both of these residues, alpha6 was incapable of associating with beta1. The alpha6 point mutants that associated with beta1 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-beta1 antibody TS2/16 to the same extent as that of the wild-type transfectant. The ability of these mutants to initiate focal-contact formation in CHO cells plated on laminin-1 substrates also appeared to be unaltered. Thus the behaviour of alpha6 mutants involving the glycine, lysine or arginine residues was indistinguishable from that of wild-type alpha6 both in inside-out and outside-in signalling. In contrast, deletion of the cytoplasmic domain of alpha6 C-terminal of the GFFKR motif resulted in a loss of responsiveness of alpha6beta1 to PMA stimulation and formation of focal contacts on laminin-1. However, this mutant was targeted to focal contacts formed by other integrins, even when they had not bound ligand. Together, these results suggest that the two phenylalanine residues of the GFFKR motif provide a site for interaction of the alpha6A subunit with beta1, whereas the cytoplasmic domain C-terminal of this motif is involved in the regulation of bidirectional signalling via alpha6Abeta1.