Identification of a talin-binding site in the integrin beta(3) subunit distinct from the NPLY regulatory motif of post-ligand binding functions - Thetalin N-terminal head domain interacts with the membrane-proximal region of the beta(3) cytoplasmic tail

Following platelet aggregation, integrin alpha(IIb)beta(3) becomes associat ed with the platelet cytoskeleton, The conserved NPLY sequence represents a potential beta-turn motif in the beta(3) cytoplasmic tail and has been sug gested to mediate the interaction of beta(3) integrins with talin, In the p resent study, we performed a double mutation (N744Q/P745A) in the integrin beta(3) subunit to test the functional significance of this beta-turn motif . Chinese hamster ovary cells were co-transfected with cDNA constructs enco ding mutant beta(3) and mild type alpha(IIb). Cells expressing either wild type (A5) or mutant (D4) alpha(IIb)beta(3) adhered to fibrinogen; however, as opposed to control A5 cells, adherent D4 cells failed to spread, form fo cal adhesions, or initiate protein tyrosine phosphorylation, To investigate the role of the NPLY motif in talin binding, we examined the ability of th e mutant alpha(IIb)beta(3) to interact with talin in a solid phase binding assay. Both mild type and mutant alpha(IIb)beta(3), purified by RGD affinit y chromatography, bound to a similar extent to immobilized talin, Additiona lly, purified talin failed to interact with peptides containing the AKWDTAN NPLYK sequence indicating that the talin binding domain in the integrin bet a(3) subunit does not reside in the NPLY motif. In contrast, specific bindi ng of talin to peptides containing the membrane-proximal HDRKEFAKFEEERARAK sequence of the beta(3) cytoplasmic tail was observed, and this interaction was blocked by a recombinant protein fragment corresponding to the 47-kDa N-terminal head domain of talin (rTalin-N), In addition, RGD affinity purif ied platelet alpha(IIb)beta(3) bound dose-dependently to immobilized rTalin -N, indicating that an integrin-binding site is present in the talin N-term inal head domain. Collectively, these studies demonstrate that the NPLY bet a-turn motif regulates post-ligand binding functions of alpha(IIb)beta(3) i n a manner independent of talin interaction. Moreover, talin was shown to b ind through its N-terminal head domain to the membrane-proximal sequence of the beta(3) cytoplasmic tail.