Identification of a Talin-binding Site in the Integrin β3 Subunit Distinct from the NPLY Regulatory Motif of Post-ligand Binding Functions

Following platelet aggregation, integrin α IIb β 3 becomes associated with the platelet cytoskeleton. The conserved NPLY sequence represents a potential β-turn motif in the β 3 cytoplasmic tail and has been suggested to mediate the interaction of β 3 integrins with talin. In the present study, we performed a double mutation (N744Q/P745A) in the integrin β 3 subunit to test the functional significance of this β-turn motif. Chinese hamster ovary cells were co-transfected with cDNA constructs encoding mutant β 3 and wild type α IIb . Cells expressing either wild type (A5) or mutant (D4) α IIb β 3 adhered to fibrinogen; however, as opposed to control A5 cells, adherent D4 cells failed to spread, form focal adhesions, or initiate protein tyrosine phosphorylation. To investigate the role of the NPLY motif in talin binding, we examined the ability of the mutant α IIb β 3 to interact with talin in a solid phase binding assay. Both wild type and mutant α IIb β 3 , purified by RGD affinity chromatography, bound to a similar extent to immobilized talin. Additionally, purified talin failed to interact with peptides containing the AKWDTANNPLYK sequence indicating that the talin binding domain in the integrin β 3 subunit does not reside in the NPLY motif. In contrast, specific binding of talin to peptides containing the membrane-proximal HDRKEFAKFEEERARAK sequence of the β 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 purified platelet α IIb β 3 bound dose-dependently to immobilized rTalin-N, indicating that an integrin-binding site is present in the talin N-terminal head domain. Collectively, these studies demonstrate that the NPLY β-turn motif regulates post-ligand binding functions of α IIb β 3 in a manner independent of talin interaction. Moreover, talin was shown to bind through its N-terminal head domain to the membrane-proximal sequence of the β 3 cytoplasmic tail.