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

Abstract

Following platelet aggregation, integrin IIb 3 becomes associated with the platelet cytoskeleton. The conserved NPLY sequence represents a potential-turn motif in the 3 cytoplasmic tail and has been suggested to mediate the interaction of 3 integrins with talin. In the present study, we performed a double mutation (N744Q/P745A) in the integrin 3 subunit to test the functional significance of this-turn motif. Chinese hamster ovary cells were co-transfected with cDNA constructs encoding mutant 3 and wild type IIb. Cells expressing either wild type (A5) or mutant (D4) IIb 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 IIb 3 to interact with talin in a solid phase binding assay. Both wild type and mutant IIb 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 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 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 IIb 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-turn motif regulates post-ligand binding functions of IIb 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 3 cytoplasmic tail.