Functional significance of the NPLY beta-turn motif in the cytoplasmic tail of the integrin beta3 subunit

Following platelet aggregation, integrin alphaIIbbeta 3 becomes associated with the platelet cytoskeleton, and this process has been implicated in several alphaIIbbeta3-dependent post-occupancy events. The conserved NPLY sequence represents a potential beta-turn motif in the cytoplasmic tail of the beta3 subunit and has been suggested to mediate cytoskeletal attachment. To further examine whether the NPLY sequence is involved in mediating cytoskeletal-dependent post-ligand binding events, we performed a double mutation (N744Q/P745A) which was predicted to alter this beta-turn into an alpha-helical structure. CHO cells were co-transfected with cDNA constructs encoding mutant beta3 and wild type alphaIIb. Flow cytometry analyses showed that cells expressing either wild-type (A5) or mutant (D4) alphaIIbbeta 3 interacted with soluble FITC-conjugated fibrinogen in the presence of an alphaIIbbeta3 activating antibody. Furthermore, both A5 and D4 cells adhered to a similar extent to immobilized fibrinogen in an RGD-dependent manner. However, as opposed to control A5 cells, adherent D4 cells failed to spread, form focal adhesions, or initiate protein tyrosine phosphorylation. Additionally, the capacity of D4 cells to support retraction of fibrin clots was substantially reduced as compared to control A5 cells. Since the NPXY motif has been implicated in mediating receptor internalization, we examined the ability of A5 and D4 cells to internalize alphaIIb beta3 and alphavbeta3 by flow cytometry. The percent internalization of alphaIIbbeta 3 and alphavbeta3 was similar in both the cell types. To investigate the role of the NPLY motif in talin binding, we examined the ability of the mutant alphaIIbbeta3 to interact with talin. In these experiments, both wild-type and mutant alpha IIbbeta3, were found to bind to a similar extent to immobilized talin. Furthermore, purified talin bound to microtiter wells coated with CHDRKEFAKFEEERARA, but not CAKWDTANNPLYK. Thus, these findings suggest that the talin binding domain in beta3 integrins does not reside in the NPLY motif. Additionally, talin binding site was localized to the positively charged amino acids R724/K725 in the membrane proximal region of the beta3 cytoplasmic domain.;Collectively, these studies demonstrate that the N744Q/P745A mutation of the integrin beta3 subunit is essential for mediating post-ligand binding events of alphaIIbbeta3 in a manner independent of talin interaction.