UC San Diego

Upon vascular injury, platelet stimulation with agonists permits αIIbβ3 integrin to bind its cognate ligands to enable stable adherences to the vessel wall and platelet aggregation. Integrins remain in a low-affinity state until intracellular signaling pathways induce a high-affinity form, a process defined as integrin activation. Binding of the cytosolic adapter protein talin1 to the integrin β cytoplasmic tail is a critical, final step in integrin activation. Previous studies implicated GTPases, Rap1a and Rap1b, in regulating talin1 binding to integrins in platelets. Structural and biochemical evidence showed a direct interaction between Rap1 and talin1 F0 domain, further confirmed in Dictyostelium. We hypothesized that Rap1 may directly interact with talin1 in platelets leading to its association with the β3 cytoplasmic tail to activate αIIbβ3. A mouse strain harboring the R35E mutation in talin1 F0 domain that blocks binding to Rap1 was generated. There was no significant defect in the ability of the Tln1R35E/R35E platelets to activate αIIbβ3 integrins. RIAM, a known Rap1 effector, and upregulations of talin2 were excluded as compensatory mechanisms. Results indicated that Rap1b binding to talin1 F0 domain makes a minimal contribution in activating platelet αIIbβ3. Remarkably, Rap1 also binds to talin1 F1 domain, a structural homolog to the F0 domain. Disrupting this interaction prevents αIIbβ3 integrin activation in a CHO cell model system. These findings suggest that talin1 may function as a direct effector of Rap1 through its F1 domain. Future studies will investigate the Rap1-talin1 F1 interaction in murine platelets by mutating the F1 domain.