Rap1 and membrane lipids cooperatively recruit talin to trigger integrin activation

Abstract

Recruitment and tethering of talin to the plasma membrane initiate the process of integrin activation. Multiple factors including Rap1, RIAM, and PIP2 bind talin and have been proposed to regulate these processes, but not systematically analyzed. By expressing specific talin mutants into talin-null fibroblasts, we show that binding of talińs F0 domain to Rap1 synergizes with membrane lipid binding of talińs F2 domain during talin membrane targeting and integrin activation, whereas the interaction of the talin rod with RIAM was dispensable. We also characterized a second Rap1 binding site within the talin F1 domain by detailed NMR analysis. Interestingly, while talin F1 exhibited significantly weaker Rap1-binding affinity than talin F0, expression of a talin F1 Rap1-binding mutant inhibited cell adhesion, spreading, talin recruitment and integrin activation similarly as the talin F0 Rap1-binding mutant. Moreover, the defects became significantly stronger when both Rap1-binding sites were mutated. In conclusion, our data suggest a model in which cooperative binding of Rap1 to the talin F0 and F1 domains synergizes with membrane PIP2 binding to spatiotemporally position and activate talin to regulate integrin activity.