Recruitment and tethering of talin to the plasma membrane initiate the process of integrin activation. Multiple factors including the Rap1 proteins, RIAM (also known as APBB1IP) and PIP2 bind talin proteins 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 the talin F0 domain to Rap1 synergizes with membrane lipid binding of the talin 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 to 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 talins to regulate integrin activity.

中文翻译：

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Rap1 和膜脂协同招募 talin 来触发整合素激活。


talin 的募集和束缚到质膜上启动整合素激活过程。包括 Rap1 蛋白、RIAM（也称为 APBB1IP）和 PIP2 在内的多种因子与踝蛋白结合，并已被提议调节这些过程，但尚未进行系统分析。通过将特定的talin突变体表达到talin无效的成纤维细胞中，我们发现talin F0结构域与Rap1的结合在talin膜靶向和整合素激活过程中与talin F2结构域的膜脂结合协同作用，而talin杆与RIAM的相互作用是可有可无。我们还通过详细的 NMR 分析表征了 talin F1 结构域内的第二个 Rap1 结合位点。有趣的是，虽然talin F1表现出比talin F0显着弱的Rap1结合亲和力，但talin F1 Rap1结合突变体的表达抑制细胞粘附、扩散、talin募集和整合素激活，与talin F0 Rap1结合突变体类似。此外，当两个 Rap1 结合位点发生突变时，缺陷变得明显更强。总之，我们的数据提出了一个模型，其中 Rap1 与踝蛋白 F0 和 F1 结构域的协同结合与膜 PIP2 与时空位置的结合产生协同作用，并激活踝蛋白以调节整合素活性。