Max-Planck-Institute of Biochemistry, Department of Molecular Medicine, 82152 Martinsried, Germany.
Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Ave., Cleveland, OH 44195, USA.
J Cell Sci. 2019 Nov 1;132(21):jcs235531. doi: 10.1242/jcs.235531.
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 PIP 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 PIP binding to spatiotemporally position and activate talins to regulate integrin activity.
招募并将 talin 连接到质膜上，从而启动整合素激活过程。多种因素包括 Rap1 蛋白、RIAM（也称为 APBB1IP）和 PIP 结合 talin 蛋白，据推测它们可以调节这些过程，但尚未得到系统分析。通过将特定的 talin 突变体表达到 talin 缺失型成纤维细胞中，我们表明，talin F0 结构域与 Rap1 的结合与 talin F2 结构域与质膜脂质的结合在 talin 靶向质膜和整合素激活过程中协同作用，而 talin 杆与 RIAM 的相互作用是可有可无的。我们还通过详细的 NMR 分析，对 talin F1 结构域中的第二个 Rap1 结合位点进行了表征。有趣的是，尽管 talin F1 与 Rap1 的结合亲和力明显弱于 talin F0，但表达 talin F1 Rap1 结合突变体可抑制细胞黏附、铺展、talin 募集和整合素激活，与 talin F0 Rap1 结合突变体相似。此外，当两个 Rap1 结合位点都发生突变时，缺陷变得更加明显。总之，我们的数据表明，Rap1 与 talin F0 和 F1 结构域的协同结合与质膜 PIP 的结合共同作用，以时空方式定位并激活 talin，从而调节整合素的活性。
