Structural Basis for Talin-Mediated Integrin Activation

血小板受体α-IIb-β3属于一类细胞黏附受体，即整合素。Alpha-IIb-Beta3介导血小板聚集，这对止血是必不可少的，但也可能导致急性闭塞性血栓的形成，导致心脏病发作和中风。α-IIb-beta3的核心功能是其经历激活的能力，即从低亲和力/亲和力状态转变为配体识别的高亲和力状态。α-IIb-β3的小细胞质表面 α-IIb-β3通过一种独特的称为“内向外”的信号传递过程来调节这种转变，即在凝血酶或ADP等激动剂刺激血小板时，α-IIb-β3的细胞质表面发生构象变化，并传播到细胞外区域，使其以高亲和力结合可溶性纤维蛋白原或von Willebrand因子。在过去的十年里，直接启动这种构象变化的确切信号(S)或蛋白质(S)一直在进行深入的研究。塔林为一个这样的激活信号提供了一个模型。Talin是一种细胞骨架蛋白，直接与α-IIb-β3细胞质表面结合，激活 受体。缺失和生化分析表明，talin的N末端头域(talin-H)或其较小片段(talin-HS)特异性识别β3细胞质尾部，并负责诱导α-IIb-β3的激活。有趣的是，完整的talin对beta3尾巴的亲和力显著低于分离的talin-H，这表明talin-beta3相互作用是基于构象的调控过程。分子水平上对talin-beta3相互作用在调节α-IIb-beta3激活中的了解仍不清楚。我们建议 通过使用高度综合和系统的结构/生化方法来解决这一问题。我们将测定talin-HS结构域(27 KDa)与Beta3细胞质尾部(5 KDa)的复合体的核磁共振结构，并进行基于结构的突变，以评估相互作用在介导整合素激活中的意义。我们还将研究调节整合素激活的其他因素，如酪氨酸-磷酸化和整合素-骨架相互作用。最后，我们将研究talin-H/beta3相互作用如何经历从低亲和力状态到高亲和力状态的转变。这些研究将对理解α-IIb-β3的激活机制产生重大影响，这最终将为深入了解 血栓形成的基本过程。

Platelet receptor alpha-IIb-beta3 belongs to a class of cell adhesion receptors, the integrins. alpha-IIb-beta3 mediates platelet aggregation, which is essential for hemostasis but also can result in the formation of acute occlusive thrombi, leading to heart attacks and strokes. Central to the function of alpha-IIb-beta3 is its capacity to undergo activation, a transition from a low to a high affinity/avidity state for ligand recognition. The small cytoplasmic face of alpha-IIb-beta3 regulates such transition through a distinct process called "inside-out" signaling, i.e., upon platelet stimulation by agonists, such as thrombin or ADP, the cytoplasmic face of alpha-IIb-beta3 undergoes a conformational change that propagates to the extracellutar domain allowing it to bind soluble fibrinogen or von Willebrand Factor with high affinity. The exact signal(s) or protein(s) that directly initiates this conformational change has been under intensive investigation over the past decade. Talin provides a model for one such activating signal. Talin is a cytoskeletal protein that binds directly to the alpha-IIb-beta3 cytoplasmic face and activates the receptor. Deletion and biochemical analyses revealed that the N-terminal head domain of talin (talin-H) or its smaller fragment (talin-HS) specifically recognizes the beta3 cytoplasmic tail and is responsible for inducing the alpha-IIb-beta3 activation. Interestingly, intact talin has significantly lower affinity for the beta3 tail than the isolated talin-H, suggesting a conformation-based regulatory process for the talin-beta3 interaction. A molecular level understanding of the talin-beta3 interaction in regulating the alpha-IIb-beta3 activation remains unclear. We propose to address this issue by using highly integrated and systematic structural/biochemical approaches. We will determine NMR structure of the talin-HS domain (27 kDa) in complex with the beta3 cytoplasmic tail (5 kDa) and perform structure-based mutagenesis to evaluate the significance of the interaction in mediating the integrin activation. We will also examine other factors in regulating the integrin activation such as tyrosine-phosphorylation and integrin-skelemin interaction. Finally we will investigate how the talin-H/beta3 interaction undergoes the transition from a lower affinity state to a higher affinity state. These studies will significantly impact on understanding the mechanisms of alpha-IIb-beta3 activation, which will ultimately provide insight into a fundamental process in thrombosis.