Role of the Tail Domain in Vinculin Function

描述（由申请人提供）：本提案的重点是血管蛋白，一种细胞骨架蛋白，是局灶黏附和粘附连接的重要组成部分。Vinculin对细胞迁移，细胞存活和胚胎发生至关重要，在小鼠中Vinculin表达的破坏导致胚胎致死表型。Vinculin作为跨膜整合素受体和肌动蛋白细胞骨架之间的机械连接体，通过与细胞粘附分子的联系发挥作用。自抑制性头尾相互作用的释放被认为激活了vinculin作为支架蛋白的功能，以促进对细胞骨架结构和下游信号传导调节重要的多蛋白网络的组装。尽管vinculin与大量的细胞骨架蛋白结合，但在许多情况下，我们并不了解相互作用的性质或功能意义。例如，paxillin相互作用以及与血管蛋白尾部（Vt）结构域在血管蛋白功能中的作用尚不清楚。此外，已经提出了酸性磷脂（PL）的几种功能作用，包括酸性磷脂参与激活过程以及在激活后调节血管蛋白的功能。虽然最近的研究表明，vinculin/PL相互作用促进了肌动蛋白细胞骨架上粘附位点的解耦，但我们的初步数据表明，这些研究是在可能存在多种缺陷的突变体上进行的，包括PL结合。因此，虽然酸性PLs，特别是肌醇4,5二磷酸，被认为在血管蛋白调节和功能中起重要作用，但其作用尚不清楚。我们的工作将集中在核磁共振，生物物理，生化和细胞生物学的研究，主要集中在配体与Vt相互作用的作用。我们将研究Vt的结构特征，wt Vt与酸性PLs和paxillin的相互作用，并探讨这些相互作用在vinculin的生物学功能中的重要性。本提案的具体目的是研究：1)通过PL结合对毒蛋白的调控。2) Vt/paxillin复合物形成的结构基础及其相互作用在控制血管蛋白功能中的作用。血管蛋白是一种重要的蛋白质，参与控制细胞形状和运动，部分是通过将肌动蛋白聚合物锚定在膜上。这种功能也延伸到癌细胞，通过调节它们的运动和向身体其他部位的增殖。因此，不太令人惊讶的是，vinculin表现出肿瘤抑制蛋白的特性。vinculin的活性是通过调节其头部和尾部结构域之间的自我抑制或“自我”相互作用来控制的。这项工作的目标是表征尾部结构域与选定的蛋白质和脂质的相互作用，以更好地了解这些结合相互作用如何调节血管蛋白的活性和功能。从这些研究中获得的结果可能有助于开发新的治疗方法来对抗人类疾病中异常的血管蛋白活性。

DESCRIPTION (provided by applicant): This proposal is focused on vinculin, a cytoskeletal protein that is a prominent component of focal adhesions and adherens junctions. Vinculin is critical for cell migration, cell survival and embryogenesis and disruption of vinculin expression in mice results in an embryonic lethal phenotype. Vinculin functions as a mechanical linker between transmembrane integrin receptors and the actin cytoskeleton through association with cell adhesion molecules. Release of autoinhibitory head/tail interactions is believed to activate vinculin's function as a scaffold protein to facilitate assembly of multi-protein networks important for modulation of cytoskeleton structure and for downstream signaling. Although vinculin binds to a large number of cytoskeletal proteins, in many cases, we do not understand the nature of the interaction or functional significance. For example, the role of paxillin interactions and with the vinculin tail (Vt) domain in vinculin function is not known. In addition, several functional roles for acidic phospholipids (PL) have been proposed, including the involvement of acidic PLs in the activation process as well in regulating vinculin function once vinculin is activated. While recent studies suggest that vinculin/PL interactions promote uncoupling of adhesion sites from the actin cytoskeleton, our preliminary data indicates that these studies were conducted with mutants that are likely to multiple defects, including PL binding. Thus, while acidic PLs, specifically inositol 4, 5 bisphosphate, is believed to play an important role in vinculin regulation and function, its role remains unclear. Our efforts will focus on NMR, biophysical, biochemical and cell biological studies of vinculin, with the primarily focus on the role of ligand interactions with Vt. We will investigate the structural features of Vt, interactions of wt Vt with acidic PLs and paxillin, and explore the importance of these interactions in the biological function of vinculin. The specific aims of this proposal are to investigate the: 1) Regulation of vinculin through PL binding. 2) Structural basis for Vt/paxillin complex formation and the role of these interactions in controlling vinculin function. PUBLIC HEALTH RELEVANCE Vinculin is an essential protein involved controlling cell shape and motility, in part by anchoring actin polymers to the membrane. This function also extends to cancer cells, by regulating their movement and proliferation to other parts of the body. Hence, not too surprising, vinculin exhibits properties of a tumor suppressor protein. The activity of vinculin is controlled by regulating auto-inhibitory or `self' interactions between its head and tail domain. The goal of this effort is to characterize tail domain interactions with select proteins and lipids, to better understand how these binding interactions regulate vinculin activity and function. Results obtained from these studies may aid in developing novel therapeutic approaches for counteracting aberrant vinculin activity in human disease.