Structure and function of integrins in the kidney

描述（由申请人提供）：整合素是一种异二聚体细胞粘附受体，通过与细胞外基质（ECM）的严格调节的金属依赖性相互作用，在发育和成年肾脏中发挥重要作用，调节细胞迁移、增殖、分化、基质沉积和重塑。整合素与ECM配体的结合需要在细胞骨架talin或kindlin与β-亚基胞质结构域结合后启动的活化步骤。这种相互作用打破了胞质结构域中的胞内α盐桥，触发了穿过α跨膜（TM）和小腿结构域的构象波，以激活配体结合头部结构域。ECM配体的结合然后触发整合素的结构变化，其以相反的方向行进到细胞内部，从而改变细胞行为。晶体结构的unliganded？TM-AV <$3和？所有的一切3胞外域和这些胞外域的结构结合到短肽配体-扩散到预先形成的蛋白质晶体-鉴定了胞外域的意外弯曲构象，并阐明了配体结合的金属离子依赖性的结构基础。但是，由于这些结构缺乏关键的外表面残基，以及TM和细胞质结构域，并且未被大分子ECM配体从头占据，因此在原子水平上对双向整合素信号传导的全面理解仍然是难以捉摸的。在初步研究中，我们已经确定了1 TM-aV <$3的晶体结构，它编码胞外域的完整序列加上一个a/<$TM片段。第二，我们生产了第一个全长整合素的电子冷冻显微镜结构，在一个弯曲的构象与解决功能的TM域。第三，我们获得了整合素与大分子配体和活化mAb的复合物的衍射质量晶体。第四，我们证明了在细菌中表达的TM-胞质结构域可以在胞外域不存在的情况下通过两个TM界面之一形成异二聚体，其中一个界面存在于无活性的全长整合素中。第五，我们展示了通过荧光寿命成像显微镜评估活哺乳动物细胞中整合素构象状态的可行性。这些发现构成了新假设的基础，这些新假设在三个具体目标中进行了测试。从拟议的研究中得出的结果应该回答整合素介导的细胞粘附生物学中的核心问题，并定义双向信号传导的结构基础。 公共卫生相关性：肾脏结构和功能的形成和维持关键取决于称为整合素的细胞粘附受体家族的正常功能。肾脏发育不全、蛋白尿、有害炎症和移植排斥反应是整合素功能异常的表现。阐明整合素如何在原子水平上调节对于理解其生物学作用至关重要，这些信息可以用于设计新的治疗方法。

DESCRIPTION (provided by applicant): Integrins are a/¿ heterodimeric cell adhesion receptors that play essential roles in the developing and adult kidney, through tightly regulated metal-dependent interactions with the extracellular matrix (ECM) that modulate cell migration, proliferation, differentiation, matrix deposition and remodeling. Binding of integrins to ECM ligands requires an activation step initiated upon binding of cytoskeletal talin or kindlin to the ¿-subunit cytoplasmic domain. This interaction breaks an intracellular a/¿ salt bridge in the cytoplasmic domains, triggering a conformational wave that travels through the a/¿ transmembrane (TM) and lower leg domains to activate the ligand-binding head domain. Binding of ECM ligands then triggers structural changes in the integrin that travel in the opposite direction to the cell interior, modifying cell behavior. Crystal structures of unliganded ?TM-aV¿3 and ?TM-aII¿3 ectodomains and structures of these ectodomains bound to short peptide ligands- diffused into the preformed protein crystals- identified an unexpected bent conformation of the ectodomain and elucidated the structural basis of metal ion dependency for ligand binding. But as these structures are lacking critical exofacial residues, as well as the TM and cytoplasmic domains, and were unoccupied by macromolecular ECM ligands de novo, a comprehensive understanding of bidirectional integrin signaling at the atomic level remains elusive. In preliminary studies, we have determined the crystal structure of 1TM-aV¿3, which encodes the complete sequence of the ectodomain plus an a/¿ TM fragment. Second, we produced the first electron cryomicroscopy structure of a full-length integrin in a bent conformation with resolved features of the TM domains. Third, we obtained diffraction quality crystals of the integrin in complex with a macromolecular ligand and with an activating mAb. Fourth, we demonstrate that a/¿ TM-cytoplasmic domains expressed in bacteria can form heterodimers in the absence of the ectodomain, through one of two TM interfaces, one of which is found in the inactive full-length integrin. Fifth, we show the feasibility of assessing integrin conformational states in live mammalian cells by fluorescence lifetime imaging microscopy. These findings form the basis of new hypotheses that are tested in three specific aims. The results derived from the proposed studies should answer central questions in the biology of integrin-mediated cell adhesion and define the structural basis of bidirectional signaling. PUBLIC HEALTH RELEVANCE: Formation and maintenance of kidney structure and function critically depends on the normal function of a family of cell adhesion receptors named integrins. Kidney agenesis, proteinuria, harmful inflammation and graft rejection are manifestations of abnormal integrin function. Elucidating how integrins are regulated at an atomic level is essential in understanding their biologic role, information that could be harnessed in devising new therapeutics.