Structure and function of integrins in the kidney

描述(申请人提供)：整合素是一种异二聚体细胞黏附受体，通过严格调控与细胞外基质(ECM)的金属依赖性相互作用，调节细胞迁移、增殖、分化、基质沉积和重塑，在发育和成人肾脏中发挥重要作用。整合素与ECM配体的结合需要在细胞骨架talin或kindlin与？亚单位胞质结构域结合时启动激活步骤。这种相互作用破坏了细胞质区域内的a/？盐桥，触发了通过a/？跨膜(TM)区域和小腿区域的构象波，从而激活了配体结合的头部区域。ECM配体的结合然后触发整合素的结构变化，这些结构变化以相反的方向传播到细胞内部，改变细胞的行为。未连接的TM-AV3和TM-AII3胞外区的晶体结构以及这些胞外区与短肽配体结合的结构-扩散到预制的蛋白质晶体中-鉴定了胞外区意外的弯曲构象，并阐明了金属离子依赖于配体结合的结构基础。但是，由于这些结构缺乏关键的表面残基以及TM和细胞质结构域，并且没有被大分子ECM配体从头开始占据，因此在原子水平上对双向整合素信号的全面理解仍然是遥不可及的。在初步研究中，我们确定了1TM-AV？3的晶体结构，它编码了胞外结构域的完整序列和一个a/？TM片段。第二，我们制作了第一个全长整合素的弯曲构象的电子冷冻显微镜结构，具有TM结构域的可分辨特征。第三，我们得到了整合素与大分子配体和活化单抗形成的络合物的衍射性晶体。第四，我们证明了在细菌中表达的a/？TM-细胞质结构域可以在没有胞外结构域的情况下通过两个TM界面中的一个形成异二聚体，其中一个在不活跃的全长整合素中被发现。第五，我们证明了用荧光寿命成像显微镜评估活的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说的基础，这些假说在三个具体目标上进行了检验。从拟议的研究中得出的结果应该回答了整合素介导的细胞黏附生物学中的核心问题，并确定了双向信号的结构基础。 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一类细胞黏附受体的正常功能，该家族被称为整合素。肾发育不全、蛋白尿、有害炎症和移植物排斥反应是整合素功能异常的表现。阐明整合素是如何在原子水平上被调控的，对于理解它们的生物学作用是至关重要的，这些信息可以被用来设计新的疗法。

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.