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，在发育和成人肾脏中发挥重要作用，通过与细胞外基质（ECM）紧密调节金属依赖的相互作用，调节细胞迁移、增殖、分化、基质沉积和重塑。整合素与ECM配体的结合需要细胞骨架talin或kindlin与亚基细胞质结构域结合后的激活步骤。这种相互作用打破了细胞质域的胞内a/¿盐桥，引发了通过a/¿跨膜（TM）和下肢域的构象波，从而激活了配体结合的头域。ECM配体的结合引发了整合素的结构变化，这种变化与细胞内部的运动方向相反，从而改变了细胞的行为。无配体的晶体结构？TM-aV¿3和？TM-aII¿3外畴和这些外畴与短肽配体结合的结构-扩散到预形成的蛋白质晶体中-鉴定了外畴意想不到的弯曲构象，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难以捉摸。在初步研究中，我们已经确定了1TM-aV¿3的晶体结构，它编码完整的外结构域序列和a/¿TM片段。其次，我们制作了具有TM结构域的弯曲构象的全长整合素的第一个电子低温显微镜结构。第三，我们获得了具有大分子配体和活化单抗的整合素复合物的衍射质量晶体。第四，我们证明在细菌中表达的a/¿TM胞质结构域可以通过两个TM界面中的一个形成异源二聚体，其中一个是在无活性全长整合素中发现的。第五，我们展示了利用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，这些假设在三个具体目标中得到验证。这些研究的结果将回答整合素介导的细胞粘附生物学中的核心问题，并确定双向信号传导的结构基础。

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.