Conformational regulation in integrin bidirectional transmembrane signaling

 描述（由申请方提供）：整合素是α/β异二聚体细胞表面受体，通过其以多种构象存在的能力，调节从器官发育到免疫调节再到止血的各种生物学过程。与整联蛋白的细胞外或细胞质表面结合的蛋白质可以影响跨质膜传递的长程构象变化。从外到内的双向信号传递启动支持细胞迁移和存活的细胞信号传导级联，而特异性蛋白质与整联蛋白的胞质尾部的结合（内部信号传导）诱导胞外域内的长距离构象重排，将其从静止状态转化为活化（配体结合）感受态。尽管在过去的25年中进行了许多研究，但在结构水平上仍然不清楚由内而外和由外而外的信号传导是如何通过跨膜和胞质结构域控制的。开发无混杂副作用的新型疗法需要对这些过程进行原子水平的分子理解。我们最近发现，删除整联蛋白中α亚基的C-末端，包括血小板α IIb β 3和白细胞α L β 2，对整联蛋白响应由内而外的信号而经历构象变化的能力具有显著的出乎意料的影响。本提案的目的1将集中于定义整合素α亚基胞质结构域，特别是其不太保守的膜远端区域，在调节整合素由内而外激活中的新作用。多种独立的方法，包括全面诱变，半胱氨酸扫描可及性，二硫键交联，和全长整联蛋白的半胱氨酸荧光标记，将被用来检查整合素跨膜和胞质结构域的重组，以响应由内而外和由外而外的信号转导与胞外域的多种活性构象的协调。目的2将集中于第二个重要问题：即，观察到在临床试验中或在临床用于治疗血栓形成或癌症的大多数RGD（Arg-Gly-Asp）-模拟整联蛋白拮抗剂具有诱导血小板减少症和在某些情况下血栓形成或增强肿瘤生长和血管生成的不良副作用。这些作用似乎是由于药物诱导的构象变化导致整合素活化和能够诱导免疫应答的新表位的产生。在我们最近的结构研究的基础上，我们将在原子水平上定义当整合素结合小的或小的时发生的关键结构变化。 大的配体。研究结果将用于指导开发新的整合素拮抗剂，在不改变整合素构象的情况下阻断功能。总之，这些互补的特异性目的将揭示配体诱导的整合素构象变化的分子基础，并提供必要的双向整合素信号传导的基于结构的理解，以促进具有潜在改善的治疗功效的新型整合素拮抗剂的开发。

 DESCRIPTION (provided by applicant): Integrins are α/ß heterodimeric cell surface receptors that, via their ability to exist in multiple conformations, regulate a diverse array of biological processes ranging from organ development to immune regulation to hemostasis. Proteins binding to either the extracellular or cytoplasmic face of an integrin can effect long-range conformational changes that are transmitted across the plasma membrane. Bi-directional signal transmission from outside to inside initiates cell signaling cascades supporting cell migration and survival, while binding of specific proteins to the cytoplasmic tails of the integrin (inside-ot signaling) induces long-range conformational rearrangements within the ectodomains that convert it from a resting to an activated (ligand-binding) competent state. Despite many studies over the past 25 years, exactly how inside-out and outside-in signaling is controlled through the transmembrane and cytoplasmic domains are still not understood at the structural level. Development of novel therapeutics free of confounding side effects requires an atomic level molecular understanding of these processes. We recently found that deleting the C-terminus of the α-subunits in integrins, including the platelet αIIbß3 and leukocyte aLß2, has a dramatic ad unexpected effect on the ability of integrins to undergo conformational change in response to inside-out signals. Aim 1 of this proposal will focus on defining this novel role of integrin a-subunit cytoplasmic domains, especially their less-conserved membrane distal region, in regulating integrin inside-out activation. Multiple independent approaches, including comprehensive mutagenesis, cysteine scanning accessibility, disulfide crosslinking, and cysteine fluorescence labeling of full-length integrins, will be employed to examine restructuring of integrin transmembrane and cytoplasmic domains in response to inside-out and outside-in signaling in coordination with the multiple active conformations of the ectodomain. Aim 2 will focus on a second important issue: namely, the observation that most RGD (Arg-Gly-Asp)-mimetic integrin antagonists in clinical trials or in clinical use for treating thrombosis or cance have the undesirable side effect of inducing thrombocytopenia and in some cases, thrombosis or enhanced tumor growth and angiogenesis. These effects appear to be due to drug-induced conformational changes leading to integrin activation and creation of neo-epitopes capable of inducing an immune response. Building on our recent structural studies, we will define at the atomic level the critical structural changes that take place when an integrin binds either small or large ligands. Findings made will be used to guide the development of novel integrin antagonists that block function without altering integrin conformation. Together, these complementary Specific Aims will reveal the molecular basis for ligand-induced integrin conformational change and provide the structure-based understanding of bidirectional integrin signaling necessary to facilitate development of novel integrin antagonists with potentially improved therapeutic efficacy.