Investigation of Adhesion GPCR and Ric-8 protein control of heterotrimeric G proteins

项目摘要：这项米拉提案将结合我们长期在结构和 粘附性G蛋白偶联受体(或B2族GPCRs)及其分子伴侣的功能 蛋白质α亚基，Ric-8蛋白质。粘附性GPCRs是一个由33个成员组成的受体大家族， 将信号从一个细胞传递到另一个细胞或通过细胞膜将细胞外基质黏附事件传递到 等待G蛋白，以便在细胞内引起反应。我们的工作提出了一种粘合机制 细胞外黏附结构域锚定于蛋白质配体和细胞的GPCR激活 它包含GPCR域或七个跨膜区(7TM)，以任何数量的方式与 这个固定的锚点将粘连的GPCr的两半解离。这一解离事件揭示了之前 隐藏的GPCR/7TM结构域的拴系激动剂，可自我激活受体。这种激活模式 对于粘连gpr家族的大多数成员来说似乎是常见的，但粘连gpr的替代模式 激活也走到了这个领域的前列。我们在这个提案中的目标是扩展我们的机械性 和结构性工作，以破译各种黏附GPCR激活模式，使用生化、细胞生物学和 活体方法，包括使用为这一提议新创建的两个小鼠模型。我们将重点关注 存在于血液循环细胞中的粘附性GPCRs，例如血小板上的ADGRG1/GPR56， 以及存在于其他循环细胞类型上的其他ADGRG亚家族成员。循环细胞提供 询问粘附性GPCR作用的优势是因为解离 受体的两个片段，用于拴系激动剂激活，很容易处理。总和 我们的工作将是区分拴系激动剂依赖和非依赖的粘连模式 激活，我们认为这可能是黏附GPCR领域最重要的当代问题。 同时，我们将Ric-8A和Ric-8B蛋白的功能指定为分子伴侣 需要共同折叠所有G蛋白α亚基。对于我们的实验室来说，这是一个成熟的项目，但我们已经做出了 最近在解决Ric-8/G蛋白无鸟嘌呤核苷酸复合体结构方面的突破。我们 提出了一种新的Ric-8/G蛋白质复合结构的初步证据，这将使我们能够最终解决 主要悬而未决的问题；哺乳动物Ric-8A和Ric-8B的哪些结构元素定义了它们的 不同G蛋白亚型的特异性？结合对祖先Ric-8的单拷贝的分析 (果蝇Ric-8)似乎越过了界限，似乎折叠了所有G蛋白亚型，我们的工作将 提供了对底物特异性规则的新理解，从而为潜在靶向提供了理由 G蛋白驱动疾病背景下的Ric-8伴侣系统。

Project Summary: This MIRA proposal will combine work from our longstanding projects on the structure and function of adhesion G protein coupled receptors (or Family B2 GPCRs) and the molecular chaperones for G protein alpha subunits, Ric-8 proteins. Adhesion GPCRs are a large, 33-member family of receptors that transduce signals from cell to cell or cell to extracellular matrix adhesion events across the cell membrane to awaiting G proteins in order to elicit responses within the cell. Our work has advanced a mechanism for adhesion GPCR activation in which the extracellular adhesion domains become anchored to protein ligands and the cell that contains the GPCR or seven transmembrane domain (7TM) moves, by any number of means, in relation to this fixed anchor to dissociate the two halves of the adhesion GPCR. This dissociation event reveals a previously hidden tethered agonist of the GPCR/7TM domain that self-activates the receptor. This mode of activation seems common to most members of the adhesion GPCR family, but alternative modes of adhesion GPCR activation have also come to the fore of the field. Our goal in this proposal is to expand upon our mechanistic and structural work to decipher varied adhesion GPCR activation modes using biochemical, cell biological, and in vivo approaches, including work with two mouse models newly created for this proposal. We will focus on adhesion GPCRs that are present in cells that circulate in the blood, for example ADGRG1/GPR56 on platelets, as well as additional ADGRG subfamily members present on other circulating cell types. Circulating cells offer an advantage for interrogating adhesion GPCR action because the shear force component that dissociates the two fragments of the receptors, for the purpose of tethered-agonist-activation, is readily tractable. The sum of our work will be to distinguish tethered agonist -dependent and -independent modes of adhesion GPCR activation, which we argue is perhaps the most important contemporary question in the adhesion GPCR field. In parallel, we have assigned the function of Ric-8A and Ric-8B proteins as molecular chaperones that are required to collectively fold all G protein alpha subunits. This is a mature project for our lab, yet we have made recent breakthroughs in solving the structures of Ric-8/G protein guanine nucleotide-free complexes. We present preliminary evidence of a new Ric-8/G protein complex structure that will enable us to finally tackle a major outstanding question; What are the structural elements of mammalian Ric-8A and Ric-8B that define their specificities for different G protein subtypes? Combined with analysis of the single copy of an ancestral Ric-8 (Drosophila Ric-8) that seemingly crosses the lines and appears to fold all G protein subtypes, our work will provide new understanding of the substrate specificity rules, thereby providing a rationale for potentially targeting the Ric-8 chaperone system in contexts of G protein-driven disease.