Structural and Functional Studies of Adhesion GPCRs

项目摘要 细胞黏附和细胞信号之间的相互作用对所有器官的发育都是必不可少的。 例如大脑，以及免疫和神经系统等系统的功能。粘附力G- 蛋白偶联受体(AGPCRs)是一个研究较少的gpcr家族，被认为介导细胞间的相互作用。 沟通。它们在多种细胞功能和许多人类疾病中扮演着新的角色。 包括神经系统疾病和癌症。与其他GPCRs不同，aGPCRs有很大的胞外区 (ECR)是从它们的七次跨膜区(TM)中自动蛋白分解而来的 保守的GPCR-自蛋白分解诱导(GAIN)结构域。这两个片段即使在 乳沟。人们认为，配体与ECR结合会导致ECR脱落，并暴露出短的 先前隐藏在增益域中的多肽。这种名为Stachel的短肽作为一种 系留激动剂并激活跨膜结构域。然而，潜在的分子机制 AGPCR的激活仍然知之甚少。本申请中提出的研究的最终目标是 为了了解aGPCR激活的逐步机械细节，从配体与细胞的黏附开始 ECR，继续通过增益域将附着信号从ECR传递到TM域， 并以Stachel多肽激活TM结构域而结束。我们提出了三个具体目标，即 基于这些基本步骤中的主要未知数：首先，我们的目标是揭示是否和 细胞外黏附如何开始发出信号。其次，我们的目标是了解增益域在 将黏附信号传递到TM结构域。第三，我们的目标是揭示aGPCRTM的分子细节 结构域被Stachel多肽激活。这项研究采用了多学科的方法，其中结构 在PI的实验室中进行的功能数据与电子显微镜、激动剂/拮抗剂相补充 通过提供或执行的蛋白质工程、神经元分析和G蛋白专业知识生成 密切合作者的实验室。拟议的实验将建立在取得令人兴奋的结果的基础上，因为我们 开始了我们的实验室，包括建立一个强大的体外信号分析，配体的晶体结构- AGPCR复合体，抑制aGPCR功能的合成蛋白的工程，以及在 AGPCRs的纯化方案。我们预计，这项研究将为 AGPCR激活的机制细节，有助于建立关于细胞间的基本原则 对许多蜂窝功能至关重要的通信。

Project Summary The interplay between cellular adhesion and cellular signaling is essential for the development of all organs such as the brain, and for the functioning of systems such as the immune and nervous systems. Adhesion G- Protein Coupled Receptors (aGPCRs) are an understudied GPCR family that is thought to mediate intercellular communication. They have emerging roles in multiple cellular functions and numerous human diseases including neurological disorders and cancers. Unlike other GPCRs, aGPCRs have large extracellular regions (ECR) that are autoproteolytically cleaved from their seven-pass transmembrane regions (TM) within a conserved GPCR-Autoproteolysis INducing (GAIN) domain. The two fragments stay associated even after cleavage. It is believed that ligand binding to the ECR causes shedding of the ECR, and exposure of a short peptide that was previously hidden within the GAIN domain. This short peptide, called stachel, acts as a tethered agonist and activates the transmembrane domain. However, the molecular mechanisms underlying aGPCR activation remains poorly understood. The ultimate goal of the research proposed in this application is to understand the stepwise mechanical details of aGPCR activation that start with adhesion of the ligand to the ECR, continue with transduction of the adhesion signal from the ECR to the TM domain via the GAIN domain, and end with the activation of the TM domain by the stachel peptide. We propose three Specific Aims that are based on the major unknowns in these fundamentals steps for aGPCR activation: First, we aim to reveal if and how extracellular adhesion starts signaling. Second, we aim to understand the role of the GAIN domain in transducing the adhesion signal to the TM domain. Third, we aim to reveal the molecular details of aGPCR TM domain activation by the stachel peptide. This research has a multi-disciplinary approach where the structural and functional data performed in the PI's lab are complemented with electron microscopy, agonist/antagonist generation via protein engineering, neuronal assays and G-protein expertise provided or performed by the laboratories of close collaborators. The proposed experiments will build on exciting results obtained since we started our lab, including the establishment of a robust in vitro signaling assay, the crystal structure of a ligand- aGPCR complex, the engineering of a synthetic protein that inhibits an aGPCR function, and key advances in the purification protocols of aGPCRs. We expect that this research will provide critical insights into the mechanistic details of the aGPCR activation, helping to establish fundamental principles on intercellular communication that are vital for numerous cellular functions.