Structural and Functional Studies of Adhesion GPCRs

项目摘要 细胞粘附和细胞信号之间的相互作用对于所有器官的发育都是必不可少的 如大脑，以及免疫和神经系统等系统的功能。附着力G- 蛋白偶联受体（aGPCR）是一个研究不足的GPCR家族，被认为是介导细胞间 通信它们在多种细胞功能和许多人类疾病中具有新兴作用 包括神经系统疾病和癌症。与其他GPCR不同，aGPCR具有大的细胞外区域 (ECR)其在一个细胞内从其七次跨膜区（TM）被自蛋白水解切割， 保守的GPCR-自身蛋白水解诱导（GAIN）结构域。这两个碎片即使在 乳沟据信，配体与ECR的结合导致ECR脱落，并且暴露于短的ECR。 之前隐藏在GAIN结构域中的肽。这种短肽称为stachel， 栓系激动剂并激活跨膜结构域。然而，潜在的分子机制 aGPCR激活仍然知之甚少。本申请中提出的研究的最终目标是 为了理解aGPCR激活的逐步机械细节，其开始于配体与细胞粘附， ECR，继续通过GAIN结构域将粘附信号从ECR转导至TM结构域， 并以TM结构域被stachel肽激活而结束。我们提出三个具体目标， 基于aGPCR激活的这些基本步骤中的主要未知数：首先，我们的目标是揭示 细胞外粘附是如何启动信号的其次，我们的目标是了解GAIN域在以下方面的作用： 将粘附信号转导至TM结构域。第三，我们的目标是揭示aGPCR TM的分子细节 通过stachel肽的结构域激活。本研究采用多学科方法， 在PI实验室进行的功能数据与电子显微镜、激动剂/拮抗剂 通过蛋白质工程，神经元测定和G蛋白专业知识提供或执行的生成 密切合作者的实验室。拟议的实验将建立在令人兴奋的结果，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复合物，抑制aGPCR功能的合成蛋白的工程化，以及在 aGPCR的纯化方案。我们希望这项研究将提供关键的见解， aGPCR激活的机制细节，有助于建立细胞间的基本原则 这对许多细胞功能至关重要。

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.