Structure, Function, and Antibody-based Modulation of GPR126: Regulation of an Adhesion GPCR by its Extracellular Region

项目摘要/摘要 细胞黏附是所有器官发育的关键过程，由黏附家族介导。 细胞表面G蛋白偶联受体(AGPCRs)。AGPCRs是一大类未被研究的受体 将细胞黏附与细胞信号联系起来。AGPCRs调节器官发育、神经系统发育和 功能：它们在发育疾病、神经疾病和癌症中调节失调。AGPCRs的含义是 下一代药物针对这些适应症，然而药物开发受到不完整的阻碍 了解aGPCR功能的分子基础。AGPCR是由其神秘的多域定义的 胞外区(ECRs)，被认为是调节配体结合和受体活性的区域。我们的 假设ECR可以通过占据调控的构象来影响基础受体的信号活性。 AGPCR的7个跨膜区。这项建议旨在发现 通过ECR对aGPCR进行调控，这是aGPCR生物学中的关键空白。这将使用模型受体来完成， GPR126。GPR126与周围神经髓鞘形成和脊柱发育有关。GPR126失调是 与发育性疾病和癌症有关。GPR126有一个带有两个拼接的大型多域ECR 促进低信号活性和高信号活性的变体。我们的实验室先前确定了一个GPR126的结构 剪接变异体，代表该ECR的低活性构象。我进一步奠定了以下基础 本建议通过提纯高活性的GPR126剪接变异体，获得蛋白质晶体。我也有过 优化了信号分析，使我能够询问ECR中的点变化对GPR126的影响 信号活动。我提出了三个具体目标，将有助于审问GPR126 ECR的结构基础 功能：首先，我的目标是确定高活性GPR126剪接变异体的高分辨率结构。第二, 我将使用功能分析测试与疾病相关的GPR126变异改变活动的能力，我将 与凯利·蒙克博士的实验室合作，研究斑马鱼的变种。第三，我会发现抗体 靶向GPR126的ECR，并在功能分析中对其进行表征。这项提议将导致 对依赖于ECR的GPR126信号调节的机制描述，从而解释了调节失调 这种蛋白质的缺失可能会导致疾病。我还将生产抗体，可以用作探测的工具 GPR126在疾病模型中发挥作用。这项建议是一个多学科和协作的建议，具有很好的 培训潜力源于建议的技术范围，从结构生物学到基于细胞的分析和 抗体的发现。在Araç实验室，我将有机会与一位世界领先的 AGPCRs，并对黏附GPCR信号的分子基础进行高影响力的研究。我会扩大规模 我的技能是细胞外蛋白质的结构生物学，基于细胞的分析，并在 我的合作导师科西亚科夫博士的实验室。芝加哥大学和Araç实验室是世界级的 我有机会发展我的组合和技能，使我在教师职位上具有竞争力。

Project Summary/Abstract Cellular adhesion is a process critical for the development of all organs and is mediated by the adhesion family of cell-surface G protein-coupled receptors (aGPCRs). aGPCRs are a large, understudied group of receptors that link cell adhesion to cell signaling. aGPCRs regulate organ development, nervous system development and function; they are dysregulated in developmental disease, neurological disease, and cancer. aGPCRs stand as the next generation of drug targets for these indications, yet drug development is hindered by an incomplete understanding of the molecular basis for aGPCR function. aGPCRs are defined by their enigmatic multidomain extracellular regions (ECRs) which are hypothesized to mediate ligand binding and receptor activity. Our hypothesis is that the ECR can affect basal receptor signaling activity by occupying conformations that modulate the seven-transmembrane region of the aGPCR. This proposal aims to discover the structural basis of the regulation of an aGPCR by its ECR, the key gap in aGPCR biology. This will be done using a model receptor, GPR126. GPR126 is linked with peripheral nerve myelination and spine development. GPR126 dysregulation is associated with developmental disease and cancer. GPR126 has a large multidomain ECR with two splice variants that promote low and high signaling activity. Our lab previously determined the structure of one GPR126 splice variant which represents the low activity conformation of this ECR. I have further built the groundwork for this proposal by purifying the high activity GPR126 splice variant and obtaining protein crystals. I have also optimized a signaling assay that will allow me to interrogate the effect of point variations in the ECR on GPR126 signaling activity. I propose three Specific Aims that will help interrogate the structural basis GPR126 ECR function: First, I aim to determine a high-resolution structure of the high activity GPR126 splice variant. Second, I will test disease-associated GPR126 variations for their ability to alter activity using functional assays and I will collaborate with the laboratory of Dr. Kelly Monk to study the variants in zebrafish. Third, I will discover antibodies targeting the ECR of GPR126 and characterize them in functional assays. This proposal will result in a mechanistic description of ECR-dependent regulation of GPR126 signaling, thus explaining how dysregulation of this protein can contribute to disease. I will also produce antibodies which can be used as tools to probe GPR126 function in disease models. This proposal is a multi-disciplinary and collaborative one, with great training potential due to the span of techniques proposed, from structural biology to cell-based assays and antibody discovery. In the Araç laboratory, I will be provided the opportunity to work with a world leader in aGPCRs and to perform high-impact research on the molecular basis of adhesion GPCR signaling. I will expand my skillset into structural biology of extracellular proteins, cell-based assays, and learn antibody discovery within the lab of my Co-Mentor Dr. Kossiakoff. The University of Chicago and the Araç laboratory are world class opportunities for me to develop the portfolio and skillsets for me to be competitive for faculty positions.