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

GPR126 的结构、功能和基于抗体的调节：细胞外区域对粘附 GPCR 的调节

• 批准号: 10402808
• 负责人: Sumit Bandekar
• 金额: $ 6.76万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10402808
• 关键词: Adhesions; Adolescent; Affect; Affinity; Antibodies; Arthrogryposis; Binding; Binding Sites; Biological Assay; Biology; Biophysics; Calcium Binding; Cell Adhesion; Cell Surface Receptors; Cell surface; Cell to Cell Adhesion Signaling Pathway; Cells; Cellular Assay; Chicago; Coupling; Crystallization; Cyclic AMP; Defect; Development; Developmental Process; Disease; Disease model; Drug Targeting; Embryo; Equilibrium; Extracellular Protein; Faculty; Family; Future; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; Human; Idiopathic scoliosis; Laboratories; Learning; Left; Ligand Binding; Link; Malignant Neoplasms; Mediating; Mentors; Modeling; Molecular; Molecular Conformation; Mutation; Nerve; Organ; Orthologous Gene; Peripheral Nerves; Pharmaceutical Preparations; Positioning Attribute; Process; Proteins; RNA Splicing; Receptor Activation; Receptor Signaling; Regulation; Research; Resolution; Role; Signal Transduction; Signaling Protein; Site; Spinal; Structure; Surface; Techniques; Testing; Training; Transgenic Organisms; Transmembrane Domain; Universities; Validity of Results; Variant; Vertebral column; Work; X-Ray Crystallography; Zebrafish; angiogenesis; base; developmental disease; drug development; drug discovery; experimental study; extracellular; knockout animal; malignant neurologic neoplasms; multidisciplinary; myelination; nervous system development; nervous system disorder; next generation; organ growth; receptor; receptor function; structural biology; tool

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.

项目总结/摘要 细胞粘附是所有器官发育的关键过程，由粘附家族介导 细胞表面G蛋白偶联受体（aGPCR）。aGPCR是一个大的，未充分研究的受体组 将细胞粘附和细胞信号联系起来。aGPCR调节器官发育、神经系统发育和 功能;它们在发育疾病、神经系统疾病和癌症中失调。aGPCR代表 这些适应症的下一代药物靶点，但药物开发受到不完整的 了解aGPCR功能的分子基础。aGPCR由其神秘的多结构域定义 细胞外区（ECR），其被假设为介导配体结合和受体活性。我们 假设ECR可以通过占据调节受体信号传导的构象来影响基础受体信号传导活性， aGPCR的七跨膜区。该建议旨在发现 通过其ECR调节aGPCR，这是aGPCR生物学中的关键缺口。这将使用模型受体来完成， GPR126。GPR 126与周围神经髓鞘形成和脊柱发育有关。GPR 126失调 与发育性疾病和癌症有关。GPR 126具有大的多结构域ECR，具有两个剪接 促进低和高信号传导活性的变体。我们的实验室先前确定了一种GPR 126的结构， 剪接变体，其代表该ECR的低活性构象。我已经进一步为 该方案通过纯化高活性GPR 126剪接变体并获得蛋白质晶体。我也 我优化了一个信号分析，这将使我能够询问ECR中的点变化对GPR 126的影响 信号活动。我提出了三个具体目标，这将有助于询问结构基础GPR 126 ECR 功能：首先，我的目标是确定高活性GPR 126剪接变体的高分辨率结构。第二、 我将测试疾病相关的GPR 126变异的能力，改变活动使用功能测定，我将 与凯利·蒙克博士的实验室合作，研究斑马鱼的变异。第三，我会发现抗体 靶向GPR 126的ECR并在功能测定中表征它们。这一提议将导致 对GPR 126信号传导的ECR依赖性调节的机制描述，从而解释了调节异常如何 这种蛋白质会导致疾病。我还将生产抗体， GPR 126在疾病模型中的功能。这是一个多学科和合作的建议，具有很大的 由于所提出的技术跨度，从结构生物学到基于细胞的测定， 抗体发现在阿拉塔斯实验室，我将有机会与世界领先的 aGPCR，并对粘附GPCR信号传导的分子基础进行高影响力的研究。我会扩大 我的技能进入细胞外蛋白质的结构生物学，基于细胞的测定，并学习抗体发现内 我的共同导师科西亚科夫博士的实验室芝加哥大学和阿拉塔斯实验室是世界一流的 我有机会发展我的投资组合和技能，使我在教师职位上具有竞争力。