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: 10624817
• 负责人: Sumit Bandekar
• 金额: $ 7.18万
• 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/10624817
• 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; Collaborations; Coupling; Cyclic AMP; Defect; Development; Developmental Process; Disease; Disease model; Drug Targeting; Embryo; Equilibrium; Extracellular Domain; 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; Variant; Vertebral column; Work; X-Ray Crystallography; Zebrafish; angiogenesis; developmental disease; drug development; drug discovery; experimental study; extracellular; knockout animal; multidisciplinary; myelination; nervous system development; nervous system disorder; next generation; organ growth; receptor; receptor function; skills; 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.

项目摘要/摘要 细胞粘附是所有器官发展至关重要的过程，并由粘附家族介导 细胞表面G蛋白偶联受体（AGPCR） AGPCR是一组大型的受体 将细胞粘合剂连接到细胞信号传导。 AGPCR规范器官发育，神经系统的发展和 功能;它们在发育中的疾病，神经系统疾病和癌症中失调。 AGPCR的站立 这些适应症的下一代药物靶标，但是药物开发受到不完整的阻碍 了解AGPCR功能的分子基础。 AGPCR由其神秘的多域定义 细胞外区域（ECR），假设介导配体结合和受体活性。我们的 假设是，ECR可以通过占据调节的构象来影响基本的接收器信号传导活动 AGPCR的七跨膜区域。该建议旨在发现 通过其ECR对AGPCR进行调节，ECR是AGPCR生物学的关键差距。这将使用模型接收器完成 GPR126。 GPR126与周围神经髓鞘和脊柱发育有关。 GPR126失调是 与发育疾病和癌症有关。 GPR126具有大型多域ECR，带有两个剪接 促进低信号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 剪接变体代表该ECR的低活性构象。我进一步为 通过纯化高活性GPR126剪接变体并获得蛋白质晶体，该建议。我也有 优化了信号传导测定，该测定将使我能够询问ECR中点变化对GPR126的影响 信号活动。我提出了三个具体目标，这些目标将有助于询问结构基础GPR126 ECR 功能：首先，我旨在确定高活性GPR126剪接变体的高分辨率结构。第二， 我将测试与疾病相关的GPR126的变化，以便使用功能测定法改变活动的能力，我将 与凯利·僧侣博士的实验室合作研究斑马鱼的变体。第三，我会发现抗体 针对GPR126的ECR并在功能测定中表征它们。该建议将导致 GPR126信号传导ECR依赖性调节的机械描述，从而解释了失调的方式 该蛋白质可以导致疾病。我还将生产可用作证明工具的抗体 GPR126在疾病模型中的功能。该建议是一项多学科和协作的建议，很棒 从结构生物学到基于细胞的测定和 抗体发现。在Araç实验室中，我将有机会与世界领导者合作 AGPCR并根据粘合剂GPCR信号的分子基础进行高影响研究。我会扩展 我在细胞外蛋白质，基于细胞的测定和学习抗体发现的结构生物学中的技能 我的联合官员科西亚科夫博士的实验室。芝加哥大学和阿拉萨实验室是世界一流的 我有机会发展投资组合和技能，让我在教师职位上具有竞争力。