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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/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) 是一大类尚未得到充分研究的受体。将细胞粘附与细胞信号传导联系起来。功能；它们在发育疾病、神经系统疾病和癌症中失调。这些适应症的下一代药物靶点，但药物开发因不完整的药物开发受到阻碍 aGPCR 功能的分子基础的理解是由其神秘的多域定义的。细胞外区域（ECR）被重新捕获以介导配体结合和受体活性。假设 ECR 可以通过占据调节的构象来影响基础受体信号传导活性 aGPCR 的七跨膜区域该提案旨在发现 aGPCR 的结构基础。 aGPCR 通过其 ECR 进行调节，这是 aGPCR 生物学的关键空白，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失调有关。与发育疾病和癌症相关。GPR126 具有带有两个剪接的大型多结构域 ECR。我们的实验室之前确定了一种 GPR126 的结构。剪接变体代表了该 ECR 的低活性构象，我进一步为此奠定了基础。我也提出了通过纯化高活性 GPR126 剪接变体并获得蛋白质晶体的建议。优化了信号分析，使我能够询问 ECR 中的点变化对 GPR126 的影响我提出了三个具体目标，这将有助于探究 GPR126 ECR 的结构基础。功能：首先，我的目标是确定高活性 GPR126 剪接变体的高分辨率结构。我将使用功能测定测试与疾病相关的 GPR126 变异，了解它们改变活性的能力，并且我将与Kelly Monk博士的实验室合作研究斑马鱼的变异第三，我将发现抗体。靶向 GPR126 的 ECR 并在功能测定中对其进行表征该提案将产生测定结果。 GPR126信号传导的ECR依赖性调节的机制描述，从而解释失调是如何发生的我还将产生可用作探测工具的抗体。 GPR126在疾病模型中的功能这一提议是一项多学科协作的提议，具有重大意义。由于所提出的技术范围广泛，从结构生物学到基于细胞的测定，因此具有培训潜力在 Araç 实验室，我将有机会与世界领先者一起工作。我将扩展 aGPCR 并对粘附 GPCR 信号传导的分子基础进行高影响力的研究。我的技能包括细胞外蛋白的结构生物学、基于细胞的分析，并学习抗体发现我的合作导师 Kossiakoff 博士的实验室和 Araç 实验室都是世界一流的。我有机会发展自己的作品集和技能，使我能够在教师职位上具有竞争力。