Biochemical approaches to enlighten GPR85 function

生化方法揭示 GPR85 功能

• 批准号: 10047548
• 负责人: Aashish Manglik
• 金额: $ 15.62万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10047548
• 关键词: Antibodies; Arrestins; Binding; Biochemical; Biochemistry; Biological; Biological Models; Biological Process; Biology; Cells; Communities; Complex; Foundations; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GPR85 gene; GTP-Binding Proteins; Genome; Hippocampus (Brain); Human; Human Genome; In Vitro; Individual; Knock-out; Learning; Libraries; Ligands; Link; Location; Memory; Modern Medicine; Molecular Conformation; Neuronal Plasticity; Orphan; Output; Pharmacology; Physiological Processes; Proteins; Protocols documentation; Receptor Signaling; Role; Schizophrenia; Sequence Homology; Signal Transduction; Structure; Therapeutic; Tissues; Ursidae Family; Work; Zebrafish; beta-arrestin; experience; extracellular; innovation; insight; milligram; nanobodies; nervous system development; neurogenesis; neuropsychiatric disorder; novel strategies; overexpression; protein complex; receptor; receptor expression; small molecule; structural biology; success; tool; tool development; vertebrate genome

PROJECT SUMMARY/ABSTRACT BIOCHEMICAL APPROACHES TO ENLIGHTEN GPR85 FUNCTION. G protein-coupled receptors (GPCRs) are the most successfully targeted proteins by modern medicine, with transformative therapies that modulate essentially every physiological process. In the face of such success are the numerous “orphan” GPCRs for which a lack of tool molecules has crippled even the most basic understanding of biological function. Among the dozens of such orphan receptors, GPR85 stands out. GPR85 is one of the most conserved GPCRs in the human genome, with 94% identity between human and zebrafish homologs. Initial hints from knockout and overexpression studies suggest a critical role in neural plasticity in the hippocampus, with potential therapeutic relevance for recalcitrant neuropsychiatric diseases like schizophrenia. Despite this intriguing biological insight, the true function of GPR85 remains poorly understood due to a complete lack of validated tools, both antibodies to localize the receptor in various tissues and molecules to manipulate receptor signaling. We propose to develop new approaches to purify orphan GPCRs, using GPR85 as a model system. In preliminary work, we have established protocols to express and purify milligram quantities of GPR85. We will now generate purified GPR85 for biochemical screens to identify new small molecules and for structural studies of GPR85-G protein complexes. Using an in vitro platform for single domain antibody (nanobody) discovery, we will identify nanobodies that specifically recognize GPR85, providing new tools to interrogate GPR85 location in tissues. Finally, we will develop nanobodies that bind specific conformations of GPR85, providing needed tools to manipulate GPR85 signaling. The output of this proposal will be new tools for an enigmatic orphan GPCR, and a blueprint for biochemical interrogation for many pharmacologically “dark” GPCRs.

项目总结/摘要 激活GPR 85功能的生化途径G蛋白偶联受体 是现代医学最成功的靶向蛋白质，具有调节 每一个生理过程面对这样的成功，有许多“孤儿”GPCR， 工具分子的缺乏甚至削弱了对生物功能的最基本理解。在几十个 在这些孤儿受体中，GPR 85脱颖而出。GPR 85是人类中最保守的GPCR之一。 基因组，与94%的同源性之间的人类和斑马鱼。最初的提示从淘汰赛和 过表达研究表明，在海马神经可塑性中起关键作用，具有潜在的治疗作用。 与精神分裂症之类的神经精神疾病有关。尽管有这种有趣的生物学见解， 由于完全缺乏有效的工具，GPR 85的真正功能仍然知之甚少， 将受体定位在各种组织和分子中，以操纵受体信号。我们建议发展 使用GPR 85作为模型系统纯化孤儿GPCR的新方法。在初步工作中，我们 建立了表达和纯化毫克量的GPR 85的方案。我们现在将产生纯化的GPR 85 用于生物化学筛选以鉴定新的小分子和GPR 85-G蛋白的结构研究 配合物使用用于单结构域抗体（纳米抗体）发现的体外平台，我们将鉴定 特异性识别GPR 85的纳米抗体，提供了询问组织中GPR 85位置的新工具。 最后，我们将开发结合GPR 85特定构象的纳米抗体，提供所需的工具， 操纵GPR 85信号传导。该提案的成果将是用于神秘的孤儿GPCR的新工具， 一个生化审讯的蓝图，用于许多“黑暗”的GPCR。