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.

项目概要/摘要 阐明 GPR85 功能的生物化学方法。 G 蛋白偶联受体 (GPCR) 是现代医学最成功的靶向蛋白质，具有调节的转化疗法 基本上每个生理过程。面对如此成功的是无数“孤儿”GPCR 工具分子的缺乏甚至削弱了对生物功能最基本的理解。数十人之中 在这些孤儿受体中，GPR85 脱颖而出。 GPR85是人类中最保守的GPCR之一 基因组，人类和斑马鱼的同源性高达 94%。淘汰赛的初步提示和 过度表达研究表明在海马体的神经可塑性中发挥着关键作用，具有潜在的治疗作用 与精神分裂症等顽固性神经精神疾病的相关性。尽管有这种有趣的生物学见解， 由于完全缺乏经过验证的工具，GPR85 的真正功能仍然知之甚少，这两种抗体 将受体定位在各种组织和分子中以操纵受体信号传导。我们建议开发 使用 GPR85 作为模型系统纯化孤儿 GPCR 的新方法。在前期工作中，我们有 建立了表达和纯化毫克量 GPR85 的协议。我们现在将生成纯化的 GPR85 用于生化筛选以鉴定新的小分子以及用于 GPR85-G 蛋白的结构研究 复合物。使用单域抗体（纳米抗体）发现的体外平台，我们将鉴定 特异性识别 GPR85 的纳米抗体，为探究 GPR85 在组织中的位置提供了新工具。 最后，我们将开发结合 GPR85 特定构象的纳米抗体，为 操纵 GPR85 信号传导。该提案的成果将是用于神秘的孤儿 GPCR 的新工具，并且 许多药理学“暗”GPCR 的生化审讯蓝图。