Biochemical approaches to enlighten GPR85 function

生化方法揭示 GPR85 功能

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要/摘要启发GPR85功能的生化方法。 G蛋白偶联受体（GPCR）是现代医学最成功的靶向蛋白质，并具有调节的变革性疗法本质上是每个物理过程。面对这样的成功，有众多的“孤儿” GPCR 缺乏工具分子也使对生物功能的最基本理解也削弱了。在数十个在这种孤儿受体中，GPR85脱颖而出。 GPR85是人类最保守的GPCR之一基因组，人类和斑马鱼同源物之间具有94％的身份。最初的提示是从淘汰赛和过表达的研究表明，在海马中的神经可塑性中起关键作用，并具有潜在的治疗与精神分裂症这样的顽固性神经精神疾病的相关性。尽管这种有趣的生物学见解，但 GPR85的真实功能由于完全缺乏经过验证的工具，两种抗体仍然很少理解将受体定位在各种时间和分子中以操纵受体信号。我们建议发展使用GPR85作为模型系统净化孤儿GPCR的新方法。在初步工作中，我们有建立的协议以表达和净化毫克的GPR85。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和GPR85-G蛋白的结构研究复合物。使用单个域抗体（Nanoboty）发现的体外平台发现，我们将确定专门识别GPR85的纳米构造，提供了新工具来询问组织中的GPR85位置。最后，我们将开发纳米体，以结合GPR85的特定构象，从而为所需的工具提供操纵GPR85信号。该提案的输出将是一个神秘的孤儿GPCR的新工具，并且用于许多药物“黑暗” GPCR的生化询问的蓝图。