Biochemical approaches to enlighten GPR85 function

项目摘要/摘要 启迪GPR85功能的生化方法。G蛋白偶联受体 是现代医学最成功的靶向蛋白质，通过改变疗法调节 基本上每一个生理过程。在这样的成功面前，是许多“孤儿”的GPCR 工具分子的缺乏甚至削弱了对生物功能的最基本的理解。在几十个人中 在这些孤儿受体中，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有94%的同源性。淘汰赛的初步提示和 过度表达研究表明在海马区的神经可塑性中起关键作用，具有潜在的治疗作用 与顽固性神经精神疾病如精神分裂症的相关性。尽管有这种耐人寻味的生物学洞察力， 由于完全缺乏两种抗体的验证工具，GPR85的真正功能仍然知之甚少 在不同的组织和分子中定位受体，以操纵受体信号。我们建议开发 使用GPR85作为模型系统来净化孤立GPCRs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的方案。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并用于GPR85-G蛋白的结构研究 复合体。利用单域抗体(纳米体)的体外发现平台，我们将鉴定 专门识别GPR85的纳米体，为询问GPR85在组织中的位置提供了新的工具。 最后，我们将开发结合GPR85特定构象的纳米体，提供必要的工具来 操纵GPR85信令。这项提议的成果将是一个神秘的孤儿GPCR的新工具，以及 许多药理上“黑暗”的GPCR的生化讯问蓝图。

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.