Biochemical approaches to enlighten GPR85 function

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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.