G Protein coupled Receptors (GPCRs) regulate every aspect of human physiology. However, the endogenous ligands activating ~100 GPCRs are still unknown, hence they are named orphan GPCRs. Considering their understudied biology and established physiological roles, orphan GPCRs represent unexploited pharmacological targets. The quest for endogenous and synthetic compounds modulating their activity is significant for both understanding their biology and developing novel therapeutics. Using an innovative cell- based assay, we recently established Gi/o/z coupling for several orphan GPCRs. Building on this discovery, we optimized a platform to screen candidate ligands based on the heterologous co-expression of orphan GPCRs and a multicomponent activity biosensor. The ultimate goal of this project is to identify pharmacological modulators, endogenous ligands, and binding partners for GPR156, a clinically relevant target for the treatment of hearing and balance disorders with unexplored roles in the central nervous system. Our preliminary data support the feasibility of the approach and also establish the presence of endogenous ligands in the brain. Thus, the identification of these endogenous ligands will constitute an additional goal of our proposal that we will pursue applying innovative proximity-mediated labeling technologies. Activity of identified ligands and function of interacting proteins identified by proteomics analysis will be validated in a physiologically relevant system. In fact, GPR156 controls hair cell orientation in auditory and balance organs, with GPR156 gene mutations leading to deafness and balance disorders in patients. Finally, combining proteomics analysis and mouse genetics, we will define components of GPR156 signaling complex and determine their role in establishing planar cell polarity in the mouse inner ear.