Implementation of a method for analysis of Gi/o‐coupled orphan GPCR signaling

Abstract

G‐protein coupled receptors (GPCRs) are currently the target of almost 30% of the marketed drugs. However, only a small percentage of the whole GPCR repertoire is well characterized for both endogenous ligands and signaling pathways, with ~100 GPCRs still considered orphans (oGPCRs). Nonetheless, oGPCRs are increasingly recognized for their essential pathophysiological roles. This pool of uncharacterized GPCRs has therefore a considerable untapped potential as new pharmacological targets. Scientists exploited several indirect approaches to discover new ligand‐receptor pairs and characterize receptor‐signaling couplings, however in the last few years the number of deorphanized GPCRs has not grown as expected. According to the “GPCR database” almost two thirds of GPCRs are coupled to Gi/o/z proteins, for which currently available assays display low sensitivity. To address part of this problem, we recently used different reporter systems in combination with Gα‐protein chimeras to uncover Gα‐protein coupling by measuring the constitutive activity of a library of oGPCRs expressed in a heterologous system. Using this assay we identified 8 out of 19 oGPCRs tested as coupled to Gi/o/z proteins. In the present project, we aim at setting up a new in vitro high‐throughput screening assay for Gi/o/z‐coupled oGPCRs, combining Gi/o/z‐protein chimeras and cAMP measurements using GloSensor. Our preliminary data obtained studying traditional Gi/o/z‐coupled receptors suggest that expression of the GsGz chimera in combination with Pertussis Toxin (PTX) results in an assay displaying the highest sensitivity and signal amplitude. Considering the high expression of many oGPCRs in the brain, we hypothesized that their endogenous ligands would be also localized there. We therefore tested the activation of transfected oGPCRs using our optimized system by mouse brain extract, as a mixture of candidate ligands. The success of this preliminary analysis validates our approach for future screening purposes. Overall, implementation of this method for screening medium to large libraries of molecules would allow to deorphanize oGPCRs, providing new insights in GPCR pharmacology and the discovery of new ligand‐receptor pairs.