In vitro profiling of orphan G protein coupled receptor (GPCR) constitutive activity

Background and Purpose: Members of the GPCR family are targeted by a significant fraction of the available FDA-approved drugs. However, the physiological role and pharmacological properties of many GPCRs remain unknown, representing untapped potential in drug design. Of particular interest are similar to 100 less-studied GPCRs known as orphans because their endogenous ligands are unknown. Intriguingly, disease-causing mutations identified in patients, together with animal studies, have demonstrated that many orphan receptors play crucial physiological roles and, thus, represent attractive drug targets. Experimental Approach: The majority of deorphanized GPCRs demonstrate coupling to G(i/o). However, a limited number of techniques allow the detection of intrinsically small constitutive activity associated with G(i/o) protein activation, which represents a significant barrier in our ability to study orphan GPCR signalling. Using luciferase reporter assays, we effectively detected constitutive G(s), G(q) and G(12/13) protein signalling by unliganded receptors and introducing various G protein chimeras, we provide a novel, highly sensitive tool capable of identifying G(i/o) coupling in unliganded orphan GPCRs. Key Results: Using this approach, we measured the constitutive activity of the entire class C GPCR family that includes eight orphan receptors and a subset of 20 prototypical class A GPCR members, including 11 orphans. Excitingly, this approach illuminated the G protein coupling profile of eight orphan GPCRs (GPR22, GPR137b, GPR88, GPR156, GPR158, GPR179, GPRC5D and GPRC6A) previously linked to pathophysiological processes. Conclusion and Implications: We provide a new platform that could be utilized in ongoing studies in orphan receptor signalling and de-orphanization efforts.