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

Br J Pharmacol. 2021 Aug;178(15):2963-2975. doi: 10.1111/bph.15468. Epub 2021 May 8.


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 ~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 Gi/o . However, a limited number of techniques allow the detection of intrinsically small constitutive activity associated with Gi/o protein activation, which represents a significant barrier in our ability to study orphan GPCR signalling. Using luciferase reporter assays, we effectively detected constitutive Gs , Gq and G12/13 protein signalling by unliganded receptors and introducing various G protein chimeras, we provide a novel, highly sensitive tool capable of identifying Gi/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.

Keywords: GPCR; cell signalling; constitutive activity; molecular pharmacology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • GTP-Binding Protein alpha Subunits, Gi-Go
  • GTP-Binding Proteins
  • Humans
  • Ligands
  • Receptors, G-Protein-Coupled* / genetics
  • Receptors, G-Protein-Coupled* / metabolism
  • Signal Transduction*


  • Ligands
  • Receptors, G-Protein-Coupled
  • GTP-Binding Proteins
  • GTP-Binding Protein alpha Subunits, Gi-Go