Characterization of an Orphan G Protein-coupled Receptor, GPR20, That Constitutively Activates Gi Proteins

Abstract

GPR20 was isolated as an orphan G protein-coupled receptor from genomic DNA by PCR amplification. Although GPR20 was closely related to nucleotide or lipid receptors, the functional role of this receptor, as well as its endogenous ligand, remains unclear. Here we demonstrate that GPR20 is constitutively active in the absence of ligand, leading to continuous activation of its coupled G proteins. When GPR20 was exogenously expressed in HEK293 cells, both the basal level and the prostaglandin E 2 -induced production of cAMP were significantly decreased. A remarkable increase in [35 S]guanosine 5-(␥-thio)triphosphate (GTP␥S) binding to membrane preparations was also observed in GPR20-expressing cells. These effects of GPR20 overexpression were diminished in cells treated with pertussis toxin, suggesting that the expression of GPR20 results in the activation of G i/o proteins. Involvement of GPR20 in the activation of G i/o proteins was also supported by evidence that the disruption of a conserved DRY motif in GPR20 attenuated both [35 S]GTP␥S incorporation and inhibition of the prostaglandin E 2 -induced cAMP production. Knockdown of GPR20 in PC12h cells resulted in an elevation of the basal cAMP level, suggesting that the endogenous GPR20 achieves a constitutively or spontaneously active conformation. Furthermore, enhancement of [ 3 H]thymidine incorporation was also observed in the GPR20-silencing cells, implying that the GPR20 expression seems to attenuate PC12h cell growth. Taken together, these data indicate that GPR20 constitutively activates G i proteins without ligand stimulation. The receptor may be involved in cellular processes, including control of intracellular cAMP levels and mitogenic signaling. Mammalian G protein-coupled receptors (GPCRs)2 are a diverse superfamily of proteins with hundreds of members, and these receptors regulate many processes in vivo via interactions with a variety of ligands, including small organic molecules, lipids, protons, hormones, short and large polypeptides, glycoproteins, and even photons. Despite the vast and longstanding efforts of academic and industrial researchers to pair GPCRs with potential ligands, more than 150 nonsensory GPCRs still remain orphan receptors, for which the cognate ligands have not yet been identified (1). Because GPCRs have proven particularly amenable to modulation by small molecules and are the targets of approximately half of currently marketed prescription drugs, the nonsensory GPCRs are clearly important therapeutic targets (2). Consequently, the orphan GPCRs constitute a vast reservoir of potential drug targets for therapeutic development. Indeed, numerous research groups, including ours, work on characterizing these receptors.GPR20 is one of the orphan GPCRs that has been identified from human genomic DNA by PCR amplification using primers based on the sequences of the opioid/somatostatin-related receptors, GPR7 and GPR8 (3). The expression of human GPR20 has been detected in several brain regions, including the caudate nuclei, putamen, a...

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