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J. Biol. Chem., Vol. 283, Issue 19, 12747-12755, May 9, 2008

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Characterization of an Orphan G Protein-coupled Receptor, GPR20, That Constitutively Activates G_i Proteins^*

Momoko Hase, Takehiko Yokomizo, Takao Shimizu, and Motonao Nakamura¹

From the Department of Biochemistry and Molecular Biology, Faculty of Medicine, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 and the Department of Medical Biochemistry, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Fukuoka 812-8582, Japan

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 E₂-induced production of cAMP were significantly decreased. A remarkable increase in [³⁵S]guanosine 5'-(-thio)triphosphate (GTPS) 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 [³⁵S]GTPS incorporation and inhibition of the prostaglandin E₂-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 [³H]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.

Received for publication, November 19, 2007 , and in revised form, March 7, 2008.

^* This work was supported in part by a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to T. S. and T. Y.) and grants from the Japan Society for the Promotion of Science (Global Center of Excellence (COE) Program), the Takeda Science Foundation, the Mitsubishi Foundation, the Naito Foundation, the Uehara Memorial Foundation, the Astellas Foundation for Research on Metabolic Disorders (to T. Y.), and the Center for NanoBio Integration at the University of Tokyo. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 81-3-5802-2925; Fax: 81-3-3813-8732; E-mail: moto-nakamura{at}umin.net.

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