Agonist activation of the G protein-coupled receptor GPR35 involves transmembrane domain III and is transduced via Gα₁₃ and β-arrestin-2.

Laura Jenkins, Elisa Alvarez-Curto, Kate Campbell, Sabrina de Munnik, Meritxell Canals, Sabine Schlyer, Graeme Milligan

Index: Br. J. Pharmacol. 162 , 733-748, (2011)

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Abstract

GPR35 is a poorly characterized G protein-coupled receptor at which kynurenic acid has been suggested to be the endogenous ligand. We wished to test this and develop assays appropriate for the study of this receptor.Human and rat orthologues of GPR35 were engineered and expressed and assays developed to assess interaction with β-arrestin-2, activation of Gα₁₃ and agonist-induced internalization.GPR35-β-arrestin-2 interaction assays confirmed that both the endogenous tryptophan metabolite kynurenic acid and the synthetic ligand zaprinast had agonist action at each orthologue. Zaprinast was substantially more potent than kynurenic acid at each and both agonists displayed substantially greater potency at rat GPR35. Two novel thiazolidinediones also displayed agonism and displayed similar potency at each GPR35 orthologue. The three ligand classes acted orthosterically with respect to each other, suggesting overlapping binding sites and, consistent with this, mutation to alanine of the conserved arginine at position 3.36 or tyrosine 3.32 in transmembrane domain III abolished β-arrestin-2 recruitment in response to each ligand at each orthologue.These studies indicate that β-arrestin-2 interaction assays are highly appropriate to explore the pharmacology of GPR35 and that Gα₁₃ activation is an alternative avenue of signal generation from GPR35. Arginine and tyrosine residues in transmembrane domain III are integral to agonist recognition and function of this receptor. The potency of kynurenic acid at human GPR35 is sufficiently low, however, to question whether it is likely to be the true endogenous ligand for this receptor.© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.