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

Abstract

BACKGROUND AND PURPOSE 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. EXPERIMENTAL APPROACH 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. KEY RESULTS 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. CONCLUSIONS AND IMPLICATIONS 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.

The Antiallergic Mast Cell Stabilizers Lodoxamide and Bufrolin as the First High and Equipotent Agonists of Human and Rat GPR35

“…Human embryonic kidney (HEK) 293T cells were maintained in DMEM supplemented with 0.292 g/l L-glutamine, 10% (v/v) fetal bovine serum, and 1% penicillin/streptomycin mixture. Transient transfections using HEK293T cells were performed using polyethylenimine, with experiments carried out 48 hours after transfection (Jenkins et al, 2011). HT-29 human adenocarcinoma cells were purchased from the American Type Culture Collection (Gaithersburg, MD) and maintained in McCoy's 5A (Modified) media containing 25 mM HEPES.…”

“…All novel plasmids employed expressed human (GPR35a or GPR35b) or rat GPR35 receptor constructs that contain an enhanced yellow fluorescent protein (eYFP) fused to the C terminus and an N-terminal FLAG epitope tag as previously described (Jenkins et al, 2010(Jenkins et al, , 2011. Individual amino acid swap mutations between human and rat sequences were introduced into the FLAG-hGPR35a-eYFP or FLAG-rGPR35-eYFP constructs using the QuikChange method (Stratagene, La Jolla, CA).…”

“…The PathHunter b-arrestin-2 recruitment assay was performed using the CHO-K1 cell line stably expressing human GPR35 and b-arrestin-2 (DiscoveRx) as previously described . The bioluminescence resonance energy transfer (BRET)-based b-arrestin-2 recruitment assay was performed using HEK293T cells transfected transiently to coexpress forms of human GPR35a, human GPR35b, or rat GPR35 along with b-arrestin-2, as described by Jenkins et al, (2011).…”

“…It would, therefore, be of particular value to identify agonists with similar and high potency at the human and rodent orthologs of GPR35 and to better understand the basis of ligand selectivity between species. One common feature of human and rat GPR35 is the presence of an arginine residue in TMD III [position 3.36 in the Ballesteros and Weinstein (1995) residue numbering system] that when mutated to a nonbasic residue results in virtual complete loss of potency of kynurenic acid and a number of GPR35 surrogate agonist ligands (Jenkins et al, 2011). Importantly, arginine 3.36 is conserved in a number of other receptors, such as the lactate receptor GPR81 (Liu et al, 2009), that are activated by endogenously generated acids, suggesting that kynurenic acid and potentially other acidic ligands, interact with this residue via an ionic interaction.…”

“…Zaprinast (Taniguchi et al, 2006;Jenkins et al, 2010Jenkins et al, , 2011 has become the standard surrogate agonist of GPR35 and, akin to zaprinast, a number of mast cell stabilizers also contain an acid bioisostere. Because zaprinast is an example of a ligand that displays marked variation in potency between rodent and human GPR35 (Taniguchi et al, 2006;Jenkins et al, 2010Jenkins et al, , 2011, this also encouraged us to also explore the contributions of a series of nonmaintained arginine residues within regions of the receptor species orthologs that are predicted to define the ligand binding pocket. Cross-species alterations of such residues provided novel insights into the binding pocket.…”