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


METHODS
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.


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
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.
Molecular Pharmacology Group, Neuroscience and Molecular Pharmacology, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK.
Laura
L
Jenkins
Elisa
E
Alvarez-Curto
Kate
K
Campbell
Sabrina
S
de Munnik
Meritxell
M
Canals
Sabine
S
Schlyer
Graeme
G
Milligan
D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D001681:Biological Assay; D002478:Cells, Cultured; D003106:Colon; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000378:metabolism*; D005727:Ganglia, Spinal; D006801:Humans; D007736:Kynurenic Acid / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011688:Purinones / Q000494:pharmacology; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C000605254:ARRB2 protein, human; C000605257:Arrb2 protein, rat; D019390:Arrestins; D018691:Excitatory Amino Acid Antagonists; C111379:GPR35 protein, human; C513985:GPR35 protein, rat; D010726:Phosphodiesterase Inhibitors; D011688:Purinones; D043562:Receptors, G-Protein-Coupled; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D044365:GTP-Binding Protein alpha Subunits, G12-G13; C011145:zaprinast; D007736:Kynurenic Acid