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. 2011 Feb;162(3):733-48.
doi: 10.1111/j.1476-5381.2010.01082.x.

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

Laura Jenkins  1 , Elisa Alvarez-Curto, Kate Campbell, Sabrina de Munnik, Meritxell Canals, Sabine Schlyer, Graeme Milligan
Affiliations

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

Laura Jenkins et al. Br J Pharmacol. 2011 Feb.

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.

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Figures

Figure 1
Figure 1
Expression of FLAG-hGPR35-eYFP and FLAG-rGPR35-eYFP. (A, B) Flp-In™ T-REx™ 293 cells harbouring either FLAG-hGPR35-eYFP (A) or FLAG-rGPR35-eYFP (B) at the Flp-In™ locus were maintained in the absence (- dox) or presence of varying concentrations (ng·mL−1) of doxycycline for 24 h and then imaged. (C) Membranes prepared from cells akin to those of (A, B) were resolved by SDS-PAGE and immunoblotted with anti-FLAG (5 µg of protein was loaded per lane). Upper panel human GPR35, lower panel rat GPR35.
Figure 2
Figure 2
Orthologues of GPR35 elevate [Ca2+]i via a Gαq/Gα13 chimera but not a Gαq/Gα12 chimera. (A) HEK293T cells were transfected with either human GPR35 or rat GPR35 and either full length Gαq or a Gαq/Gα13 chimera. Single cell Ca2+ imaging studies were performed and effects of zaprinast (zap) were recorded in each case. In cells that did not respond to zaprinast (black, blue), ATP was added subsequently to activate endogenously expressed P2Y purinoceptors. (B) Similar studies were performed on cells transfected to express human GPR35 or rat GPR35 and a Gαq/Gα12 chimera. As in (A), cells transfected to express the Gαq/Gα12 chimera, although producing little response to zaprinast, were subsequently challenged with ATP to confirm that [Ca2+]i could be elevated in these cells. At least 20 cells were examined for each condition and data represent means ± SEM.
Figure 3
Figure 3
Orthologues of GPR35 promote activation of Gα13. Studies with a Gα13 antibody that interacts selectively with GTP-Gα13. Cells harbouring either FLAG-hGPR35-eYFP (A, B) or FLAG-rGPR35-eYFP (C, D) were either untreated (- dox) or treated (+ dox) with doxycycline (concentrations as noted). Cells were then transfected with either wild type Gα13 or a constitutively active Gln226Leu Gα13 variant (CAM). Cells were treated with or without 100 µM zaprinast as noted for 30 min. Samples were immunoprecipitated with an active-state Gα13 antibody and these samples (GTP-Gα13) and cell lysates (Total Gα13) were resolved by SDS-PAGE and subsequently immunoblotted with a polyclonal Gα13 antiserum. In (B and D) the relative amount of GTP-Gα13 and total levels of Gα13 were assessed by analysis of such immunoblots following different periods of exposure to 100 µM zaprinast (means ± SEM, n = 3). (E) Transfection with either wild-type or CAM Gα13 results in a substantial enhancement of expression. Cells induced to express FLAG-hGPR35-eYFP were transfected transiently with either wild-type Gα13 or the CAM variant. An empty vector transfection (-Gα13) was also performed. Lysates from cells were resolved by SDS-PAGE and immunoblotted with the polyclonal Gα13 antiserum.
Figure 4
Figure 4
Analysis of GPR35-β-arrestin-2 interactions. (A) Human and rat forms of GPR35 C-terminally tagged with the Prolink™ fragment of β-galactosidase were transiently introduced into HEK 293-BAEA cells stably expressing β-arrestin-2 linked to the remaining element of β-galactosidase. Cells were treated with varying concentrations of zaprinast for 60 min, as suggested by the manufacturer, and β-galactosidase activity reflecting complementation of the enzyme produced via GPR35-β-arrestin-2 interaction was measured. (B) FLAG-hGPR35-eYFP or FLAG-rGPR35-eYFP was co-transfected with β-arrestin-2-Renilla luciferase into HEK293T cells. BRET signals were monitored after treatment of the cells for 5 min with varying concentrations of zaprinast, kynurenic acid or kynurenic acid ethyl ester. (C) Studies akin to those of (B) were conducted using varying concentrations of compound 3 and compound 10 using FLAG-hGPR35-eYFP or FLAG-rGPR35-eYFP.
Figure 5
Figure 5
Zaprinast, kynurenic acid and compound 10 share an overlapping binding site in GPR35. (A) Kynurenic acid, zaprinast and (Z)-[4-(2,4-dioxo-thiazolidin-5-ylidenemethyl)-phenoxy] acetic acid (compound 10) are agonists at GPR35. Receptor-β-arrestin-2 interaction BRET assays were performed as in Figure 4 following co-transfection of either FLAG-rGPR35-eYFP (B, C, D) or FLAG-hGPR35-eYFP (E) and β-arrestin-2-Renilla luciferase. Concentration-response curves to zaprinast were performed in the presence of varying fixed concentrations of kynurenic acid (B) or concentration-response curves to kynurenic acid were performed in the presence of varying fixed concentrations of zaprinast (C). In (D and E), concentration-response curves to zaprinast were performed in the presence of varying fixed concentrations of compound 10. Tables provide the estimated pEC50 values for each condition. Data represent means ± SEM.
Figure 6
Figure 6
Mutation of arginine 3.36 eliminates agonist function of zaprinast, kynurenic acid and compound 10. (A, B) Receptor-β-arrestin-2 interaction BRET assays in response to varying concentrations of kynurenic acid (A) or zaprinast (B) were performed as in Figure 4 using either wild-type or Arg3.36Ala mutants of either FLAG-hGPR35-eYFP or FLAG-rGPR35-eYFP. The effects of the Arg3.36Ala mutations on response to a single concentration (10−4 M) of compound 10 that was maximally effective at the wild-type GPR35 orthologues was also assessed (C). Total levels of GPR35 expressed, as monitored by eYFP fluorescence above non-transfected cells, were unaltered by the Arg3.36Ala mutation (D), while cell surface delivery was monitored by anti-FLAG elisa (E) of the forms of GPR35. * Less than wild-type P < 0.05. (F) The amino acid sequence of a section of transmembrane domain III from both human and rat GPR35 is shown with Arg3.36 highlighted.
Figure 7
Figure 7
The role of tyrosine 3.32 of GPR35 in ligand function. Receptor-β-arrestin-2 interaction BRET assays were performed in response to varying concentrations of zaprinast (A, C) or kynurenic acid (B) following co-transfection of FLAG-rGPR35-eYFP, FLAG-Tyr3.32Ala rGPR35-eYFP or FLAG-Tyr3.32Leu rGPR35-eYFP (A, B) of the equivalent forms of human GPR35 (C). Effects of mutation of Tyr 3.32 on the response to 10−4 M compound 10 was also studied (D). Total expressed levels, as monitored by eYFP fluorescence (E), of the various forms of GPR35 were unaltered by these mutations, as was cell surface delivery monitored by anti-FLAG elisa (F). (G) The amino acid sequence of a section of transmembrane domain III from both human and rat GPR35 is shown with Tyr3.32 highlighted.
Figure 8
Figure 8
Agonist-induced internalization of GPR35 is correlated with agonist potency in receptor-β-arrestin-2 interaction BRET assays. (A) Biotinylation studies demonstrated expression at the cell surface of FLAG-hGPR35-eYFP following treatment with doxycycline (compare – dox with – drug). Following treatment with increasing concentrations of zaprinast (zap), and single concentrations of either compound 3 or compound 10 for 30 min, but not with kynurenic acid (KNA), cell surface (biotinylated) but not total levels (total extract) of GPR35 were reduced (8 µg of protein from total extract was loaded per lane). Results from a representative experiment are shown. in (B). Cell surface levels in such experiments were quantified. Data represent means ± SEM, n = 3. (C) Cells induced to express similar levels of FLAG-hGPR35-eYFP and FLAG-rGPR35-eYFP were exposed to varying concentrations of zaprinast for 30 min. Cell surface anti-FLAG elisa studies were quantified to measure internalization of the GPR35 orthologues. A representative experiment of 3 performed in triplicate is shown.
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