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. 2020 Jan 31;9:e50519. doi: 10.7554/eLife.50519

Figure 5. GPR88 biases the signaling of multiple GPCRs.

Figure 5.

We evaluated the consequences of GPR88 co-expression on the signaling of adenosine A2A, dopamine D1, dopamine D2, muscarinic M1, muscarinic M4, vasopressin V2R and CXCR4 receptors in HEK293FT cells. BRET1 assay was used to assess the activation of the (A) G protein dependent pathway (cAMP sensor: CAMYEL; calcium sensor: aequorin-GFP) or (B) the recruitment of Ypet β-arrestin 2 (β-arr2) by Venus-tagged receptors in presence of increasing amounts of GPR88 cDNA transfected. (A) GPR88 co-expression blunted the G protein mediated signaling of all GPCRs from which it comes close (panels c-f; dopamine D2: H3,12=9.6, p=0.0223; muscarinic M1: H3,16=13.2, p=0.0041; M4 calcium: H3,35=24.0, p=0.0000; M4 cAMP: H3,12=12.9, p=0.0048), except the adenosine A2A receptor (panel a; H3,12=1.2, p=0.7602). GPR88 had no significant impact on, or even facilitated, G protein dependent signaling of receptors for which we failed to evidence proximity to it, namely D1 (panel b, increased activity in presence of GPR88; H3,20=9.7; p=0.0211), V2R and CXCR4 receptors (panels g,h; V2R: H3,16=1.2, p=0.7602; CXCR4: H3,12=7.6, p=0.0546). (B) In contrast, GPR88 co-expression compromised the ability of all GPCRs tested to recruit Ypet-β-arr2 when activated by their agonist, independently from previously evidenced close proximity to GPR88 (panels a-g; A2A: H3,12=9.7, p=0.0211; D1: H3,12=9.6, p=0.223; D2: H3,16=13.2, p=0.0080; M1: H3,15=10.4, p=0.0151; M4: H3,16=13.2, p=0.0041; V2R: H3,16=10.9, p=0.0123; CXCR4: H3,16=13.2, p=0.0041). Focusing on CXCR4, co-expressing GPR88 diminished the probability of Ypet-β-arr2 recruitment at this receptor under both stimulated and basal conditions (panel h). Specific agonists used to stimulate GPCRs were: CGS21680 (A2A, 10 μM), SKF81297 (D1, 10 µM), quinpirole (D2, 10 µM), carbachol (M1 and M4, 10 µM), AVP (V2R, 10 μM) and SDF1 (CXCR4, 125 µM). Data are presented as mean ± SEM of n = 3–4 independent experiments (performed in triplicates). BRET1 values are presented as induced BRET (normalized as the percentage of maximal BRET values in absence of GPR88) normalized by Venus/Rluc8 BRET ratio. Asterisks: Kruskal-Wallis ANOVA, multiple comparison of mean ranks, *p<0.05, **p<0.01.