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

Figure 2. GPR88 dampens µOR-mediated signaling in vitro: ERK phosphorylation and effects of µOR expression levels.

(A) Upper panel: in HEK293FT cells, DAMGO stimulated µOR (0.5 µg cDNA) activity, resulting in an increase in the phospho-ERK/ERK total (pERK/tERK) ratio peaking 15 min after DAMGO stimulation; this response was suppressed when GPR88 (1 µg cDNA) was co-expressed with µOR. Lower panel: representative western blotting images. (B) Upper panel: addition of pertussis toxin (PTX, 0.1 ng/µl, overnight) blocked the early phase of DAMGO-induced response, demonstrating its dependence on Gi/o protein recruitment, but failed to inhibit a later component of ERK phosphorylation. GPR88 co-expression completely blocked DAMGO-induced phosphorylation of ERK. Lower panel: representative western blotting images. Levels of phosphorylated-ERK (pERK) and total-ERK (tERK) were normalized to the loading control protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are presented as mean ± SEM of n = 5 independent experiments. Kruskal-Wallis ANOVA, multiple comparison of mean ranks *p<0.05, **p<0.01 (see gels in Figure 2—figure supplement 1 and statistics in Supplementary file 1). (C) Increasing the amount of µOR (wild-type, ng of cDNA) expressed in HEK293FT cells allowed to overcome the inhibitory effects of GPR88 (wild-type) on DAMGO (µOR agonist, 10 μM)-induced inhibition of cAMP production (CAMYEL), and a complete rescue of signaling was observed for the highest doses of µOR cDNA transfected (GPR88 effect: F2,21=86.7, p=0.0018; µOR effect: F7,147=90.9, p=0.0000; GPR88 x µOR interaction: F7,147=3.1, p=0.00030). Although increasing the amount of GPR88 (30 to 50 ng of cDNA) shifted the µOR dose response to the right, it was not able to prevent full restoration of µOR signaling at high doses of µOR. (D) Increasing the amount of µOR (wild-type) expressed in HEK293FT cells allowed only a partial overcoming of the inhibitory effects of GPR88 (wild-type) on DAMGO (µOR agonist, 10 μM)-induced Ypet-β-arr2 recruitment; no further recruitment was detected for doses of µOR cDNA over 50 ng (GPR88 effect: F2,6=86.7, p=0.000037; µOR effect: F6,36=32.3, p=0.0000; GPR88 x µOR interaction: F6,36=15.7, p=0.0000). Increasing the amount of GPR88 (from 30 to 50 ng of cDNA transfected) nearly suppressed β-arr2 recruitment at µOR. Data for 15 ng of µOR cDNA transfected are framed in gray, to allow comparison with Figure 1E. Data are presented as mean ± SEM of n = 3–12 independent experiments (performed in triplicates). BRET1 values are presented as induced BRET (normalized as the percentage of maximal BRET values in absence of GPR88) by Venus/Rluc8 BRET ratio. ANOVA (repeated measure), stars: GPR88 effect, daggers: GPR88xµOR interaction; one symbol: p<0.05, two symbols: p<0.01, three symbols: p<0.001. Effects of GPR88 activation by synthetic agonist Compound 19 on its inhibitory action at µOR signaling in vitro are presented in Figure 2—figure supplement 2.

Figure 2.

Figure 2—figure supplement 1. Gels from western blot experiments; ERK phosphorylation assay in HEK293FT cells.

Figure 2—figure supplement 1.

Figure 2—figure supplement 2. Effects of Compound 19 on GPR88 signaling pathways and GPR88-mediated blunting of G-protein dependent signaling and β-arrestin recruitment by µOR.

Figure 2—figure supplement 2.

(A) Activation of GPR88 by Compound 19 induced a dose-dependent inhibition of cAMP production (cAMP sensor: CAMYEL, Gαi/o GPR88 coupling). Compound 19 failed to induce detectable recruitment of Ypet-β-arr2 by Venus-GPR88 receptor at a high dose of 10 µM. (B) Co-expressing increasing amounts of GPR88 blunted DAMGO (10 µM)-induced inhibition of cAMP production by µOR. In an attempt to visualize the putative effects of GPR88 activation by Compound 19 on this inhibitory action, we subtracted the percentage of cAMP inhibition induced by Compound 19 to the percentage of cAMP inhibition produced by both DAMGO (10 µM) and Compound 19 (10 µM) in presence of µOR and increasing amounts of GPR88. When Compound 19 was added to DAMGO, GPR88 tended to further dampen µOR-induced inhibition of cAMP. (C) Co-expressing increasing amounts of GPR88 with µOR blunted DAMGO (10 µM)-induced Ypet-β-arr2 recruitment at µOR; addition of Compound 19 (10 µM) did not affect this inhibitory effect; Compound 19 had no influence on Ypet-β-arr2 recruitment by µOR in the absence of DAMGO. Data are presented as mean ± SEM of n = 3–4 independent experiments (performed in triplicates). BRET1 values are presented as net BRET (normalized as the percentage of maximal BRET values) or induced BRET (normalized as the percentage of maximal BRET values in absence of GPR88) by Venus/Rluc8 BRET ratio.