Fig. 3. GPR139 inhibits MOR signaling.

(A) Experimental design for evaluating MOR signaling via its effector GIRK. MOR activation leads to Gβγ subunit release, which opens GIRK channels to produce membrane hyperpolarization (Vm) that is measured with voltage sensitive dye. (B) Coexpression of GPR139 inhibits MOR-mediated kinetics of membrane potential change in response to morphine (0.1 μM). (C) Quantification shows GPR139 reduces morphine effects on Vm amplitude. (D) Co-immunoprecipitation of MOR-FLAG and myc-GPR139 following their coexpression. (E) Experimental design for evaluating cell surface abundance of MOR. HiBiT-tagged MOR complements the LargeBiT (LgBiT) nanoluciferase enzyme only at the plasma membrane. (F) Quantification of the maximal cell surface content of HiBiT-MOR indicates that GPR139 inhibits MOR surface localization only at high (12X) expression levels. (G) Experimental design for evaluating agonist-induced β-arrestin recruitment to MOR. Recruitment of β-arrestin2-LgBiT to SmBiT-MOR generates a functional nanoluciferase enzyme. (H) Effect of GPR139 coexpression on the kinetics of β-arrestin2-LgBiT recruitment induced by DAMGO (10 μM). (I) Quantification shows that low level GPR139 coexpression increases the extent of β-arrestin2 recruitment to MOR. (J) Experimental design for evaluating MOR signaling to G proteins by BRET assay that monitors MOR-mediated release of Gβγ subunits. (K) Effect of GPR139 coexpression on the kinetics of G protein activation by MOR in response to morphine (1 μM) application. (L) Quantification shows GPR139 coexpression reduces maximal BRET response of MOR-Gαo signaling. All experiments were performed in HEK293T cells. In all panels, means are shown from 3-5 independent experiments with 3-4 replicates each ± S.E.M. Significance tested by one-way ANOVA with Dunnett’s post-hoc test. *** p<0.001, ** p<0.01, * p<0.05. Arrows denote application of opioids.