FIGURE 9.

GHS-R1a-dependent constitutive activity at G_q and G₁₃ and selectivity of agonists and inverse agonists. A and B, BRET signal measured in HEK293T cells co-expressing either Gα_q-Rluc8 (A) or Gα₁₃-Rluc8 (B) and GFP10-Gγ₂ and Gβ₁ in the absence or presence of increasing amounts of HA-GHS-R1a (vectors encoding N-terminally the HA-tagged GHS-R1a ranging from 0.001 to 4 μg/well) and in the absence of ligand. Data represent the mean ± S.E. of at least three independent experiments. Statistical significance between cells expressing or not the HA-GHS-R1a was assessed using a one-way ANOVA followed by Tukey's test (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Each transfection condition was controlled for Gα_q-Rluc8 (C) and Gα_q-Rluc8 (D) total expression levels by measuring luminescence intensity (C and D) and cell surface expression of HA-GHS-R1a quantified by ELISA using an anti-HA antibody (E and F). Results are expressed as the mean ± S.E. of at least three independent experiments. G and H, BRET signal promoted by ligands measured in HEK293T cells co-expressing either Gα_q-Rluc8 (G) or Gα13-Rluc8 (H), GFP10-Gγ₂ and Gβ1, in the presence of the HA-GHS-R1a, and stimulated or not with 10 μm ligands. Results are expressed as the difference in BRET signals measured in the presence and in the absence of ligand. Values are mean ± S.E. of at least four independent experiments. Statistical significance between stimulated and non-stimulated cells was assessed using a paired Student's t test (***, p < 0.001; **, p < 0.01; *, p < 0.05).