FIGURE 4.

GHS-R1b modulates GHS-R1a-mediated inhibition of adenylyl cyclase in HEK-293T cells. cAMP accumulation was determined in HEK-293T cells transfected with GHS-R1a-YFP cDNA (1 μg) (A) or co-transfected with GHS-R1a-YFP cDNA (1 μg) and increasing amounts of GHS-R1b-Rluc cDNA (0.05–0.6 μg) to obtain a 0.5, 1.4, and 5 GHS-R1b-Rluc/GHS-R1a-YFP ratio (B). Cells were incubated overnight with vehicle or PTX (10 ng/ml) or for 2 h with CTX (100 ng/ml) and pretreated (15 min) with vehicle or the GHS-R1a antagonist YIL781 (2 μm), followed by activation (15 min) with ghrelin in the absence or presence of 0.5 μm forskolin (FK). Values are means ± S.E. of five to six experiments per treatment and expressed as decreases of forskolin-induced cAMP accumulation (100%, dotted line). Statistical differences of the effect of differently treated cells under different transfection conditions were analyzed by ANOVA followed by Bonferroni's corrections. *, p < 0.05; ***, p < 0.001 compared with the effect of forskolin alone.