Fig. 3
Sustained inhibition of the adenylyl cylase (AC) by Y2R stimulation can be abolished by PTX-treatment. a Concentration-response curves of transiently transfected HEK293-hY2R were measured by cAMP–accumulation assay without first stimulation (w/o; black line) and after stimulation with 10 nM, 100 nM and 1 μM NPY, respectively (brown, red, olive-green). AC inhibition caused by Gαi activity was measured in the presence of 5 μM forskolin. The maximum cAMP response is reduced in stimulated cells. b Data were verified by testing native Gαi-signaling with SMS-KAN cells that endogenously express Y2R. We observed the same reduction of the maximal inducible adenylyl cyclase response induced by forskolin in stimulated cells (olive-green). Data were validated with c) an internalization deficient mutant S374T376T379D-Y2R. d Concentration-response curves of transiently co-transfected HEK293-hY2R/hY1R were measured by cAMP–accumulation assay. Cells without first stimulation (NPY, black line; F7, P34-NPY, orange line; Ahx5–24-NPY, blue line) or with first stimulation with 1 μM F7, P34-NPY, a selective Y1R peptide (FP), followed by stimulation with Ahx5–24-NPY, a selective Y2R agonist (dashed blue line) exhibited no significant loss in AC-activity. First stimulation with 1 μM Ahx5–24-NPY, followed by stimulation with F7, P34-NPY (FP, dashed orange line) revealed an obvious inhibition of AC-activity. Data were normalized to Ahx5–24-NPY as control curve (100%, blue line). e Concentration-response curves of transiently co-transfected HEK293-hY2R with melanocortin 1 receptor were measured by cAMP–accumulation assay and had only slight impact on Gαs-signaling, as the inhibitory effect of Y2R is still present after stimulation with 1 μM NPY (left panel) but is completely abolished in presence of PTX and thus preventing Gαi- signaling (right panel). f cAMP-accumulation assay of transiently transfected HEK293-hY2R treated with varying concentrations of pertussis toxin (PTX) caused inhibition of AC-activity compared to control curve (black line). g AC-activity is rescued comparing ± PTX- treated cells after first stimulation with different NPY concentration and reached again the cAMP level of control (w/o). Experiments represent data n ≥ 3; significance was determined by one-way ANOVA, Tukey post test, ns: not significant, ***: P < 0.0001, **: P < 0.001