Figure 5 From: Engineered G protein coupled receptors reveal independent regulation of internalization, desensitization and acute signaling Desensitization of cAMP inhibition and superactivation of AC after pretreatment with spiradoline (A) Spiradoline (1 nM pretreatment) inhibited forskolin-induced cAMP formation in HEK293 cells transiently expressing Rog, Rog-A, Rog-μ, and Rog-μA. Data are expressed as inhibition of forskolin-induced cAMP. The baseline (0) represents maximum forskolin-induced cAMP accumulation in control cells. Pretreatment with 1 nM spiradoline for 10 min causes a shift in the dose-response curve for later spiradoline treatment in Rog and Rog-μA cells, but not Rog-A. Spiradoline pretreatment lowered the maximal response of Rog-μ to subsequent spiradoline treatment. (B) HEK293 cells transiently expressing receptors were treated 18 h with 10 nM spiradoline, and assayed for cAMP accumulation in response to a 15-min treatment with 10 μM forskolin. Spiradoline pretreatment significantly increased forskolin-induced cAMP in cells expressing KOR, Rog and Rog-A. Pretreatment of cells expressing Rog-μ and Rog-μA had not effect on response to forskolin. Data are expressed relative to the amount of cAMP accumulated after 10 μM forskolin treatment in cells pretreated with vehicle. Bars represent mean ± SEM for six replicates per condition. (C) HEK293 cells transiently expressing receptors were treated 18 h with 10 nM spiradoline, and assayed for loss of cell-surface expression by ELISA. Long-term spiradoline treatment significantly reduces cell-surface expression of all receptors, but the degree of internalization does not differ among different receptors. Bars represent mean ± SEM for three replicates per condition.Back to article page