Mutations in the N-terminal domain of MC4R result in reduced constitutive activity. (A) Basal activities of WT and mutated MC4R were assayed by analyzing their ability to activate the expression of a cAMP-induced luciferase reporter gene under basal unstimulated conditions or in response to AGRP. The data are normalized to maximal stimulation obtained in presence of 8Br-cAMP (1 μM) and to Renilla luciferase activity for assessment of transfection efficiency. (B) Ratios of cAMP accumulation (measured by CatchPoint assays) to membrane expression (measured by ELISA) were determined on the same batch of transiently transfected cells for WT and mutant receptors. Data are expressed as percentage of WT activity as the means of quadruplicate determinations (± SEM) and are the averages of 3 independent experiments. Inset: There were no differences in membrane expression between the WT and mutant receptors. The ratio of basal activity to membrane expression was significantly reduced for all N-terminal domain mutants (P < 0.05). (C) The ratio of basal activity to cell-surface expression was measured in the WT and R18C receptors under acute (2-hour incubation) and chronic (overnight incubation) basal conditions. (D–F) Basal activity relative to cell-surface expression of WT and R18C in transiently transfected HEK293 cells. Increasing cell-surface expression was obtained by transfecting 0.1, 0.2, and 0.5 μg DNA in 6-well tissue culture dishes. WT MC4R-transfected cells showed a linear increase in basal activity with increasing membrane expression (slope = 2.2; r² = 0.98). In contrast, R18C, R7H, and T11S show a lower increase in basal activity despite an increase in cell-surface expression similar to the WT receptor.