FIGURE 2.
α-MSH-induced activation of pertussis toxin-sensitive G proteins in HEK293-Ex-MC4R or HEK293-Ex-MC4R-D90N cells. A, 20 μg of total membrane fractions derived from HEK293-Ex-MC4R or HEK293-Ex-MC4R-D90N cells were incubated with 0. 1 nm non-hydrolysable GTPγ35S for 5, 15, or 30 min at 30 °C. Signals from non-stimulated membranes are subtracted from the signals of stimulated (1 μm α-MSH) cells. Results are expressed as the mean ± S.E. of two independent experiments performed in quadruplicate. B, incorporation of GTPγ35S (30 min) was monitored as described under A. Additionally, membranes derived from control HEK293-Ex-MC4R cells or from cells treated with the Gi/o-specific inhibitor PTX (50 ng/ml) for 16–24 h at 37 °C were stimulated with 1 μm somatostatin (SST) to activate endogenously expressed somatostatin-2 receptor subtypes as a control for a Gi/o-coupled receptor. Results are expressed as the mean ± S.E. of four independent experiments carried out in quadruplicate. Asterisks indicate a significant (**, p < 0.01) difference between PTX-treated and non-treated cells.