Figure 3.

Inhibition by α_s-A³⁶⁶S/G²²⁶A/E²⁶⁸A depends on concentrations of transfected α_s and LH-R DNA as well as concentration of agonist ligand. (A and B) COS-7 cells were transfected with control plasmids (open symbols) or a plasmid containing LH-R DNA (filled symbols; 0.04–0.16 μg per 1.6 × 10⁶ cells per 60-mm dish, as indicated) and cotransfected with a second control plasmid (squares) or a plasmid encoding the α_s triple mutant (0.6 μg (circles) or 2.4 μg (triangles) per 1.6 × 10⁶ cells per 60-mm dish). After labeling with [³H]adenine for 24 hr either at 37°C (A) or at 33°C (B), cells were further incubated either at 37°C or at 33°C for 25 min with 100 μM IBMX and 75 ng/ml hCG (filled symbols) or no ligand (open symbols), and cAMP accumulation was measured. (C) Membranes (0.25 mg/ml) of COS-7 cells transfected with plasmids encoding either HA–α_s-wild type or HA–α_s-triple mutant (2.4 μg per 1.6 × 10⁶ cells per 60-mm dish) and incubated either at 37°C or at 33°C (as indicated) for the ensuing 24 hr were solubilized with 1% CHAPS on ice for 60 min. Extracts (each representing 12.5 μg of membrane protein) were subjected to SDS/PAGE and immunoblotted with 12CA5 antibody (see Materials and Methods). (D) Cells were transfected with plasmids encoding the LH-R (0.2 μg per 1.6 × 10⁶ cells per 60-mm dish) with (filled symbols) or without (open symbols) the α_s triple mutant (1.6 μg per 1.6 × 10⁶ cells per 60-mm dish). After labeling with [³H]adenine for 24 hr at 33°C, cells were further incubated at 33°C for 25 min with 100 μM IBMX and the indicated concentration of hCG, and accumulation of cAMP was measured. Values represent means ± SD of triplicate determinations. Each set of results is representative of at least two additional experiments.