Figure 4.

α_s-A³⁶⁶S/G²²⁶A/E²⁶⁸A does not inhibit agonist ligands that act by activating G_q or G_i. (A) COS-7 cells were transfected with control plasmid pcDNAI or plasmids encoding HA–α_s-A³⁶⁶S/G²²⁶A/E²⁶⁸A (1.2 μg per 1.6 × 10⁶ cells per 60-mm dish) and the M1 muscarinic receptor (0.2 μg per 1.6 × 10⁶ cells per 60 mm dish) and labeled with myo-[³H]inositol. Cells were incubated for 45 min with 5 mM LiCl and 200 μM carbachol (shaded bars) or no drug (hatched bars) and, phosphatidylinositol accumulation was measured. Values represent means ± SD of triplicate determinations. (B) COS-7 cells were transfected with vector plasmid pcDNAI or plasmids encoding wild-type HA–α_s-A³⁶⁶S/G²²⁶A/E²⁶⁸A (triple mutant, 1.2 μg per 1.6 × 10⁶ cells per 60-mm dish) and the D2 dopamine receptor (0.2 μg per 1.6 × 10⁶ cells per 60-mm dish) plus HA–MAPK (0.4 μg per 1.6 × 10⁶ cells per 60-mm dish). Cells were incubated for 8 min with 10 μM quinpirole (shaded bars) or no drug (hatched bars), and HA-MAPK activity was measured; the small circles indicate the values of duplicate determinations. Each set of results is representative of at least two additional experiments.