Fig. 4

Activity of PTX-insensitive Gα_i1 chimeras upon receptor activation. HEK293 cells were co-transfected with D₂R and various Gα_i1 constructs (0.2 μg/mL each), treated with PTX (100 ng/mL, 16 h), and then assayed for forskolin-induced [³H]cAMP accumulation in the absence or presence of 100 nM quinpirole. A Expression of the PTX-insensitive Gα_i1 chimeric mutants was confirmed by immunoblotting with 20 μg of total protein. B Forskolin-stimulated cAMP levels are expressed as a percentage of the response normalized against Gα_i1-CI. C Quinpirole-induced activity is expressed as a percentage of inhibition of the forskolin response. Data shown are mean ± SEM (n = 3). Bonferroni t test, p < 0.05; *, significantly lower than the control; #, significantly higher than the control; †, significant inhibition upon receptor activation. D Rationale of the subunit dissociation assay. Activated Gα_i1 dissociates with Gβγ, resulting in a drop in Gα_i1 intensity in immunodetection after co-immunoprecipitation with the Flag-tagged Gβ. Gα_i1 activation by GTPγS, but not AlF₄⁻, requires guanine nucleotide exchange. E–G HEK293 cells were transiently co-transfected with 0.2 μg/mL each of Flag-tagged Gβ₁, HA-tagged Gγ₂, and either vector (V), Gα_i1 or Chi1. E Expressions of the G proteins were confirmed by immunoblotting with 20 μg of the total proteins. F 500 μg of the total proteins of the lysate were incubated with or without AlF₄⁻ (30 μM AlCl₃ plus 10 mM NaF) or 100 μM of GTPγS at 37 °C for 15 min prior to immunoprecipitation by anti-Flag affinity gel. G Quantification of the co-immunoprecipitation results. Results are expressed as a percentage of Gα_i1 or Chi1 pull-down by Flag-Gβ₁. Graph is shown as mean ± SEM (n = 3). Student t test, p < 0.05; †, significantly different