Figure 6. EP₁ receptor activation uncouples the β₂ AR from G_s .

β₂AR coupling to G_s was assessed by measuring isoproterenol-stimulated [³⁵S]GTPγS binding in membranes from COS-7 cells transfected with G_αs and the β₂AR, or with G_αs and β₂AR plus EP₁ (A) or mouse primary ASM cells (B). Reactions were performed with partially purified cell membranes. The [³⁵S]GTPγS bound to G_αs was recovered by immunoprecipitation with G_αs antibody as described in Methods. (A) The presence of 17-PTP in the reaction significantly reduced [³⁵S]GTPγS binding stimulated by isoproterenol in the transfected COS-7 cells, while the β₂AR expressed without the EP₁ had the greatest stimulation by isoproterenol. No effect of 17-PTP was observed on cells transfected to only express β₂AR. **P = 0.05 versus β₂AR only; ^##P = 0.02 versus 17-PTP–untreated cells coexpressing β₂AR and EP₁ receptor. Shown are mean ± SEM from 6 experiments. (B) The presence of 17-PTP in the reaction decreased isoproterenol-stimulated [³⁵S]GTPγS binding in ASM cell membranes. *P < 0.05 versus isoproterenol in the absence of 17-PTP. Shown are mean ± SEM from 6 experiments. In both types of experiments, basal (non-agonist) binding was ~65% of isoproterenol-stimulated binding.