Abstract

β₂-Adrenoceptor-mediated activation of G_s and adenylyl cyclase or other receptor-mediated G protein activations is believed to occur by receptor-catalyzed replacement of GDP with GTP on the α-subunit of the G protein. Here we showed that a β₂-adrenoceptor-G_s system, heterologously expressed in cyc^- or human embryonic kidney (HEK)-293 cells, can be activated in the presence of GDP or its phosphorylation-resistant analog, guanosine 5′-O-(2-thiodiphosphate) (GDPβS). The potency and maximal ability of GDP to activate G_s and adenylyl cyclase were identical to those of GTP. GDP-mediated activation of adenylyl cyclase, similar to that mediated by GTP, was concentration-dependent, required high magnesium concentrations, was inhibited by inverse agonists, and was correlated with the efficacy of receptor ligands used to stimulate the receptor. UDP did not block the GDP-mediated activation, although it completely blocked the formation of a small amount of GTP (∼5% GDP) from GDP. Moreover, the activation of G_s in the presence of GDP was insensitive to cholera toxin treatment of the cells, whereas that observed in the presence of GTP was amplified by the treatment, which showed that the activation observed in the presence of GDP was not mediated by GTP. Therefore, we concluded that GDP itself could mediate β-adrenoceptor-induced activation of G_s-adenylyl cyclase system as much as GTP. We discuss the results in the context of the current paradigm of receptor-mediated G protein activation and propose an additional mode of activation for β₂-adrenoceptor-G_s adenylyl cyclase system where nucleotide exchange is not necessary and GDP and GTP play identical roles in receptor-induced G_s protein activation.