Guanine Nucleotide Exchange-Independent Activation of G_sProtein by β₂-Adrenoceptor

Abstract

␤ 2 -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 ␣-subunit of the G protein.Here we showed that a ␤ 2 -adrenoceptor-G s system, heterologously expressed in cyc Ϫ or human embryonic kidney (HEK)-293 cells, can be activated in the presence of GDP or its phosphorylation-resistant analog, guanosine 5Ј-O-(2-thiodiphosphate) (GDP␤S). 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 concentrationdependent, 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 (ϳ5% 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 ␤-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 ␤ 2 -adrenoceptor-G sadenylyl cyclase system where nucleotide exchange is not necessary and GDP and GTP play identical roles in receptorinduced G s protein activation.Heterotrimeric G proteins and their cognate heptahelical membrane receptors constitute the largest family of transmembrane signaling proteins. According to the accepted model of G protein activation, the GTP-bound form of G protein is active, whereas the GDP-bound form is inactive and the receptor-mediated exchange of GDP with GTP on the nucleotide-binding site is necessary and sufficient to initiate the activation process. In turn, deactivation is achieved by the hydrolysis of bound GTP to GDP. Fine-tuning of the rates of the elementary steps involved in this activation-deactivation cycle determines the average activity of the G protein (i.e., the amount of accumulated G␣-GTP), thus the strength of the signal transmitted to the inside of the cell (for review see Gilman, 1987;Hamm, 1998). This scenario implies that GTP is the natural activator of the G proteins, whereas GDP is their universal inhibitor, and that GTPase reaction is the off-switch for the G protein-mediated signal transduction. Therefore, the presence of excess GDP is expected to inhibit receptor-induced G protein activation, at least partly, by competing with GTP and the potency of this inhibitory effect should be correlated with the ability of the receptor's ligand to displace GDP from G␣. Viewed from a more rigorous standpoint, the sensitivity o...

Rapid-mix flow cytometry measurements of subsecond regulation of G protein-coupled receptor ternary complex dynamics by guanine nucleotides

“…Our data show that GDP is capable of inducing separation of ternary complex components, and is consistent with a number of literature reports where GDP was shown to induce effector activation [52][53][54]. In one study, GDP was reported to be capable of activating Gα s to the degree that subsequent interaction with adenylyl cyclase was measured [54].…”

“…In one study, GDP was reported to be capable of activating Gα s to the degree that subsequent interaction with adenylyl cyclase was measured [54]. It has been suggested that the affinity of a GTP-bound Gα s for adenylyl cyclase is ten times higher than its GDP bound counterpart [55].…”

Can a GDP-Liganded G-Protein Be Active?: Fig. 1.

“…6 (Seifert et al, 1998). GTP was found to be approximately 3 orders of magnitude more potent than GDP in inducing agonist displacement than GDP under conditions in which hydrolysis of GTP to GDP or transphosphorylation of GDP to GTP is less likely to occur than under the conditions used by Ugur et al (2005). Nevertheless, these experimental conditions also apply to the studies with cholera toxin-treated membranes; thus, this possibility does not seem to explain the differences between GDP and GTP observed in the latter experiments.…”

Mechanistic pathways and biological roles for receptor-independent activators of G-protein signaling

“…This concept is also supported by the kinetic coupling model for the action of RGS proteins indicating that in the presence of RGS proteins, GTP hydrolysis is too rapid to allow dissociation of Gα and Gβγ prior to effector activation (Ross and Wilkie, 2000). An interesting thought was presented by Ugur and Onaran with their observation that the β2-adrenergic receptor could activate adenylyl cyclase without causing nucleotide exchange on Gsα (Ugur et al, 2005).…”