Receptor-stimulated dissociation of GTP[S] from Gi-proteins in membranes of HL-60 cells

Mechanisms of G protein activation via the D2 dopamine receptor: evidence for persistent receptor/G protein interaction after agonist stimulation

“…We observe that in CHO cell membranes transfected with the dopamine D 2 receptor, [ 35 S]GTP γ S dissociation occurs under basal conditions (absence of agonist) and that the dissociation is markedly increased in rate and extent in the presence of dopamine. This agrees well with previous studies on the formyl peptide receptor ( Kupprion et al ., 1993 ), where a 50% decrease in bound [ 35 S]GTP γ S was observed over a 30 min period upon addition of 1 μ M non‐radiolabelled GTP γ S. In that study, the dissociation was rapid and maximal in its extent in the first 10 min. Agonist independent [ 35 S]GTP γ S dissociation observed for cannabinoid receptors ( Breivogel et al ., 1998 ) showed a rapid dissociation phase with a t 1/2 of 6.8 min.…”

“…Studies carried out in the 1980s showed that the binding of [ 35 S]GTP γ S to purified G α o and G α i2 subunits of G proteins and to purified heterotrimeric G o protein was irreversible in the presence of millimolar concentrations of Mg 2+ ( Sternweis and Robishaw, 1984 ; Higashijima et al ., 1987 ). More recent studies carried out in membrane systems containing GPCRs have shown, however, that [ 35 S]GTP γ S binding is reversible and that dissociation may be accelerated in the presence of an agonist ( Kupprion et al ., 1993 ; Breivogel et al ., 1998 ; Waelbroeck, 2001 ; Alt et al ., 2002 ). In this study, we have used the D 2 dopamine receptor stably transfected in CHO cells to further investigate the dissociation of [ 35 S]GTP γ S from G proteins.…”

“…The [ 35 S]GTP γ S binding was seen to be irreversible in the presence of millimolar concentrations of Mg 2+ , but became reversible when the Mg 2+ concentration was lowered. Since then studies carried out in native membrane systems containing GPCRs have suggested that [ 35 S]GTP γ S binding is reversible and that the [ 35 S]GTP γ S binding reaches an equilibrium dependent on the association and dissociation of [ 35 S]GTP γ S. In support of this, agonists have been shown to stimulate the dissociation of [ 35 S]GTP γ S binding via muscarinic receptors in atrial porcine membranes ( Hilf et al ., 1989 ), the formyl peptide receptor in HL‐60 cells ( Kupprion et al ., 1993 ), cannabinoid receptors in rat cerebellar membranes ( Breivogel et al ., 1998 ), β 2 ‐adrenergic receptors in Sf9 cells ( Wenzel‐Seifert and Seifert, 2000 ), muscarinic M1 receptors in CHO cells ( Waelbroeck, 2001 ), 5 ‐HT 1A receptors in CHO cells ( Newman–Tancredi et al ., 2002 ), and opioid receptors in SH‐SY5Y cells ( Alt et al ., 2002 ).…”

The olfactory G protein Gαolf possesses a lower GDP‐affinity and deactivates more rapidly than Gsαshort: consequences for receptor‐coupling and adenylyl cyclase activation

“…1996b; Wenzel‐Seifert and Seifert 2000). This background, and the fact that in contrast to generally held belief GTPγS does not bind quasi ‐irreversibly to G proteins (Kupprion et al . 1993; Wenzel‐Seifert and Seifert 2000), complicates the analysis of GTPγS binding data.…”

“…G protein deactivation is accomplished by the GTPase activity of G α (Gilman 1987; Birnbaumer et al . 1990) or by nucleotide dissociation from G α (Cassel and Selinger 1977; Kupprion et al . 1993; Seifert et al .…”