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

Background and purpose: The aim of this report is to study mechanisms of G protein activation by agonists. Experimental approach: The association and dissociation of guanosine 5′‐O‐(3‐[35S]thio)triphosphate ([35S]GTPγS) binding at G proteins in membranes of CHO cells stably transfected with the human dopamine D2short receptor was studied in the presence of a range of agonists. Key results: Binding of [35S]GTPγS was dissociable in the absence of agonist and dissociation was accelerated both in rate and extent by dopamine, an effect which was blocked by the dopamine D2 receptor antagonist raclopride and by suramin, which inhibits receptor/G protein interaction. A range of agonists of varying efficacy increased the rate of dissociation of [35S]GTPγS binding, with the more efficacious agonists resulting in faster dissociation. Agonists were able to dissociate about 70% of the pre‐bound [35S]GTPγS, leaving a component which may not be accessible to the agonist‐bound receptor. The dissociable component of the [35S]GTPγS binding was reduced with longer association times and increased [35S]GTPγS concentrations. Conclusions and implications: These data are consistent with [35S]GTPγS binding being initially to receptor‐linked G proteins and then to G proteins which have separated from the agonist bound receptor. Under the conditions used typically for [35S]GTPγS binding assays, therefore, much of the agonist‐receptor complex remains in proximity to G proteins after they have been activated by agonist. British Journal of Pharmacology (2007) 151, 125–133. doi:10.1038/sj.bjp.0707197