Mechanisms of G protein activation via the D 2 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‐[ 35 S]thio)triphosphate ([ 35 S]GTPγS) binding at G proteins in membranes of CHO cells stably transfected with the human dopamine D 2short receptor was studied in the presence of a range of agonists. Key results: Binding of [ 35 S]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 D 2 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 [ 35 S]GTPγS binding, with the more efficacious agonists resulting in faster dissociation. Agonists were able to dissociate about 70% of the pre‐bound [ 35 S]GTPγS, leaving a component which may not be accessible to the agonist‐bound receptor. The dissociable component of the [ 35 S]GTPγS binding was reduced with longer association times and increased [ 35 S]GTPγS concentrations. Conclusions and implications: These data are consistent with [ 35 S]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 [ 35 S]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