Abstract

Previous studies had shown that the amplification factors for cannabinoid receptors, defined as the number of total G proteins activated per occupied receptor, differs between several rat brain regions. In this study, we sought to determine which specific Gi/Go_α subunits were activated by CB1 receptors in several rat brain regions and if this coupling might explain the regional differences in receptor/G protein amplification factors. Furthermore, we examined whether cannabinoid agonists might activate different subtypes of G_α subunits with varying degrees of efficacy and/or potency. Activation of specific G proteins by cannabinoid receptors was evaluated by the ability of the agonist WIN 55212-2 to stimulate incorporation of [α-³²P]azidoanilido-GTP into G_α subunits in membranes. Photolabeled G proteins were either directly resolved using urea/SDS-polyacrylamide gel electrophoresis or first immunoprecipitated with specific antisera for different G_α subunits before electrophoresis. Individual G_α subunits were separated into distinct bands on a single gel and the amount of agonist-induced increase in radioactivity was quantified by densitometry. Stimulation of CB1 receptors by WIN 55212-2 resulted in the activation of a distinct pattern of at least five different G_iα/G_oα subunits in several brain regions. Furthermore, although the pattern of G proteins activated by WIN 55212-2 appeared to be similar across brain regions, slight differences were observed in both the percentage of increase and the amount of the individual G_α subunits activated. Most importantly, the amount of WIN 55212-2 required to half-maximally activate individual G proteins in the cerebellum varied over a 30-fold range for different G_α subunits. These results suggest that cannabinoid receptors activate multiple G proteins simultaneously in several brain regions and both the efficacy and potency of cannabinoid agonists to activate individual G_α subunits may vary considerably.