Cannabinoid receptor agonist efficacy for stimulating [35S]GTPγS binding to rat cerebellar membranes correlates with agonist-induced decreases in GDP affinity

The relationship between GDP and cannabinoid-stimulated [S-35]guanosine-5'-O-(3-thiotriphosphate) ([S-35]GTP gamma S) binding was investigated in rat cerebellar membranes. Kinetic analyses showed that [35S]GTP gamma S binding reached steady-state levels and that the association rate was increased by the agonist WIN 55212-2 proportional to the concentration of GDP. Dissociation of [S-35]GTP gamma S occurred with two rates (t(1/2) = 7 and 170 min), and WIN 55212-2 increased the proportion of sites exhibiting the faster rate. Without GDP, [S-35]GTP gamma S bound to membranes with high and low affinity, and WIN 55212-2 had no effect. With 30 mu M GDP, [S-35]GTP gamma S bound to low and intermediate affinity sites, and WIN 55212-2 induced high affinity [S-35]GTP gamma S binding without affecting low affinity sites, GDP competed for high affinity [S-35]GTP gamma S binding with high and intermediate affinity in the absence of WIN 55212-2 and with high and low affinity in the presence of WIN 55212-2. Cannabinoid ligands displayed differential abilities to maximally stimulate [S-35]GTP gamma S binding in the presence of GDP. Efficacy differences among ligands increased with increasing GDP concentrations. GDP competition curves revealed that agonists induced low affinity GDP Ki values that were proportional to agonist E-max values, indicating that agonist efficacy is determined by displacement of GDP from G-proteins.