The assay for [35S]GTPγS binding was performed as previously described by Kearn et al. (1999). Membranes (final concentration, 5 μg of protein per incubation mixture) were added to TME buffer containing 0.1% fatty acid-free bovine serum albumin, 10 μmol/L GDP, and 150 mmol/L NaCl. [35S]GTPγS (final concentration, 0.65 nmol/L) was added, and the incubation was continued for 30 min at 37°C using the Multiscreen Filtration System with Durapore filters (pore size, 1.2 μm; Millipore, Bedford, MA, USA). Non-specific binding was determined in the presence of 10 μmol/L Gpp(NH)p and accounted for <15% of the total binding. Bound [35S]GTPγS was separated from free [35S]GTPγS by filtration followed by washing the filters four times with cold TME buffer containing NaCl and GDP. The cannabinoid CB1 receptor agonist WIN 55,212 was added in 1 μL of dimethyl sulfoxide at concentrations of 0, 0.1, 0.3, 0.6, 1, 2, 3, 6, 10, 20 and 30 μmol/L. In each experiment, the agonist-dependent [35S]GTPγS binding was calculated as a percent of agonist-independent binding. The EC50 values and maximal agonist-induced increase in binding (Emax) of [35S]GTPγS were determined by fitting the data to a sigmoidal concentration-response curve using nonlinear regression (Prism; GraphPad, San Diego, CA, USA).
