Evaluation of agonist efficacy and receptor reserve for alpha(2D)-adrenergic receptor regulated g protein activation in PC12 cell membranes

The receptor-coupling efficiency for epinephrine (EPI) stimulated heterotrimeric G protein activation was studied at the G protein level in membranes prepared from PC12 cells expressing cloned alpha(2D)-adrenergic receptors (alpha(2D)-AR). After pretreatment with different concentrations of N-ethoxycarbonyl-1,2-dihydroquinoline, which irreversibly inactivates alpha(2D)-AR, the portion of alpha(2D)-ARs remaining active (q) was estimated from EPI-stimulated [S-35]GTP gamma S binding. This function-derived estimate was close to the actual remaining number of receptors, as determined in saturation-binding studies using the selective alpha(2)-AR antagonist [H-3]rauwolscine in the same membranes. The agonist dissociation constant (K-A) derived from EPI-stimulated [S-35]GTP gamma S binding via Furchgott analysis was similar to the EC(50) of EPI in the same assay, but 40-fold lower than its K-i measured from EPI competition for [H-3]rauwolscine-binding sites in the presence of GTP gamma S and Na+. The occupancy-response relationship, calculated using K-i rather than K-A, was markedly nonlinear, consistent with the high expression of alpha(2D)-AR in these membranes, A nonlinear occupancy-response relationship was more directly confirmed by measuring the maximal level (i.e., full occupancy level) of G protein activation at graded densities of alpha(AD)-AR after N-ethoxycarbonyl-1,2-dihydroquinoline treatment, Determination of the number of G-proteins activated per receptor yielded lower values at higher receptor densities, indicating that overexpression of receptors can reduce their efficiency, Our results indicate the potential utility of using GTP-binding studies to assess agonist efficacy at the G protein level under conditions where receptor occupation can also be directly measured.