title: Evaluation of agonist efficacy and receptor reserve for alpha 2D-adrenergic receptor regulated G protein activation in PC12 cell membranes.
abstract: 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 [35S]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 [3H]rauwolscine in the same membranes. The agonist dissociation constant (KA) derived from EPI-stimulated [35S]GTP gamma S binding via Furchgott analysis was similar to the EC50 of EPI in the same assay, but 40-fold lower than its Ki measured from EPI competition for [3H]rauwolscine-binding sites in the presence of GTP gamma S and Na+. The occupancy-response relationship, calculated using Ki rather than KA, 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.
authors.affiliation: Department of Pharmaceutical Sciences, Northeastern University, Boston, Mass. 02115, USA.
authors.forename: W N
authors.initials: WN
authors.lastname: Tian
authors.forename: E
authors.initials: E
authors.lastname: Duzic
authors.forename: R C
authors.initials: RC
authors.lastname: Deth
mesh_terms: D000316:Adrenergic alpha-Agonists / Q000494:pharmacology*; D000818:Animals; D004789:Enzyme Activation; D004837:Epinephrine / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D016716:PC12 Cells; D011804:Quinolines / Q000494:pharmacology; D051381:Rats; D018341:Receptors, Adrenergic, alpha-2 / Q000187:drug effects* / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D000316:Adrenergic alpha-Agonists; D011804:Quinolines; D018341:Receptors, Adrenergic, alpha-2; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C000470:EEDQ; D019204:GTP-Binding Proteins; D004837:Epinephrine