Quantitative stoichiometry of G-proteins activated by μ-opioid receptors in postmortem human brain

Paradoxically, the potencies (EC50) of agonists stimulating [S-35]GTPgammaS binding are several orders of magnitude lower than their affinities in receptor binding assays. We have investigated the quantitative stoichiometry of mu-opioid receptor-G-protein coupling in postmortem human brain. [D-Ala(2) N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO) displaced [H-3]naloxone binding in a biphasic pattern. The ratio between K-ilow and EC50 of DAMGO stimulating [S-35]GTPgammaS binding was lower than one. The K-A of DAMGO was calculated following mu-opioid receptor alkylation by beta-funaltrexamine from [S-35]GTPgammaS binding data using the "nested hyperbolic method", yielding K-A/EC50>1. Thus, only 1.2 +/- 0.2% of mu-opioid receptors was needed to be occupied to achieve the half-maximal effect of DAMGO. The estimated ratio between the G-proteins activated by 10 muM DAMGO (determined by isotopic dilution curves) and the occupied-mu-opioid receptors was 1304. In conclusion, we have determined the stoichiometric and the kinetic parameters in the mu-opioid receptor-G-protein system. (C) 2002 Elsevier Science B.V All rights reserved.