Quantitative stoichiometry of G-proteins activated by &mgr;-opioid receptors in postmortem human brain.

摘要

Paradoxically, the potencies (EC(50)) of agonists stimulating [35S]GTPgammaS binding are several orders of magnitude lower than their affinities in receptor binding assays. We have investigated the quantitative stoichiometry of &mgr;-opioid receptor-G-protein coupling in postmortem human brain. [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO) displaced [3H]naloxone binding in a biphasic pattern. The ratio between K(i-low) and EC(50) of DAMGO stimulating [35S]GTPgammaS binding was lower than one. The K(A) of DAMGO was calculated following &mgr;-opioid receptor alkylation by beta-funaltrexamine from [35S]GTPgammaS binding data using the "nested hyperbolic method", yielding K(A)/EC(50)>1. Thus, only 1.2+/-0.2% of &mgr;-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 &mgr;M DAMGO (determined by isotopic dilution curves) and the occupied-&mgr;-opioid receptors was 1304. In conclusion, we have determined the stoichiometric and the kinetic parameters in the &mgr;-opioid receptor-G-protein system.