Quantitative stoichiometry of G-proteins activated by mu-opioid receptors in postmortem human brain.
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 mu-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 mu-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 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 microM 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.
D000478:Alkylation; D000701:Analgesics, Opioid / Q000378:metabolism / Q000494:pharmacology; D001418:Baclofen / Q000494:pharmacology; D001667:Binding, Competitive / Q000187:drug effects; D001923:Brain Chemistry / Q000502:physiology*; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D005033:Ethylmaleimide / Q000378:metabolism / Q000494:pharmacology; D018755:GABA Agonists / Q000494:pharmacology; D020555:GTP-Binding Protein Regulators / Q000502:physiology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D009270:Naloxone / Q000378:metabolism; D009271:Naltrexone / Q000031:analogs & derivatives* / Q000494:pharmacology; D009292:Narcotic Antagonists / Q000494:pharmacology; D017450:Receptors, Opioid, mu / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology
D000701:Analgesics, Opioid; D004791:Enzyme Inhibitors; D018755:GABA Agonists; D020555:GTP-Binding Protein Regulators; D009292:Narcotic Antagonists; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D009270:Naloxone; D009271:Naltrexone; C024524:beta-funaltrexamine; D019204:GTP-Binding Proteins; D001418:Baclofen; D005033:Ethylmaleimide