Quantitative stoichiometry of G-proteins activated by mu-opioid receptors in postmortem human brain |
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Authors: | González-Maeso Javier Rodríguez-Puertas Rafael Meana J Javier |
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Affiliation: | Department of Pharmacology, University of the Basque Country, E-48940 Leioa, Vizcaya, Spain. Javier.Maeso@mssm.edu |
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Abstract: | 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. |
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Keywords: | [35S]GTPγS binding μ-Opioid receptor G-protein GABAB receptor Brain, human Receptor reserve Signal transduction |
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