Quantitative stoichiometry of G-proteins activated by μ-opioid receptors in postmortem human brain
Quantitative stoichiometry of G-proteins activated by μ-opioid receptors in postmortem human brain
复制标题
DOI：
10.1016/s0014-2999(02)02242-2
复制
发表时间：
2002-09-27
影响因子：
5
通讯作者：
Meana, JJ
中科院分区：
文献类型：
--
作者：
González-Maeso, J;Rodríguez-Puertas, R;Meana, JJ
矛盾的是，在受体结合试验中，刺激[S-35]GTPgammaS结合的激动剂的效价（EC50）比它们的亲和力低几个数量级。我们研究了死后人脑中mu-阿片受体- g蛋白偶联的定量化学计量学。[D-Ala(2) N-Me-Phe(4),Gly(5)-ol]脑啡肽（DAMGO）以双相模式取代[H-3]纳洛酮结合。DAMGO刺激[S-35]GTPgammaS结合的k - low与EC50之比小于1。根据[S-35]GTPgammaS结合数据，采用“嵌套双曲曲线法”，用β -funaltrexamine将mu-阿片受体烷基化，计算DAMGO的K-A，得到K-A/EC50>1。因此，只需要占用1.2 +/- 0.2%的mu-阿片受体就能达到DAMGO的半最大效果。经10 muM DAMGO活化的g蛋白（通过同位素稀释曲线确定）与被占领的μ -阿片受体的比值估计为1304。总之，我们确定了mu-阿片受体- g蛋白系统的化学计量学和动力学参数。(C) 2002 Elsevier Science b.v.版权所有
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.