Nonselective coupling of the human mu-opioid receptor to multiple inhibitory G-protein isoforms.
Nonselective coupling of the human mu-opioid receptor to multiple inhibitory G-protein isoforms.
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DOI：
10.1046/j.1432-1327.1999.00301.x
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发表时间：
1999-04
期刊：
影响因子：
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通讯作者：
G. Gaibelet;E. Meilhoc;J. Riond;I. Saves;T. Exner;L. Liaubet;B. Nürnberg;J. Masson;L. Emorine
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文献类型：
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作者：
G. Gaibelet;E. Meilhoc;J. Riond;I. Saves;T. Exner;L. Liaubet;B. Nürnberg;J. Masson;L. Emorine
人μ-阿片受体在酿酒酵母中表达。[3 H]二丙诺啡与酵母原生质球的结合是特异性且可饱和的（Kd = 1 nm，Bmax = 0.2-1 pmol x mg-1膜蛋白）。具有不同阿片选择性（μ、δ和κ）的拮抗剂和激动剂对[3 H]二丙诺啡结合的抑制作用与哺乳动物组织中的效力顺序相同。拮抗剂的亲和力是相同的酵母原生质球在参考组织，而激动剂，除了埃托啡和丁丙诺啡，低10倍至100倍。添加从牛脑中纯化的异源三聚体Gi，o-蛋白将μ-阿片受体转变为对激动剂的高亲和力状态。使用单独纯化的与β-γ-二聚体重新关联的α-亚基，我们表明，alphao 1，alphao 2，alphai 1，alphai 2和alphai 3以相同的效率重建高亲和力激动剂结合。这表明负责G-蛋白偶联的μ-阿片受体的结构决定簇不能赋予对Gi，o家族任何成员的高度特异性。阿片样物质刺激后，在专门组织中观察到的阿片样物质的选择性作用可能是细胞特异性因子调节G蛋白活性的结果，这应方便地使用本文所述的重建试验进行分析。
The human mu-opioid receptor was expressed in Saccharomyces cerevisiae. Binding of [3H]diprenorphine to yeast spheroplasts was specific and saturable (Kd = 1 nm, Bmax = 0.2-1 pmol x mg-1 of membrane proteins). Inhibition of [3H]diprenorphine binding by antagonists and agonists with varying opioid selectivities (mu, delta and kappa) occurred with the same order of potency as in mammalian tissues. Affinities of antagonists were the same with yeast spheroplasts as in reference tissues whereas those of agonists, except etorphine and buprenorphine, were 10-fold to 100-fold lower. Addition of heterotrimeric Gi,o-proteins purified from bovine brain shifted the mu-opioid receptor into a high-affinity state for agonists. Using individually purified Galpha-subunits re-associated with betagamma-dimers, we showed that alphao1, alphao2, alphai1, alphai2 and alphai3 reconstituted high-affinity agonist binding with equal efficiency. This suggests that the structural determinants of the mu-opioid receptor responsible for G-protein coupling are not able to confer a high degree of specificity towards any member of the Gi,o family. The selective effects of opioid observed in specialized tissues upon opioid stimulation may be a result of regulation of G-protein activity by cell-specific factors which should conveniently be analysed using the reconstitution assay described here.