Enhanced spontaneous activity of the mu opioid receptor by cysteine mutations: characterization of a tool for inverse agonist screening.
Enhanced spontaneous activity of the mu opioid receptor by cysteine mutations: characterization of a tool for inverse agonist screening.
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DOI：
10.1186/1471-2210-3-14
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发表时间：
2003-12-01
期刊：
影响因子：
--
通讯作者：
Massotte, Dominique
中科院分区：
文献类型：
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作者：
Brillet, Karl;Kieffer, Brigitte L.;Massotte, Dominique
背景：对于许多G蛋白偶联受体，自发或组成活性的概念已被广泛接受和验证，并且这种不依赖于配体的活性也被认为在某些病理中起作用。已经报道了mu阿片受体的构成活性。在某些情况下，受体基础活性的增加是由慢性吗啡给药引起的，这表明构成性活性可能有助于药物耐受和依赖的发展。组成活性突变体是收集有关受体激活机制和自发受体活性可能的生理相关性的信息的绝佳工具。这些突变体的高基础活性水平也允许更容易识别逆激动剂，定义为能够抑制自发受体活性的配体，并导致更好地理解其调节作用以及可能的体内使用。结果：人mu阿片受体（hMOR）半胱氨酸348和353突变为丙氨酸，并在HEK 293细胞中稳定表达Ala348,353。三磷酸鸟苷(打进35)。S结合实验显示，Ala348,353的hMOR基础活性明显高于hMOR，表明突变受体具有组成性活性。三磷酸鸟苷(打进35)。S结合被cyprodime或CTOP降低，表明这两种配体都具有逆激动剂性质。除内源性阿片肽外，所有测试的激动剂对Ala348,353 hMOR的结合亲和力均高于对hMOR的结合亲和力。除了CTOP和cyprodime对双突变体具有较高的亲和力外，拮抗剂的亲和力几乎保持不变。激动剂DAMGO和吗啡对[S-35] GTP中Ala348,353 hMOR受体的效力增强。年代实验。最后，拮抗剂纳洛酮、昔普罗迪或CTOP预处理显著提高了Ala348,353 hor的表达。结论：综上所述，我们的数据表明，双C348/ 353A突变导致hMOR的组成活性构象，该构象仍然被激动剂激活。这是第一个关于具有筛选逆激动剂潜力的稳定CAM的报道。
Background: The concept of spontaneous- or constitutive- activity has become widely accepted and verified for numerous G protein- coupled receptors and this ligand- independent activity is also acknowledged to play a role in some pathologies. Constitutive activity has been reported for the mu opioid receptor. In some cases the increase in receptor basal activity was induced by chronic morphine administration suggesting that constitutive activity may contribute to the development of drug tolerance and dependence. Constitutively active mutants represent excellent tools for gathering information about the mechanisms of receptor activation and the possible physiological relevance of spontaneous receptor activity. The high basal level of activity of these mutants also allows for easier identification of inverse agonists, defined as ligands able to suppress spontaneous receptor activity, and leads to a better comprehension of their modulatory effects as well as possible in vivo use.Results: Cysteines 348 and 353 of the human mu opioid receptor ( hMOR) were mutated into alanines and Ala348,353 hMOR was stably expressed in HEK 293 cells. [S-35] GTP.S binding experiments revealed that Ala348,353 hMOR basal activity was significantly higher when compared to hMOR, suggesting that the mutant receptor is constitutively active. [S-35] GTP.S binding was decreased by cyprodime or CTOP indicating that both ligands have inverse agonist properties. All tested agonists exhibited binding affinities higher for Ala348,353 hMOR than for hMOR, with the exception of endogenous opioid peptides. Antagonist affinity remained virtually unchanged except for CTOP and cyprodime that bound the double mutant with higher affinities. The agonists DAMGO and morphine showed enhanced potency for the Ala348,353 hMOR receptor in [S-35] GTP.S experiments. Finally, pretreatment with the antagonists naloxone, cyprodime or CTOP significantly increased Ala348,353 hMOR expression.Conclusion: Taken together our data indicate that the double C348/ 353A mutation results in a constitutively active conformation of hMOR that is still activated by agonists. This is the first report of a stable CAM of hMOR with the potential to screen for inverse agonists.