Pharmacology of a constitutively active muscarinic receptor generated by random mutagenesis.
Pharmacology of a constitutively active muscarinic receptor generated by random mutagenesis.
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发表时间：
1995-12
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通讯作者：
T. Spalding;E. Burstein;H. Bráuner-Osborne;D. Hill-Eubanks;M. Brann
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作者：
T. Spalding;E. Burstein;H. Bráuner-Osborne;D. Hill-Eubanks;M. Brann
我们已经分离出突变的m5毒蕈碱受体，在没有激动剂的情况下介导强大的功能反应。这个具有组成活性的受体是从含有随机引入突变的第6跨膜结构域受体文库中分离出来的，它含有取代酪氨酸的丝氨酸465和取代脯氨酸的苏氨酸486。尽管这些单独的残基在其他g蛋白偶联受体中并不保守，但预计它们位于第六跨膜结构域和最后一个细胞外环之间的连接处。突变受体（CAm5）进行了详细的药理学分析。所有被测试的拮抗剂（阿托品、苯基喹啉酸酯、n -甲基东莨菪碱、4-二苯基乙酰氧基- n -甲基哌啶和吡伦齐平）都能完全抑制CAm5的组成性和激动剂诱导的活性，表明这些配体是负拮抗剂（逆激动剂）。这些配体在突变型和野生型受体上的效力相似，表明该受体的拮抗剂结合位点没有改变。通过功能反应和放射性配体结合测量，突变体对激动剂碳醇、槟榔碱和McN-A-343的敏感性增加。这些影响可以通过一个模型来解释和预测，在这个模型中，突变的主要作用是改变存在于受体活性和非活性状态之间的自发平衡。
We have isolated a mutant m5 muscarinic receptor that mediates robust functional responses in the absence of agonists. This constitutively active receptor was isolated from a library of receptors containing randomly introduced mutations in the sixth transmembrane domain and contains the substitutions serine 465 for tyrosine and threonine 486 for proline. Although these individual residues are not conserved in other G-protein-coupled receptors, they are predicted to be at the junction between the sixth transmembrane domain and the last extracellular loop. The mutant receptor (CAm5) was subjected to detailed pharmacological analysis. All of the antagonists tested (atropine, quinuclidinyl benzilate, N-methyl scopolamine, 4-diphenylacetoxy-N-methylpiperidine and pirenzepine) fully suppressed both the constitutive and agonist-induced activities of CAm5 revealing that these ligands are negative antagonists (inverse agonists). The potency of these ligands was similar at the mutant and wild-type receptors, suggesting that the antagonist binding site of this receptor is unchanged. The mutant had increased sensitivity to the agonists carbachol, arecoline, and McN-A-343 as measured both by functional response and by radioligand binding. These effects are explained and predicted by a model in which the primary effect of the mutations is to alter a spontaneous equilibrium existing between the active and inactive states of the receptor.