AMINO-TERMINAL POLYMORPHISMS OF THE HUMAN BETA(2)-ADRENERGIC RECEPTOR IMPART DISTINCT AGONIST-PROMOTED REGULATORY PROPERTIES
AMINO-TERMINAL POLYMORPHISMS OF THE HUMAN BETA(2)-ADRENERGIC RECEPTOR IMPART DISTINCT AGONIST-PROMOTED REGULATORY PROPERTIES
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DOI：
10.1021/bi00198a006
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发表时间：
1994-08-16
期刊：
影响因子：
2.9
通讯作者：
LIGGETT, SB
中科院分区：
文献类型：
--
作者：
GREEN, SA;TURKI, J;LIGGETT, SB
我们最近描述了人类β(2)-肾上腺素能受体的三个自然发生的多态性，这些突变是由编码该受体胞外N-末端的氨基酸16和27的错义突变引起的。我们通过定点突变和这些受体在中国仓鼠成纤维细胞中的重组表达，研究了这些多态的功能后果。这些多态包括16位氨基酸的Gly取代Arg(Arg 16--&Gt；Gly)，27位的Glu取代27位的Gln(Gln27--&Gt；Glu)，以及这两种取代的组合。3种突变型受体均表现出与G(S)正常的激动剂结合和功能偶联，导致腺苷环化酶活性增强。然而，这些突变显著改变了激动剂促进受体表达下调的程度。在10亩M异丙肾上腺素作用24小时后，野生型β(2)AR的受体密度下降了26+/-3%。相比之下，Arg16-->；Gly减少了41%+/-3%。另一方面，Gln27-->；Glu被发现完全抵抗下调监管。与野生型β(2)AR(39+/-4%)相比，Arg16->；Gly+Gln27-->；Glu也经历了更多的下调。不可逆烷基化后新受体的合成速率在这些受体之间没有差别，激动剂促进的受体内化到细胞内池的速率也没有差别。Gln27-->；Glu细胞mRNB。在激动剂暴露过程中，野生型β(2)AR和其他突变的受体mRNAs没有改变，推测这些多态的异常下调模式可能是由于受体蛋白的降解改变所致。在蛋白质印迹图上，Gln27-->；Glu受体与野生型β(2)AR相比，似乎没有以完全成熟的形式表达，这表明该受体的构象变化导致了激动剂暴露时受体降解的抑制。这些结果表明，N端的β(2)AR的常见多态赋予了不同的激动剂促进的调节表型，并可能解释了观察到的体内反应和调节的异质性。
We have recently delineated three naturally occurring polymorphisms of the human beta(2)-adrenergic receptor caused by missense mutations encoding for amino acids 16 and 27 of the extracellular N-terminus of the receptor. We have studied the functional consequences of these polymorphisms by site-directed mutagenesis and the recombinant expression of these receptors in Chinese hamster fibroblasts. The polymorphisms consist of substitutions of Gly for Arg at amino acid 16 (Arg 16-->Gly), Glu for Gln at amino acid 27 (Gln27-->Glu), and a combination of both substitutions. All three mutated receptors displayed normal agonist binding and functional coupling to G(s), resulting in the stimulation of adenylyl cyclase activity. However, these mutations markedly altered the degree of agonist-promoted downregulation of receptor expression. After 24-h exposure to 10 mu M isoproterenol, wild-type beta(2)AR underwent a 26 +/- 3% reduction in receptor density. In contrast, Arg16-->Gly underwent a 41 +/- 3% reduction. Gln27-->Glu, on the other hand, was found to be completely resistant to downregulation. Arg16-->Gly + Gln27-->Glu also underwent an increased downregulation compared to wild-type beta(2)AR (39 +/- 4%). The rates of new receptor synthesis after irreversible alkylation were not different between these receptors, nor were the rates of agonist-promoted receptor internalization to the intracellular pool. Gln27-->Glu cellular mRNB. minimally increased during agonist exposure, and wild-type beta(2)AR and the other mutated receptor mRNAs did not change, which infer that the aberrant downregulation patterns of these polymorphisms may be due to the altered degradation of receptor protein. On Western blots, the Gln27-->Glu receptor did not appear to be expressed in the fully mature form as compared to wild-type beta(2)AR, suggesting that conformational alterations of this receptor lead to depressed receptor degradation during agonist exposure. These results indicate that common polymorphisms of the beta(2)AR at the N-terminus impart distinct phenotypes of agonist-promoted regulation and may explain the observed heterogeneity of in vivo responsiveness and regulation of beta(2)AR.