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首页> 外文期刊>Biochemistry >Complexity of Agonist-and Cyclic AMP-Mediated Downregulation of the Human #beta#_1-Adrenergic Receptor: Role of Internalization, Degradation, and mRNA Destabilization
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Complexity of Agonist-and Cyclic AMP-Mediated Downregulation of the Human #beta#_1-Adrenergic Receptor: Role of Internalization, Degradation, and mRNA Destabilization

机译：激动剂和环AMP介导的人类＃beta＃_1-肾上腺素受体下调的复杂性：内化，降解和mRNA不稳定的作用。

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Prolonged agonist exposure often induces downregulation of G protein-coupled receptors (GPCRs). Although downregulation of the prototypical #beta#_2-adrenergic receptor (#beta#_2AR) has been extensively studied, the underlying mechanisms have yet to be resolved. As even less is known about the #beta#_1-subtype, we investigated the downregulation of human #beta#_1AR stably expressed in Chinese hamster fibroblasts in response to the agonist isoproterenol or the cell-permeable, chlorophenylthio-cAMP (CPT-cAMP). While either effector mediated decreases in both #beta#_1AR binding activity and steady-state #beta#-1AR mRNA levels, there were significant diffeences in their actions. Whereas agonist-mediated downregulation of #beta#_1AR followed first-order kinetics, that induced by CPT-cAMP was delayed for several hours and aprox 50% of the former. Furthermore, agonist bot not CPT-cAMP induced #beta#_1AR internalization, and inhibiting internalization also supressed agonist-mediated downregulation. The latter, however, was more sensitive than the former to agonist concentration (EX_50 of 0.3 vs 48 nM). Thus, at < = 1 nM agonist, downregulation occurred without internalization and with a pattern similar to that mediated by CPT-cAMP. The amounts of #beta#_1AR downregulated or internalized were proportional to initial receptor levels but reached saturation at approx 2 and 3 pmol/mg of protein, respectively. The fate of #beta#_1AR protein during downregulation was determined by immunoblotting with anti-C-terminal antibodies. In agonist-treated cells, #beta#_1AR protein disappeared with time and withot any immunoreactive degradation products. Agonist-mediated downregulation of the human #beta#-1AR appears to be a complex process that conists of both agonist-and cAMP-specific components. The former involves both receptor internalization and degradation whereas the latter involves a reduction in receptor mRNA.
机译：长时间的激动剂暴露通常会导致G蛋白偶联受体（GPCR）的下调。尽管已经广泛研究了原型＃beta＃_2-肾上腺素能受体（＃beta＃_2AR）的下调，但是其潜在机制尚未解决。由于对＃beta＃_1-亚型的了解甚少，我们研究了在中国仓鼠成纤维细胞中稳定表达的人＃beta＃_1AR的下调，以响应激动剂异丙肾上腺素或可渗透细胞的氯苯基硫代cAMP（CPT-cAMP） 。尽管任一效应子介导的＃beta＃_1AR结合活性和稳态＃beta＃-1AR mRNA水平均下降，但它们的作用存在显着差异。激动剂介导的＃beta＃_1AR下调遵循一级动力学，而CPT-cAMP诱导的下调则延迟了数小时，而前者约为前者的50％。此外，激动剂bot不是CPT-cAMP诱导＃beta＃_1AR内在化，抑制内在化也抑制了激动剂介导的下调。但是，后者对激动剂的浓度比前者更为敏感（EX_50为0.3 vs 48 nM）。因此，在≤1 nM激动剂的情况下，发生下调而没有内在化，并且具有类似于CPT-cAMP介导的模式。下调或内在化的＃beta＃_1AR的量与初始受体水平成正比，但分别达到约2 pmol / mg和3 pmol / mg蛋白质时达到饱和。 ＃beta＃_1AR蛋白在下调过程中的命运通过抗C末端抗体的免疫印迹来确定。在经激动剂处理的细胞中，＃beta＃_1AR蛋白随时间消失，并且没有任何免疫反应性降解产物。激动剂介导的人类＃beta＃-1AR下调似乎是一个复杂的过程，包含激动剂和cAMP特异性成分。前者涉及受体内在化和降解，而后者涉及受体mRNA的减少。

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