beta(2)-adrenergic receptor desensitization, internalization, and phosphorylation in response to full and partial agonists
beta(2)-adrenergic receptor desensitization, internalization, and phosphorylation in response to full and partial agonists
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DOI：
10.1074/jbc.272.38.23871
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发表时间：
1997-09-19
影响因子：
4.8
通讯作者：
Clark, RB
中科院分区：
文献类型：
--
作者：
January, B;Seibold, A;Clark, RB
以前的研究表明，部分激动剂比完全激动剂对β(2)-肾上腺素能受体(βAR)的脱敏作用更少；然而，在完整细胞中，这一分子基础尚未被研究。在目前的工作中，我们测定了一系列βAR激动剂引起的脱敏、内化和磷酸化的速率，这些研究是以高表达βAR的HEK-293细胞为对象，分别将血凝素和6-组氨酸表位引入到N和C末端，这种修饰的βAR在激动剂K-d、偶联效率和脱敏方面与野生型受体相同。β-AR激动剂激活腺酰环化酶的偶联效率，非诺特罗为4.2%，沙丁胺醇为4.9%，多巴丁胺为2.5%，麻黄素为1.1%。在这些激动剂的浓度下，受体占有率为90%，激动剂诱导脱敏的速率和程度(0-30分钟)与偶联效率顺序相同，即肾上腺素大于或等于非诺特罗；沙丁胺醇；多巴酚丁胺和GT；麻黄素对这些激动剂的β-AR内化速度也遵循与脱敏相同的顺序，表现出轻微的滞后，与内化和脱敏一样，β-AR的磷酸化表现出对激动剂强度的依赖关系。最强的两种激动剂肾上腺素和非诺特罗仅在1分钟后引起βAR磷酸化水平增加11-13倍，而较弱的激动剂多巴酚丁胺和麻黄碱仅引起3-4倍的增加，与Forsklin激活cAMP依赖的蛋白激酶所引起的水平相似。随着治疗时间的延长，β-AR磷酸化水平随强激动剂的增加而降低，但随弱部分激动剂的增加而逐渐增加，30分钟后肾上腺素升高的倍数(6.2+/-0.82)与麻黄碱(5.0+/-0.96)无明显差异，显著低于沙丁胺醇(10.4+/-1.7)。总之，我们的结果表明，激动剂强度与激动剂诱导的脱敏、内化和快速的磷酸化起始阶段之间具有良好的正比关系。这些数据支持这一假说，即激动剂偶联效率的提高主要通过增加βAR的βArk磷酸化速率来影响脱敏。
Previous studies indicated that partial agonists cause less desensitization of the beta(2)-adrenergic receptor (beta AR) than full agonists; however, the molecular basis for this in intact cells has not been investigated, In the present work, we have determined the rates of desensitization, internalization, and phosphorylation caused by a series of beta AR agonists displaying a 95-fold range of coupling efficiencies, These studies were performed with HEK-293 cells overexpressing the beta AR with hemagglutinin and 6-histidine epitopes introduced into the N and C termini, respectively, This modified beta AR behaved identically to the wild type receptor with regard to agonist K-d, coupling efficiency, and desensitization. The coupling efficiencies for beta AR agonist activation of adenylyl cyclase relative to epinephrine (100%) were 4.2% for fenoterol, 4.9% for albuterol, 2.5% for dobutamime, and 1.1% for ephedrine, At concentrations of these agonists yielding >90% receptor occupancy, the rate and extent (0-30 min) of agonist-induced desensitization of beta AR activation of adenylyl cyclase followed the same order as coupling efficiency, i.e. epinephrine greater than or equal to fenoterol > albuterol > dobutamine > ephedrine, The rate of internalization of the beta AR with respect to these agonists also followed the same order as the desensitization and exhibited a slight lag, Like internalization and desensitization, beta AR phosphorylation exhibited a dependence on agonist strength. The two strongest agonists, epinephrine and fenoterol, provoked 11-13-fold increases in the level of beta AR phosphorylation after just 1 min, whereas the weak agonists dobutamine and ephedrine caused only 3-4-fold increases, similar to levels induced by cAMP-dependent protein kinase activation with forskolin. With longer treatment times, the level of beta AR phosphorylation declined with strong agonists, but it progressively increased with the weaker partial agonists, such that after 30 min the -fold elevation with epinephrine (6.2 +/- 0.82) was not appreciably different from ephedrine (5.0 +/- 0.96) and significantly less than that caused by albuterol (10.4 +/- 1.7), In summary, our results demonstrate an excellent proportionality between the agonist strength and agonist-induced desensitization, internalization, and the rapid initial phase of phosphorylation. The data support the hypothesis that increasing agonist-coupling efficiency primarily affects desensitization by increasing the rate of beta ARK phosphorylation of the beta AR.