Liggett S B, Freedman N J, Schwinn D A, Lefkowitz R J
Department of Medicine (Pulmonary), University of Cincinnati Medical Center, OH 45267.
Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3665-9. doi: 10.1073/pnas.90.8.3665.
The physiological significance of multiple G-protein-coupled receptor subtypes, such as the beta-adrenergic receptors (beta ARs), remains obscure, since in many cases several subtypes activate the same effector and utilize the same physiological agonists. We inspected the deduced amino acid sequences of the beta AR subtypes for variations in the determinants for agonist regulation as a potential basis for subtype differentiation. Whereas the beta 2AR has a C terminus containing 11 serine and threonine residues representing potential sites for beta AR kinase phosphorylation, which mediates rapid agonist-promoted desensitization, only 3 serines are present in the comparable region of the beta 3AR, and they are in a nonfavorable context. The beta 3AR also lacks sequence homology in regions which are important for agonist-mediated sequestration and down-regulation of the beta 2AR, although such determinants are less well defined. We therefore tested the idea that the agonist-induced regulatory properties of the two receptors might differ by expressing both subtypes in CHW cells and exposing them to the agonist isoproterenol. The beta 3AR did not display short-term agonist-promoted functional desensitization or sequestration, or long-term down-regulation. To assign a structural basis for these subtype-specific differences in agonist regulation, we constructed a chimeric beta 3/beta 2AR which comprised the beta 3AR up to proline-365 of the cytoplasmic tail and the C terminus of the beta 2AR. When cells expressing this chimeric beta 3/beta 2AR were exposed to isoproterenol, functional desensitization was observed. Whole-cell phosphorylation studies showed that the beta 2AR displayed agonist-dependent phosphorylation, but no such phosphorylation could be demonstrated with the beta 3AR, even when beta AR kinase was overexpressed. In contrast, the chimeric beta 3/beta 2AR did display agonist-dependent phosphorylation, consistent with its functional desensitization. In addition to conferring functional desensitization and phosphorylation to the beta 3AR, the C-terminal tail of the beta 2AR also conferred agonist-promoted sequestration and long-term receptor down-regulation.
多种G蛋白偶联受体亚型，如β-肾上腺素能受体（βARs），其生理意义仍不清楚，因为在许多情况下，几种亚型激活相同的效应器并利用相同的生理激动剂。我们检查了βAR亚型推导的氨基酸序列中激动剂调节决定因素的变化，作为亚型分化的潜在基础。β2AR的C末端含有11个丝氨酸和苏氨酸残基，代表βAR激酶磷酸化的潜在位点，该磷酸化介导快速的激动剂促进的脱敏作用，而β3AR的可比区域仅存在3个丝氨酸，且它们处于不利的环境中。β3AR在对激动剂介导的β2AR隔离和下调很重要的区域也缺乏序列同源性，尽管这些决定因素的定义不太明确。因此，我们通过在CHW细胞中表达两种亚型并将它们暴露于激动剂异丙肾上腺素，来测试这两种受体的激动剂诱导调节特性可能不同的想法。β3AR没有表现出短期激动剂促进的功能脱敏或隔离，也没有长期下调。为了为激动剂调节中这些亚型特异性差异确定结构基础，我们构建了一种嵌合β3/β2AR，它由直到细胞质尾巴脯氨酸-365的β3AR和β2AR的C末端组成。当表达这种嵌合β3/β2AR的细胞暴露于异丙肾上腺素时，观察到功能脱敏。全细胞磷酸化研究表明，β2AR表现出激动剂依赖性磷酸化，但即使βAR激酶过表达，β3AR也未显示出这种磷酸化。相反，嵌合β3/β2AR确实表现出激动剂依赖性磷酸化，与其功能脱敏一致。除了赋予β3AR功能脱敏和磷酸化外，β2AR的C末端尾巴还赋予激动剂促进的隔离和长期受体下调。
