STRUCTURAL BASIS FOR RECEPTOR SUBTYPE-SPECIFIC REGULATION REVEALED BY A CHIMERIC-BETA(3)/BETA(2)-ADRENERGIC RECEPTOR
STRUCTURAL BASIS FOR RECEPTOR SUBTYPE-SPECIFIC REGULATION REVEALED BY A CHIMERIC-BETA(3)/BETA(2)-ADRENERGIC RECEPTOR
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DOI：
10.1073/pnas.90.8.3665
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发表时间：
1993-04-15
影响因子：
11.1
通讯作者：
LEFKOWITZ, RJ
中科院分区：
文献类型：
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作者：
LIGGETT, SB;FREEDMAN, NJ;LEFKOWITZ, RJ
多种G蛋白偶联受体亚型（如β-肾上腺素能受体（β AR））的生理意义仍然不清楚，因为在许多情况下，几种亚型激活相同的效应子并利用相同的生理激动剂。我们检查了推导的β AR亚型的氨基酸序列，以确定激动剂调节的决定因素的变化，作为亚型分化的潜在基础。而β 2 AR的C末端含有11个丝氨酸和苏氨酸残基，代表β AR激酶磷酸化的潜在位点，其介导快速激动剂促进的脱敏，而β 3 AR的可比区域中仅存在3个丝氨酸，并且它们处于不利的环境中。β 3 AR在激动剂介导的螯合和下调β 2 AR的重要区域中也缺乏序列同源性，尽管这些决定簇不太明确。因此，我们测试的想法，激动剂诱导的两种受体的调节特性可能会有所不同，表达两种亚型的CHW细胞，并将它们暴露于激动剂异丙肾上腺素。β 3 AR没有显示短期激动剂促进的功能性脱敏或隔离，或长期下调。为了给这些亚型特异性激动剂调节差异分配结构基础，我们构建了嵌合β 3/β 2 AR，其包括β 3 AR至胞质尾区的脯氨酸-365和β 2 AR的C末端。当表达这种嵌合β 3/β 2AR的细胞暴露于异丙肾上腺素时，观察到功能性脱敏。全细胞磷酸化研究表明，β 2 AR显示激动剂依赖性磷酸化，但没有这样的磷酸化可以证明与β 3 AR，即使当β AR激酶过表达。相比之下，嵌合β 3/β 2 AR确实显示激动剂依赖性磷酸化，与其功能性脱敏一致。除了赋予β 3 AR功能性脱敏和磷酸化外，β 2 AR的C末端尾部还赋予激动剂促进的螯合和长期受体下调。
The physiological significance of multiple G-protein-coupled receptor subtypes, such as the beta-adrenergic receptors (betaARs), 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 betaAR subtypes for variations in the determinants for agonist regulation as a potential basis for subtype differentiation. Whereas the beta2AR has a C terminus containing 11 serine and threonine residues representing potential sites for betaAR kinase phosphorylation, which mediates rapid agonist-promoted desensitization, only 3 serines are present in the comparable region of the beta3AR, and they are in a nonfavorable context. The beta3AR also lacks sequence homology in regions which are important for agonist-mediated sequestration and down-regulation of the beta2AR, 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 beta3AR 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 beta3/beta2AR which comprised the beta3AR up to proline-365 of the cytoplasmic tail and the C terminus of the beta2AR. When cells expressing this chimeric beta3/beta2AR were exposed to isoproterenol, functional desensitization was observed. Whole-cell phosphorylation studies showed that the beta2AR displayed agonist-dependent phosphorylation, but no such phosphorylation could be demonstrated with the beta3AR, even when betaAR kinase was overexpressed. In contrast, the chimeric beta3/beta2AR did display agonist-dependent phosphorylation, consistent with its functional desensitization. In addition to conferring functional desensitization and phosphorylation to the beta3AR, the C-terminal tail of the beta2AR also conferred agonist-promoted sequestration and long-term receptor down-regulation.