Trester-Zedlitz Michelle, Burlingame Al, Kobilka Brian, von Zastrow Mark
Department of Psychiatry and Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California 94143, USA.
Biochemistry. 2005 Apr 26;44(16):6133-43. doi: 10.1021/bi0475469.
Posttranslational modifications (PTMs) of the beta-2 adrenoceptor (B2AR) play a fundamental role in receptor regulation by agonists. We have examined the effects of several agonists on net levels of B2AR palmitoylation and phosphorylation using epitope tagging in stably transfected human embryonal kidney (HEK) 293 cells, immunoaffinity purification, and mass spectrometry combined with the method of stable isotope labeling by amino acids in cell culture (SILAC). Palmitoylation of Cys341 was confirmed and did not change detectably after 30 min exposure of cells to saturating concentrations of dopamine, epinephrine, or isoproterenol. However, all of these agonists produced a marked increase in net phosphorylation. Phosphorylation of the third cytoplasmic loop was increased to a similar degree by all three agonists, whereas differences between agonists were observed in net phosphorylation of the carboxyl-terminal cytoplasmic domain (isoproterenol approximately epinephrine >> dopamine). Interestingly, agonist-induced phosphorylation of the carboxyl-terminal cytoplasmic domain was observed exclusively in a proximal portion (between residues 339-369). None of the agonists produced detectable phosphorylation in a distal portion of the cytoplasmic tail, which contains all sites of agonist-induced phosphorylation identified previously by in vitro reconstitution. These results provide insight to agonist-dependent regulation of the B2AR in intact cells, suggest the existence of significant differences in regulatory phosphorylation events occurring between in vitro and in vivo conditions, and outline a general analytical approach to investigate regulated PTM of receptors in mammalian cells.
β2肾上腺素能受体（B2AR）的翻译后修饰（PTM）在激动剂对受体的调节中起重要作用。我们使用表位标签技术，在稳定转染的人胚肾（HEK）293细胞中，结合免疫亲和纯化、质谱分析以及细胞培养中氨基酸稳定同位素标记法（SILAC），研究了几种激动剂对B2AR棕榈酰化和磷酸化净水平的影响。Cys341的棕榈酰化得到证实，在细胞暴露于饱和浓度的多巴胺、肾上腺素或异丙肾上腺素30分钟后，其棕榈酰化水平未出现可检测到的变化。然而，所有这些激动剂均使净磷酸化水平显著增加。所有三种激动剂均使第三胞质环的磷酸化程度增加到相似水平，而在羧基末端胞质结构域的净磷酸化方面观察到激动剂之间存在差异（异丙肾上腺素≈肾上腺素>>多巴胺）。有趣的是，仅在近端部分（339 - 369位氨基酸之间）观察到激动剂诱导的羧基末端胞质结构域磷酸化。没有一种激动剂在胞质尾的远端部分产生可检测到的磷酸化，而远端部分包含先前通过体外重组鉴定的所有激动剂诱导的磷酸化位点。这些结果为完整细胞中B2AR的激动剂依赖性调节提供了见解，表明体外和体内条件下发生的调节性磷酸化事件存在显著差异，并概述了一种研究哺乳动物细胞中受体调节性PTM的通用分析方法。
