Jewell-Motz E A, Small K M, Theiss C T, Liggett S B
Departments of Medicine and Molecular Genetics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA.
J Biol Chem. 2000 Sep 15;275(37):28989-93. doi: 10.1074/jbc.M005381200.
The alpha(2A)-adrenergic receptor (AR) undergoes rapid agonist-promoted desensitization due to phosphorylation by G protein-coupled receptor kinases (GRKs) 2 and 3 at serines in the third intracellular loop of the receptor. In contrast, the alpha(2C)AR fails to display such desensitization or phosphorylation, which has been presumed to be due to this receptor lacking GRK phosphorylation sites. However, the alpha(2C)AR has multiple serines and threonines in putative favorable motifs within its third intracellular loop. We considered that the conformation of the third intracellular loop imposed by agonists binding to the transmembrane-spanning domains could be the basis of this subtype-specific property, rather than the presence or absence of phosphoacceptors per se. To address this, alpha(2A)/alpha(2C) third loop chimeric receptors were constructed. In whole cell phosphorylation studies, the alpha(2A) with the alpha(2C) third loop receptor underwent agonist-promoted phosphorylation while the alpha(2C) with the alpha(2A) third loop receptor did not, indicating that the agonist interaction with the parent receptor backbone establishes the phosphorylation phenotype. We postulated then that agonists with diverse structures that distinctly interact with alpha(2)AR should display different degrees of phosphorylation independent of receptor activation. Indeed, several full and partial agonists were identified, which evoked phosphorylation that was not related to intrinsic activity as established by [(35)S]guanosine 5'-3-O-(thio)triphosphate binding. Taken together, it appears that phosphorylation of the alpha(2)AR evoked by agonist is highly sensitive to the conformation of the third intracellular loop induced/stabilized by agonist to such an extent that these properties dictate the extent of phosphorylation of the loop when phosphoacceptors are present, and are the basis for subtype-specific phosphorylation.
α(2A)-肾上腺素能受体（AR）由于G蛋白偶联受体激酶（GRK）2和3在受体第三个细胞内环的丝氨酸处进行磷酸化，从而经历快速的激动剂促进的脱敏。相比之下，α(2C)AR未能表现出这种脱敏或磷酸化，据推测这是由于该受体缺乏GRK磷酸化位点。然而，α(2C)AR在其第三个细胞内环的假定有利基序中有多个丝氨酸和苏氨酸。我们认为，激动剂与跨膜结构域结合所导致的第三个细胞内环的构象可能是这种亚型特异性特性的基础，而不是磷酸化受体本身的存在与否。为了解决这个问题，构建了α(2A)/α(2C)第三个环的嵌合受体。在全细胞磷酸化研究中，具有α(2C)第三个环的α(2A)受体经历了激动剂促进的磷酸化，而具有α(2A)第三个环的α(2C)受体则没有，这表明激动剂与亲本受体骨架的相互作用决定了磷酸化表型。然后我们推测，与α(2)AR有不同相互作用的不同结构的激动剂应该表现出不同程度的磷酸化，而与受体激活无关。事实上，鉴定出了几种完全激动剂和部分激动剂，它们引起的磷酸化与通过[(35)S]鸟苷5'-3-O-(硫代)三磷酸结合确定的内在活性无关。综上所述，激动剂引起的α(2)AR磷酸化对激动剂诱导/稳定的第三个细胞内环的构象高度敏感，以至于这些特性决定了存在磷酸化受体时环的磷酸化程度，并且是亚型特异性磷酸化的基础。
