Eason M G, Moreira S P, Liggett S B
Department of Pulmonary Medicine, University of Cincinnati College of Medicine, Ohio 45267-0564.
J Biol Chem. 1995 Mar 3;270(9):4681-8. doi: 10.1074/jbc.270.9.4681.
During short term agonist exposure, the alpha 2A-adrenergic receptor (alpha 2AAR) undergoes rapid functional desensitization caused by phosphorylation of the receptor by the beta-adrenergic receptor kinase (beta ARK). This signal quenching is similar in nature to that found with a number of G-protein coupled receptors in which agonist-promoted desensitization is due to beta ARK phosphorylation; like these other receptors, the precise molecular determinants of the receptor required for beta ARK phosphorylation are not known. To delineate such a motif in the human alpha 2AAR (alpha 2C10), we constructed six mutated receptors consisting of deletions or substitutions of Ser-296-299 in the EESSSS sequence of the third intracellular loop of the receptor. These were expressed in Chinese hamster ovary and COS-7 cells, and agonist-promoted desensitization and receptor phosphorylation were assessed. Deletion of the EESSSS sequence and substitution of alanine for all four serines resulted in a total loss of phosphorylation and desensitization. Mutant receptors that retained two of the original serines (AASS and SSAA) underwent agonist-promoted phosphorylation of 55 +/- 7% and 57 +/- 8% of the phosphorylation found for wild type alpha 2C10. Additional substitution mutants (SSSA and SAAA) underwent 77 +/- 1% and 27 +/- 4% of wild type phosphorylation, respectively. Thus, substitution of alanine for each additional serine decreased overall phosphorylation as compared with wild type alpha 2C10 by approximately 25%, which is consistent with all 4 serines being phosphorylated. Mutated receptors that only partially phosphorylated (as compared with wild type) failed to undergo agonist-promoted desensitization. Thus, beta ARK-mediated phosphorylation of alpha 2C10 occurs at Ser-296-299 in the third intracellular loop, and this represents the critical step in rapid agonist-promoted desensitization. A number of other G-protein coupled receptors that undergo desensitization have a sequence motif similar to that which we have found for beta ARK-mediated phosphorylation of alpha 2C10, suggesting that these receptors may also be substrates for beta ARK.
在短期激动剂暴露期间，α2A - 肾上腺素能受体（α2AAR）会经历由β - 肾上腺素能受体激酶（βARK）对受体进行磷酸化所导致的快速功能脱敏。这种信号淬灭本质上与许多G蛋白偶联受体中所发现的情况相似，在这些受体中，激动剂促进的脱敏是由于βARK磷酸化；与这些其他受体一样，βARK磷酸化所需的受体精确分子决定因素尚不清楚。为了在人α2AAR（α2C10）中描绘这样一个基序，我们构建了六个突变受体，这些受体由受体第三细胞内环的EESSSS序列中的Ser - 296 - 299缺失或替换组成。它们在中国仓鼠卵巢细胞和COS - 7细胞中表达，并评估激动剂促进的脱敏和受体磷酸化情况。EESSSS序列的缺失以及用丙氨酸替换所有四个丝氨酸导致磷酸化和脱敏完全丧失。保留两个原始丝氨酸的突变受体（AASS和SSAA）的激动剂促进磷酸化分别为野生型α2C10磷酸化的55±7%和57±8%。另外的替换突变体（SSSA和SAAA）的磷酸化分别为野生型磷酸化的77±1%和27±4%。因此，与野生型α2C10相比，用丙氨酸替换每个额外的丝氨酸会使总体磷酸化降低约25%，这与所有4个丝氨酸都被磷酸化一致。仅部分磷酸化（与野生型相比）的突变受体未能经历激动剂促进的脱敏。因此，βARK介导的α2C10磷酸化发生在第三细胞内环的Ser - 29
