Kwatra M M, Benovic J L, Caron M G, Lefkowitz R J, Hosey M M
Department of Biological Chemistry and Structure, University of Health Sciences, Chicago Medical School, Illinois 60064.
Biochemistry. 1989 May 30;28(11):4543-7. doi: 10.1021/bi00437a005.
Previous studies have demonstrated that muscarinic cholinergic receptors (mAChR) become markedly phosphorylated when intact cardiac cells are stimulated with a muscarinic agonist. This process appears to be related to the process of receptor desensitization. However, the mechanism of agonist-induced phosphorylation of mAChR is not known. In situ phosphorylation studies suggested that agonist-induced phosphorylation of mAChR may involve the participation of a receptor-specific kinase and/or require agonist occupancy. These observations regarding phosphorylation and desensitization of mAChR are similar to observations made for beta-adrenergic receptors. Recent studies have indicated that homologous desensitization of beta-adrenergic receptors may be due to the phosphorylation of these receptors by a novel protein kinase that only recognizes the agonist-occupied form of the receptors. As muscarinic receptors are structurally homologous to beta-adrenergic receptors, we have initiated studies to identify the protein kinase responsible for the phosphorylation of muscarinic receptors by determining whether the chick heart muscarinic receptor would serve as a substrate for the beta-adrenergic receptor kinase (beta-AR kinase). We report that the purified and reconstituted chick heart muscarinic receptor serves as an excellent substrate in vitro for the beta-AR kinase. Phosphorylation of mAChR receptors by the beta-AR kinase was only observed in the presence of a muscarinic receptor agonist and was prevented in the presence of antagonist. Both the extent of phosphorylation (3-4 mol of P/mol of receptor) and the phosphoamino acid composition of the mAChR after incubation in vitro with beta-AR kinase were similar to the characteristics of agonist-induced phosphorylation of mAChR in situ.(ABSTRACT TRUNCATED AT 250 WORDS)
先前的研究表明，当完整的心脏细胞用毒蕈碱激动剂刺激时，毒蕈碱胆碱能受体（mAChR）会显著磷酸化。这一过程似乎与受体脱敏过程有关。然而，激动剂诱导的mAChR磷酸化机制尚不清楚。原位磷酸化研究表明，激动剂诱导的mAChR磷酸化可能涉及受体特异性激酶的参与和/或需要激动剂占据。这些关于mAChR磷酸化和脱敏的观察结果与β-肾上腺素能受体的观察结果相似。最近的研究表明，β-肾上腺素能受体的同源脱敏可能是由于一种新型蛋白激酶对这些受体的磷酸化，该激酶只识别激动剂占据形式的受体。由于毒蕈碱受体在结构上与β-肾上腺素能受体同源，我们已开始研究，通过确定鸡心脏毒蕈碱受体是否可作为β-肾上腺素能受体激酶（β-AR激酶）的底物，来鉴定负责毒蕈碱受体磷酸化的蛋白激酶。我们报告，纯化并重组的鸡心脏毒蕈碱受体在体外是β-AR激酶的优良底物。β-AR激酶对mAChR受体的磷酸化仅在毒蕈碱受体激动剂存在时观察到，而在拮抗剂存在时则被阻止。体外与β-AR激酶孵育后，mAChR的磷酸化程度（每摩尔受体3 - 4摩尔磷）和磷酸氨基酸组成与原位激动剂诱导的mAChR磷酸化特征相似。（摘要截短于250字）
