ACTIVATION OF A GTP-BINDING PROTEIN AND A GTP-BINDING-PROTEIN-COUPLED RECEPTOR KINASE (BETA-ADRENERGIC-RECEPTOR KINASE-1) BY A MUSCARINIC RECEPTOR M2 MUTANT LACKING PHOSPHORYLATION SITES
ACTIVATION OF A GTP-BINDING PROTEIN AND A GTP-BINDING-PROTEIN-COUPLED RECEPTOR KINASE (BETA-ADRENERGIC-RECEPTOR KINASE-1) BY A MUSCARINIC RECEPTOR M2 MUTANT LACKING PHOSPHORYLATION SITES
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DOI：
10.1111/j.1432-1033.1994.tb20050.x
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发表时间：
1994-12-01
期刊：
影响因子：
--
通讯作者：
SADEE, W
中科院分区：
文献类型：
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作者：
KAMEYAMA, K;HAGA, K;SADEE, W
使用杆状病毒/昆虫细胞培养系统表达和纯化人类毒蕈碱乙酰胆碱受体 m2 亚型（m2 受体）的突变体，该突变体缺乏第三细胞内环的大部分。正如先前将磷酸化位点分配到第三个细胞内环的中心部分所预期的那样，该突变体未被β-肾上腺素能受体激酶磷酸化。然而，m2受体突变体能够以激动剂依赖性方式刺激含有m2磷酸化位点的谷胱甘肽S-转移酶融合蛋白的β-肾上腺素能受体激酶1介导的磷酸化。用鸟嘌呤核苷酸结合调节蛋白（G 蛋白）G(0) 和 G(12) 重建的突变型和野生型 m2 受体均表现出鸟嘌呤核苷酸敏感的高亲和力激动剂结合，通过与氨基甲酰胆碱结合的 [H-3]奎宁环基苯甲酸的置换来评估，并且均刺激鸟苷 5'-3-O-[S-35]硫代三磷酸([S-35]GTP[S]) 在氨基甲酰胆碱和 GDP 存在下结合。对于突变型和野生型 m2 受体，氨基甲酰胆碱对 [H-3]奎宁环基苯苯甲酸结合的影响的 K-i 值无法区分。此外，通过[H-3]qunuclidinyl benzilate 或[S-35]GTP[S] 的结合评估，野生型m2 受体被β-肾上腺素能受体激酶-1 磷酸化并不影响m2 与G 蛋白的相互作用。这些结果表明（a）m2受体既充当β-肾上腺素能受体激酶的激活剂又充当β-肾上腺素能受体激酶的底物，并且（b）m2受体的第三细胞内环的大部分不参与与G蛋白的相互作用，并且其被β-肾上腺素能受体激酶磷酸化不会使受体和重构的脂质囊泡中的G蛋白解偶联。
A mutant of the human muscarinic acetylcholine receptor m2 subtype (m2 receptor), lacking a large part of the third intracellular loop, was expressed and purified using the baculovirus/insect cell culture system. The mutant was not phosphorylated by beta-adrenergic-receptor kinase, as expected from the previous assignment of phosphorylation sites to the central part of the third intracellular loop. However, the m2 receptor mutant was capable of stimulating beta-adrenergic-receptor-kinase-1-mediated phosphorylation of a glutathione S-transferase fusion protein containing the m2 phosphorylation sites in an agonist-dependent manner. Both mutant and wild-type m2 receptors reconstituted with the guanine-nucleotide-binding regulatory proteins (G protein), G(0) and G(12), displayed guanine-nucleotide-sensitive high-affinity agonist binding, as assessed by displacement of [H-3]quinuclidinyl-benzilate binding with carbamoylcholine, and both stimulated guanosine 5'-3-O-[S-35]thiotriphosphate ([S-35]GTP[S]) binding in the presence of carbamoylcholine and GDP. The K-i values of carbamoylcholine effects on [H-3]quinuclidinyl-benzilate binding were indistinguishable for the mutant and wild-type m2 receptors. Moreover, the phosphorylation of the wild-type m2 receptor by beta-adrenergic-receptor kinase-1 did not affect m2 interaction with G proteins as assessed by the binding of [H-3]qunuclidinyl benzilate or [S-35]GTP[S]. These results indicate that (a) the m2 receptor serves both as an activator and as a substrate of beta-adrenergic-receptor kinase, and (b) a large part of the third intracellular loop of the m2 receptor does not contribute to interaction with G proteins and its phosphorylation by beta-adrenergic-receptor kinase does not uncouple the receptor and G proteins in reconstituted lipid vesicles.