Nakata H, Kameyama K, Haga K, Haga T
Department of Biochemistry, Faculty of Medicine, University of Tokyo, Japan.
Eur J Biochem. 1994 Feb 15;220(1):29-36. doi: 10.1111/j.1432-1033.1994.tb18595.x.
Muscarinic acetylcholine receptors (mAChR, human m2 subtype) expressed in Sf9 (Spodoptera frugiperda) cells using the baculovirus system were purified and subjected to phosphorylation by a mAChR kinase, which was partially purified from porcine cerebrum. Two bands with apparent molecular masses of 59 kDa and 39 kDa as determined by SDS/PAGE were found to be phosphorylated in an agonist-dependent manner. Both bands were labeled by the irreversible muscarinic ligand [3H]propylbenzilylcholine mustard. Molecular masses of the [32P]phosphorylated or [3H]propylbenzilylcholine-mustard-labeled bands decreased following treatment with N-glycanase. The 59-kDa and 39-kDa bands were converted to 52-kDa and 32-kDa bands, respectively, indicating that both the 59-kDa and 39-kDa bands contain the amino-terminal region where glycosylation sites are present. The ratio of incorporated [32P]phosphate and bound [3H]propylbenzilylcholine mustard was essentially the same for the 59-kDa and 39-kDa bands, indicating that all the phosphorylation sites reside in the sequence of 39 kDa from the amino-terminal region. The amounts of incorporated [32P]phosphate were estimated to be 10-11/receptor, with 7-8 serine and 3-4 threonine, but no phosphorylated tyrosine residues. Further treatment of [32P]phosphorylated or [3H]propylbenzilylcholine-mustard-labeled receptors with V8 protease indicated that the phosphorylation sites were not present in 30-kDa amino-terminal segment. These results indicate that the phosphorylation sites are localized in the range 30-39 kDa from the amino terminus, which consists of primarily the central part of the third intracellular loop. Consistent with this conclusion, a fusion protein containing glutathione S-transferase linked to a peptide corresponding to residues 227-324 of the central part of the third intracellular loop was found to be phosphorylated by the mAChR kinase in a heparin-sensitive manner.
利用杆状病毒系统在草地贪夜蛾（Sf9）细胞中表达的毒蕈碱型乙酰胆碱受体（mAChR，人m2亚型）被纯化，并由从猪大脑中部分纯化得到的mAChR激酶进行磷酸化。通过SDS/PAGE测定，发现两条表观分子量分别为59 kDa和39 kDa的条带以激动剂依赖的方式被磷酸化。两条带均被不可逆的毒蕈碱配体[3H]丙基苯甲酰胆碱氮芥标记。用N-糖苷酶处理后，[32P]磷酸化或[3H]丙基苯甲酰胆碱氮芥标记条带的分子量降低。59-kDa和39-kDa条带分别转变为52-kDa和32-kDa条带，表明59-kDa和39-kDa条带均包含存在糖基化位点的氨基末端区域。59-kDa和39-kDa条带掺入的[32P]磷酸盐与结合的[3H]丙基苯甲酰胆碱氮芥的比例基本相同，表明所有磷酸化位点均位于从氨基末端区域起39 kDa的序列中。估计掺入的[32P]磷酸盐量为10 - 11个/受体，其中有7 - 8个丝氨酸和3 - 4个苏氨酸，但没有磷酸化的酪氨酸残基。用V8蛋白酶对[32P]磷酸化或[3H]丙基苯甲酰胆碱氮芥标记的受体进行进一步处理表明，磷酸化位点不存在于30-kDa的氨基末端片段中。这些结果表明，磷酸化位点位于从氨基末端起30 - 39 kDa的范围内，该区域主要由第三个细胞内环的中央部分组成。与此结论一致，发现一种含有与第三个细胞内环中央部分残基227 - 324对应的肽连接的谷胱甘肽S-转移酶的融合蛋白，能被mAChR激酶以肝素敏感的方式磷酸化。
