Ruiz-Gómez A, Vaello M L, Valdivieso F, Mayor F
Departamento de Biología Molecular, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Spain.
J Biol Chem. 1991 Jan 5;266(1):559-66.
The postsynaptic glycine receptor purified from rat spinal cord is rapidly and specifically phosphorylated by protein kinase C. The target for phosphorylation is the strychnine-binding subunit of the receptor (molecular mass of approximately 48 kDa), which is phosphorylated on serine residues to a final stoichiometry of approximately 0.8 mol of phosphate/mol of subunit. The 48-kDa phosphoprotein was analyzed by proteolytic cleavage and peptide mapping in order to localize the site of phosphorylation within the receptor molecule. Examination of the 32P-labeled receptor fragments generated by digestion with N-chlorosuccinimide, cyanogen bromide, and endoproteinase lysine C and of the deduced amino acid sequence of the 48-kDa protein (Grenningloh, G., Rienitz, A., Schmitt, B., Methfessel, C., Zensen, M., Beyreuther, K., Gundelfinger, E. D., and Betz, H. (1987) Nature 328, 215-220) indicates that the phosphorylation site is located in a region corresponding to the major intracellular loop of the predicted structure of the glycine receptor subunit and suggests serine 391 as the phosphorylated residue. In fact, a synthetic peptide corresponding to residues 384-392 of the 48-kDa subunit was specifically phosphorylated by protein kinase C. Moreover, tryptic digests of this phosphopeptide and of the phosphorylated 48-kDa subunit of the glycine receptor migrated to the same position in two-dimensional peptide mapping. Furthermore, antibodies elicited against peptide 384-392 were shown to inhibit the protein kinase C-dependent phosphorylation of the 48-kDa polypeptide. Interestingly, the relative position of the phosphorylated domain is similar to those known or proposed to be phosphorylated in other ligand-gated ion channel receptor subunits, thus suggesting further the existence of a homologous regulatory region in these receptor proteins.
从大鼠脊髓中纯化得到的突触后甘氨酸受体可被蛋白激酶C快速且特异性地磷酸化。磷酸化的靶点是受体的士的宁结合亚基（分子量约为48 kDa），该亚基在丝氨酸残基上被磷酸化，最终化学计量比约为0.8摩尔磷酸/摩尔亚基。通过蛋白水解切割和肽图谱分析对48 kDa的磷蛋白进行分析，以确定受体分子内的磷酸化位点。用N-氯代琥珀酰亚胺、溴化氰和内蛋白酶赖氨酸C消化产生的32P标记的受体片段以及推导得到的48 kDa蛋白的氨基酸序列（Grenningloh, G., Rienitz, A., Schmitt, B., Methfessel, C., Zensen, M., Beyreuther, K., Gundelfinger, E. D., and Betz, H. (1987) Nature 328, 215 - 220）表明，磷酸化位点位于与甘氨酸受体亚基预测结构的主要细胞内环相对应的区域，并提示丝氨酸391为磷酸化残基。事实上，与48 kDa亚基的384 - 392位残基相对应的合成肽被蛋白激酶C特异性地磷酸化。此外，该磷酸肽和甘氨酸受体的磷酸化48 kDa亚基的胰蛋白酶消化产物在二维肽图谱中迁移到相同位置。此外，针对384 - 392肽段产生的抗体被证明可抑制48 kDa多肽的蛋白激酶C依赖性磷酸化。有趣的是，磷酸化结构域的相对位置与其他配体门控离子通道受体亚基中已知或推测被磷酸化的位置相似，因此进一步表明这些受体蛋白中存在同源调节区域。
