Shigeno C, Hiraki Y, Westerberg D P, Potts J T, Segre G V
Endocrine Unit, Massachusetts General Hospital, Boston.
J Biol Chem. 1988 Mar 15;263(8):3872-8.
In the preceding article, we described physicochemical and kinetic properties of parathyroid hormone (PTH) receptors in clonal rat osteosarcoma cells (ROS 17/2.8) using photoaffinity ligand labeling and showed that the physiologically relevant receptor-ligand complex has an apparent Mr = 80,000. In this study, the photoaffinity labeled Mr = 80,000 receptor was localized exclusively on the cell surface plasma membrane and its glycoprotein nature was demonstrated through the use of lectin affinity-chromatography and specific exo- and endoglycosidases. Rinsing ROS cells, preincubated in the dark with 125I-labeled [Nle8, N-epsilon-(4-azido-2-nitrophenyl)Lys13,Nle18,Tyr34]bovine PTH-(1-34)-NH2 (NAP-NlePTH) (4 h, 15 degrees C, equilibrium conditions) with acidic phosphate-buffered saline (pH 2.5, 30 s, 4 degrees C) before photolysis resulted in selective and nearly total disappearance of the labeled Mr = 80,000 receptor. PTH receptor integrity to acid rinsing and photolysis was shown by relabeling the Mr = 80,000 receptor after a second incubation of these cells with 125I-labeled NAP-NlePTH, followed by photolysis. Adsorption of Triton X-100-solubilized, 125I-labeled NAP-NlePTH receptors to wheat germ agglutinin-agarose is nearly complete and highly selective, and elution with N-acetylglucosamine resulted in virtually total recovery of the labeled receptors from the column. The wheat germ agglutinin-retarded PTH receptors show increased electrophoretic mobility upon treatment with neuraminidase which was inhibited by simultaneous addition of 2,3-dehydro-3-desoxy-N-acetylneuraminic acid, a specific neuraminidase inhibitor. Endoglycosidase F treatment of the Mr = 80,000 receptors generated a single, labeled polypeptide with a Mr = 59,000 which migrated as a narrow band. PTH receptors on ROS 17/2.8 cells appear to be monomeric plasma membrane glycoproteins with an apparent Mr of 80,000 which contain a Mr = 59,000 polypeptide backbone and a polymeric arrangement of N-acetylglucosamine with N-acetylneuraminic acid as major terminal sugar residues.
在前一篇文章中，我们利用光亲和配体标记法描述了克隆大鼠骨肉瘤细胞（ROS 17/2.8）中甲状旁腺激素（PTH）受体的物理化学和动力学特性，并表明生理相关的受体-配体复合物的表观分子量为80,000。在本研究中，光亲和标记的分子量为80,000的受体仅定位在细胞表面质膜上，并且通过凝集素亲和层析和特异性外切糖苷酶及内切糖苷酶的使用证明了其糖蛋白性质。在用酸性磷酸盐缓冲盐水（pH 2.5，30秒，4℃）冲洗在黑暗中用125I标记的[Nle8，N-ε-（4-叠氮基-2-硝基苯基）Lys13，Nle18，Tyr34]牛PTH-（1-34）-NH2（NAP-NlePTH）预孵育的ROS细胞（4小时，15℃，平衡条件）后进行光解，导致标记的分子量为80,000的受体选择性且几乎完全消失。在用125I标记的NAP-NlePTH对这些细胞进行第二次孵育然后光解后，通过重新标记分子量为80,000的受体，显示了PTH受体对酸冲洗和光解的完整性。Triton X-100溶解的、125I标记的NAP-NlePTH受体对麦胚凝集素-琼脂糖的吸附几乎是完全的且具有高度选择性，用N-乙酰葡糖胺洗脱导致标记的受体几乎从柱中完全回收。用神经氨酸酶处理后，麦胚凝集素阻滞的PTH受体显示出电泳迁移率增加，同时加入2,3-脱氢-3-脱氧-N-乙酰神经氨酸（一种特异性神经氨酸酶抑制剂）可抑制这种增加。用内切糖苷酶F处理分子量为80,000的受体产生了一条单一的、标记的多肽，其分子量为59,000，迁移为一条窄带。ROS 17/2.8细胞上的PTH受体似乎是单体质膜糖蛋白，表观分子量为80,000，其包含分子量为59,000的多肽骨架以及以N-乙酰葡糖胺与N-乙酰神经氨酸作为主要末端糖残基的聚合物排列。
