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Studies on structure-function of the thyrotropin receptor : molecular biological analysis of post-translational modifications

促甲状腺素受体的结构功能研究：翻译后修饰的分子生物学分析

基本信息

批准号：
09671064
负责人：
NAGAYAMA Yuji
金额：
$ 1.98万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671064/
关键词：
thyrotropin receptor post-translational modification acylation glycosylation subunit structure 免疫沈降法

项目摘要

1. Glvcosylation of thyrotropin (TSH) receptor - TSH receptor ectodomain is heavily glycosylated with N-linked carbohydrates. In immunoprecipitation, two types of TSH receptor can be dctected - a precursor form with high mannose type carbohydrates and a mature protein with complex type carbohydrates. Using a glycosylation inhibitor tunicamycin and a series of mutant CHO cells (CHO-Lec) defective in the different steps of glycosylation processing, we found that acquisition of carbohydrates is essential for proper folding of TSH receptor in endoplasmic reticulum and its processing is involved in cell surface targeting in Golgi apparatus.2. Acylation of TSH receptor - We demonstrate a cysteine residue at amino acid 699 of the C-terminal cytoplasmic tail of the receptor is palmitoylated. Stuides with a mutant TSH receptor lacking palmitoylationreveal that palmitoylation plays a pivotal role in intracellular trafficking of the receptor, but not in high affinity TSH binding, Gs coupling, homologous desensitization or internalization.3. Subunit structure of TSH receptor - TSH receptor is well known to cleave into two subunits, A and B.However, we found that there may be two cleavage sites, releasing not only two subunits but also a small peptide (-7-8 kDa). Even extensive mutagenesis studies around possible cleavage sites failed to identify the exact cleavage site, suggesting proteolytic enzyme(s) may recognize the specific three dimensional structure, not specific amino acids, of the receptor.

1.促甲状腺激素（TSH）受体的糖基化- TSH受体胞外域被N-连接的碳水化合物严重糖基化。在免疫沉淀中，可以检测到两种类型的TSH受体-具有高甘露糖型碳水化合物的前体形式和具有复合型碳水化合物的成熟蛋白。利用糖基化抑制剂衣霉素和一系列糖基化加工缺陷的突变CHO细胞（CHO-Lec），我们发现糖基化的获得对于内质网中TSH受体的正确折叠是必不可少的，并且其加工参与高尔基体的细胞表面靶向.促甲状腺激素受体的酰化-我们证明了在受体的C-末端胞质尾的氨基酸699处的半胱氨酸残基是棕榈酰化的。对缺乏棕榈酰化的突变型TSH受体的研究表明，棕榈酰化在受体的胞内转运中起关键作用，但在高亲和力TSH结合、Gs偶联、同源脱敏或内化中不起作用. TSH受体的亚基结构-众所周知，TSH受体切割成两个亚基，A和B。然而，我们发现可能有两个切割位点，不仅释放两个亚基，而且释放一个小肽（~ 7 - 8 kDa）。即使围绕可能的切割位点进行广泛的诱变研究也未能鉴定出确切的切割位点，这表明蛋白水解酶可能识别受体的特定三维结构，而不是特定的氨基酸。