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

1. 促甲状腺激素（TSH）受体的糖基化- TSH受体外结构域与n -链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杂型碳水化合物的成熟蛋白。利用糖基化抑制剂tunicamycin和一系列在糖基化加工的不同步骤中存在缺陷的突变CHO细胞（CHO- lec），我们发现碳水化合物的获取对于内质网中TSH受体的适当折叠是必不可少的，其加工参与了高尔基体的细胞表面靶向。TSH受体的酰化-我们证明了受体c端胞质尾部699氨基酸的半胱氨酸残基是棕榈酰化的。对缺乏棕榈酰化的突变TSH受体的研究表明，棕榈酰化在受体的细胞内运输中起关键作用，但在高亲和力的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受体可裂解为两个亚基，A和b。然而，我们发现可能存在两个裂解位点，不仅释放两个亚基，还释放一个小肽（-7-8 kDa）。即使围绕可能的切割位点进行了广泛的诱变研究，也未能确定确切的切割位点，这表明蛋白水解酶可能识别受体的特定三维结构，而不是特定氨基酸。

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.