Haraguchi K, Saito T, Endo T, Onaya T
Third Department of Internal Medicine, University of Yamanashi Medical School, Japan.
Life Sci. 1994;55(12):961-8. doi: 10.1016/0024-3205(94)00542-7.
In order to understand the function of the first extracellular loop of the human thyrotropin receptor (hTSHR), each of two peptides of nine amino acids was inserted into the first extracellular loop of hTSHR. hTSHR cDNA was subcloned into the eukaryotic expression vector, pRc/CMV (hTSHR/pRc/CMV). B-hTSHR/pRc/CMV, a mutant hTSHR cDNA which encodes a hydrophilic peptide insert (AGTTRRVAI) and C-hTSHR/pRc/CMV which encodes a hydrophobic peptide insert (ATVLVVPMI) between +486 Ileu and +487 Asp of hTSHR were transfected into Chinese hamster ovary cells to generate the B-1 and C-6 cell lines, respectively. Neither thyrotropin (TSH) nor thyroid stimulating antibody (TSAb) stimulated cAMP production by B-1 or C-6 cells. An 125I-TSH binding assay showed that neither cell line bound TSH. Our data demonstrated that these mutations impaired both TSH binding and cAMP production. This evidence suggests that the first extracellular loop of hTSHR may have a crucial role in the TSH- and TSAb-dependent signal transduction.
为了解人促甲状腺激素受体（hTSHR）第一个细胞外环的功能，将两条九氨基酸肽分别插入hTSHR的第一个细胞外环中。hTSHR cDNA亚克隆至真核表达载体pRc/CMV（hTSHR/pRc/CMV）。将B-hTSHR/pRc/CMV（一种编码亲水性肽插入片段AGTTRRVAI的突变hTSHR cDNA）和C-hTSHR/pRc/CMV（在hTSHR的+486位异亮氨酸和+487位天冬氨酸之间编码疏水性肽插入片段ATVLVVPMI）分别转染至中国仓鼠卵巢细胞，以分别产生B-1和C-6细胞系。促甲状腺激素（TSH）和甲状腺刺激抗体（TSAb）均未刺激B-1或C-6细胞产生环磷酸腺苷（cAMP）。125I-TSH结合试验表明，这两种细胞系均不结合TSH。我们的数据表明，这些突变损害了TSH结合和cAMP产生。这一证据提示，hTSHR的第一个细胞外环可能在TSH和TSAb依赖性信号转导中起关键作用。
