Hidaka A, Ban T, Panesar N S, Minegishi T, Kohn L D, Tahara K
Section on Cell Regulation, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland.
Thyroid. 1994 Winter;4(4):447-57. doi: 10.1089/thy.1994.4.447.
Surprisingly, thyrotropin (TSH) can increase cAMP and inositol phosphate (IP) levels in Cos-7 cells transfected with the lutropin (LH)/choriogonadotropin (CG) receptor (LH/CGR) as well as LH or CG, as evidenced by similar EC50 and maximal stimulation values. Additionally surprising, TSH activation is evident, despite markedly reduced levels of high affinity TSH binding by comparison to CG (Hidaka A, et al. 1993 Biochem Biophys Res Commun 196:187-195). In this report, we questioned whether the unusual TSH activity, as well as the discrepancy between TSH activity and binding, might reflect the existence of distinct agonist and binding sites on the LH/CGR extracellular domain and the ability of TSH to interact with the former despite a minimal interaction with the latter. We evaluated this possibility by using two chimeras spanning the extracellular domain of the TSHR and the LH/CGR:Mc1 + 2, where residues 8-165 of the TSHR are substituted, and Mc2 + 3 + 4, where residues 90-370 are replaced with the corresponding peptide segment from the LH/CGR. After transfection in Cos-7 cells, Mc2 + 3 + 4 exhibits higher affinity for CG than wild-type LH/CGR, but has no CG agonist response in assays measuring cAMP or inositol phosphate (IP) levels. Conversely, the Mc1 + 2 chimera exhibits significantly decreased affinity for CG, but CG agonist activity is comparable to wild-type LH/CGR in cAMP and IP assays. These data show that the extracellular domain of the LH/CGR does have distinct sites for CG binding and agonist activity: the C-terminus in Mc2 + 3 + 4 is important for high affinity CG binding, whereas the N-terminus in Mc1 + 2 is able to exhibit a CG agonist response, despite low affinity binding. When evaluated using TSH, Mc1 + 2, with the C-terminus of the TSHR present, exhibits high affinity TSH binding comparable to wild-type TSHR. Unexpectedly, Mc1 + 2, with the substitution of the N-terminus of the extracellular domain of the LH/CGR, exhibits even better TSH agonist activity than wild-type TSHR, not a loss of activity. Thus, the N-terminus of the extracellular domain of the LH/CGR can couple TSH binding to signal transduction events even better than the N-terminus of the TSHR. This may, in part, explain why TSH has an unusual agonist activity in cells transfected with LH/CGR, despite relatively low affinity binding. Although distinct agonist and binding sites exist in the linear sequence of the extracellular domain, the activity of the two sites is interdependent.(ABSTRACT TRUNCATED AT 400 WORDS)
令人惊讶的是，促甲状腺激素（TSH）可使转染了促黄体生成素（LH）/绒毛膜促性腺激素（CG）受体（LH/CGR）以及LH或CG的Cos-7细胞中的环磷酸腺苷（cAMP）和肌醇磷酸（IP）水平升高，相似的半数有效浓度（EC50）和最大刺激值证明了这一点。另外令人惊讶的是，尽管与CG相比，TSH高亲和力结合水平显著降低，但TSH激活仍然明显（日高A等人，1993年，《生物化学与生物物理研究通讯》196:187 - 195）。在本报告中，我们质疑TSH这种不寻常的活性以及TSH活性与结合之间的差异，是否可能反映了LH/CGR细胞外结构域上存在不同的激动剂和结合位点，以及TSH尽管与后者的相互作用极小，但仍能与前者相互作用。我们通过使用两种跨越促甲状腺激素受体（TSHR）和LH/CGR细胞外结构域的嵌合体来评估这种可能性：Mc1 + 2，其中TSHR的第8 - 165位残基被替换；以及Mc2 + 3 + 4，其中第90 - 370位残基被LH/CGR的相应肽段取代。在Cos-7细胞中进行转染后，Mc2 + 3 + 4对CG的亲和力高于野生型LH/CGR，但在测量cAMP或肌醇磷酸（IP）水平的实验中没有CG激动剂反应。相反，Mc1 + 2嵌合体对CG的亲和力显著降低，但在cAMP和IP实验中，其CG激动剂活性与野生型LH/CGR相当。这些数据表明，LH/CGR的细胞外结构域确实存在不同的CG结合位点和激动剂活性位点：Mc2 + 3 + 4中的C末端对于高亲和力CG结合很重要，而Mc1 + 2中的N末端尽管结合亲和力低，但仍能表现出CG激动剂反应。当用TSH进行评估时，存在TSHR C末端的Mc1 + 2表现出与野生型TSHR相当的高亲和力TSH结合。出乎意料的是，替换了LH/CGR细胞外结构域N末端的Mc1 + 2表现出比野生型TSHR更好的TSH激动剂活性，而不是活性丧失。因此，LH/CGR细胞外结构域的N末端能比TSHR的N末端更好地将TSH结合与信号转导事件偶联起来。这可能部分解释了为什么TSH在转染了LH/CGR的细胞中具有不寻常的激动剂活性，尽管其结合亲和力相对较低。尽管在细胞外结构域的线性序列中存在不同的激动剂和结合位点，但这两个位点的活性是相互依赖的。（摘要截断于400字）
