Akamizu T, Inoue D, Kosugi S, Ban T, Kohn L D, Imura H, Mori T
Department of Laboratory Medicine, Kyoto University School of Medicine, Japan.
Endocr J. 1993 Jun;40(3):363-72. doi: 10.1507/endocrj.40.363.
A series of chimeric TSH-LH/CG receptors were constructed by substituting homologous segments of the extracellular domain of the rat TSH receptor with corresponding segments of rat LH/CG receptor: C1 (amino acids 37-123 substituted), C2 (91-112), C3 (173-234), C4 (233-266), C5 (268-304), C6 (112-305) and C7 (36-404). After transfection in Cos-7 cell, TSH- and LH/CG-receptor activities of these chimeras were evaluated and compared with those of deletion mutants involving the same residues [Kosugi et al. Thyroid 1:321 (1991)]. Western blot analyses revealed that most of the chimeric receptor proteins were normally synthesized and integrated in the membrane of transfected Cos-7 cells: an antibody to a TSH receptor specific synthetic peptide (residues 352-366) identified 170-190kDa and 90-100kDa TSH receptor structures in the plasma membrane fractions of Cos-7 cells transfected with wild-type TSH receptor cDNA and the C1 to C6 chimeras, but not C7 or wild LH/CG receptor cDNA. Despite this, no receptor except C5 exhibited any significant TSH receptor activities either in [12I]TSH binding or in cAMP responses to TSH and thyroid-stimulating antibodies (TSAbs) from Graves' patients. The chimeric receptor C5 exhibited only low affinity TSH binding (Kd = 3.5 x 10(-8) M), as did its counterpart the M2C mutant with residues 268-304 deleted. However, unlike M2C, C5 demonstrated a significant cAMP response to TSH as well as to TSAbs. The cAMP increase in response to TSH in the wild type receptor was observed at 10(-11) M TSH. In C5 the response was first evident at 10(-10) M TSH, but the maximum cAMP stimulation by TSH and TSAbs in C5 (EC50 = 6.7 x 10(-10) M) was approximately the same as the wild type receptor (EC50 = 1.5 x 10(-10) M). Inhibition of either TSH- or TSAb- stimulated cAMP increase by thyroid-stimulating blocking antibodies (TSBAbs) was also preserved in C5. These results suggest that amino acids 268-304 do not include an important determinant required for signal transduction, since a significant cAMP response to TSH and TSAbs was observed in the C5 receptor with these residues substituted. Additionally, these residues appear to be involved in ligand high affinity binding because high affinity TSH binding was lost in the chimeric receptor C5.
通过用大鼠促黄体生成素/绒毛膜促性腺激素（LH/CG）受体的相应片段替换大鼠促甲状腺激素（TSH）受体细胞外结构域的同源片段，构建了一系列嵌合TSH-LH/CG受体：C1（氨基酸37 - 123被替换）、C2（91 - 112）、C3（173 - 234）、C4（233 - 266）、C5（268 - 304）、C6（112 - 305）和C7（36 - 404）。在Cos-7细胞中进行转染后，评估了这些嵌合体的TSH和LH/CG受体活性，并与涉及相同残基的缺失突变体的活性进行了比较[小杉等人。《甲状腺》1:321（1991）]。蛋白质免疫印迹分析显示，大多数嵌合受体蛋白在转染的Cos-7细胞膜中正常合成并整合：用野生型TSH受体cDNA以及C1至C6嵌合体转染的Cos-7细胞的质膜部分中，针对TSH受体特异性合成肽（残基352 - 366）的抗体鉴定出170 - 190kDa和90 - 100kDa的TSH受体结构，但未在转染C7或野生LH/CG受体cDNA的细胞中鉴定到。尽管如此，除了C5之外，没有其他受体在[12I]TSH结合或对来自格雷夫斯病患者的TSH和甲状腺刺激抗体（TSAbs）的cAMP反应中表现出任何显著的TSH受体活性。嵌合受体C5仅表现出低亲和力的TSH结合（Kd = 3.5×10（-8）M），其对应物M2C突变体（缺失残基268 - 304）也是如此。然而，与M2C不同，C5对TSH以及TSAbs表现出显著的cAMP反应。在野生型受体中，在10（-11）M TSH时观察到对TSH的cAMP增加。在C5中，反应在10（-10）M TSH时首次明显，但C5中TSH和TSAbs对cAMP的最大刺激（EC50 = 6.7×10（-10）M）与野生型受体（EC50 = 1.5×10（-10）M）大致相同。甲状腺刺激阻断抗体（TSBAbs）对TSH或TSAb刺激的cAMP增加的抑制在C5中也得以保留。这些结果表明，氨基酸268 - 304不包含信号转导所需的重要决定因素，因为在这些残基被替换的C5受体中观察到了对TSH和TSAbs的显著cAMP反应。此外，这些残基似乎参与配体的高亲和力结合，因为嵌合受体C5中失去了高亲和力的TSH结合。
