Chimeric studies of the extracellular domain of the rat thyrotropin (TSH) receptor: amino acids (268-304) in the TSH receptor are involved in ligand high affinity binding, but not in TSH receptor-specific signal transduction.
Chimeric studies of the extracellular domain of the rat thyrotropin (TSH) receptor: amino acids (268-304) in the TSH receptor are involved in ligand high affinity binding, but not in TSH receptor-specific signal transduction.
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大鼠促甲状腺素 (TSH) 受体胞外结构域的嵌合研究：TSH 受体中的氨基酸 (268-304) 参与配体高亲和力结合，但不参与 TSH 受体特异性信号转导。
作者：
T. Akamizu;D. Inoue;S. Kosugi;T. Ban;L. Kohn;H. Imura;T. Mori
用大鼠促黄体生成素受体胞外区的同源片段替换大鼠促黄体生成素受体的相应片段，构建了一系列嵌合的促黄体生成素受体：C1(氨基酸37-123取代)、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，检测了这些嵌合体的TSH和LH/CG受体活性，并与含有相同残基的缺失突变体进行了比较[Kosugi et al.甲状腺1：321(1991年)]。Western印迹分析表明，大多数嵌合受体蛋白被正常合成并整合到转染的Cos-7细胞的细胞膜上：抗TSH受体特异性合成肽(残基352-366)的抗体在野生型TSH受体cDNA和C1至C6嵌合体的COS-7细胞质膜部分中检测到170-190 kDa和90-100 kDa的TSH受体结构，但不能检测到C7或野生的LH/CG受体基因。尽管如此，除了C5，在[12I]TSH结合或对来自Graves病患者的TSH和甲状腺刺激抗体(TSAbs)的cAMP反应中，没有任何受体显示出任何显著的TSH受体活性。嵌合受体C5只表现出低亲和力的TSH结合(Kd=3.5×10(-8)M)，与其对应的缺失残基268-304的M2C突变体也是如此。然而，与M2C不同的是，C5对TSH和TSAb均表现出明显的cAMP反应。在10(-11)M TSH时，野生型受体对TSH的反应使cAMP升高。在C5，TSH和TSAb对cAMP的最大刺激作用在10(-10)M时出现，但其对cAMP的最大刺激作用(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(TSBAbbs)对TSH或TSAb刺激的cAMP增加的抑制作用也保留在C5中。这些结果表明，氨基酸268-304不包括信号转导所需的一个重要决定因素，因为在C5受体中观察到对TSH和TSAbs的显著cAMP反应，这些残基被取代。此外，这些残基似乎参与了配体的高亲和力结合，因为高亲和力TSH结合在嵌合受体C5中丢失。
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.
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促甲状腺素黄体生成素/绒毛膜促性腺激素受体胞外域嵌合体作为促甲状腺素受体功能的探针。
DOI：
10.1073/pnas.88.3.902
发表时间：
1991
影响因子：
11.1
作者：
Nagayama,Y;Wadsworth,HL;Chazenbalk,GD;Russo,D;Seto,P;Rapoport,B
通讯作者：
Rapoport,B
人促甲状腺素受体胞外域羧基末端一半在信号转导中的作用。
影响因子：
4.8
作者：
Nagayama,Y;Rapoport,B
通讯作者：
Rapoport,B
影响因子：
56.9
作者：
MCFARLAND, KC;SPRENGEL, R;SEEBURG, PH
通讯作者：
SEEBURG, PH
影响因子：
56.9
作者：
PARMENTIER, M;LIBERT, F;VASSART, G
通讯作者：
VASSART, G