Multimeric complex formation by the thyrotropin receptor in solubilized thyroid membranes
Multimeric complex formation by the thyrotropin receptor in solubilized thyroid membranes
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DOI：
10.1210/en.137.9.3915
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发表时间：
1996-09-01
期刊：
影响因子：
4.8
通讯作者：
Davies, TF
中科院分区：
文献类型：
--
作者：
Graves, PN;Vlase, H;Davies, TF
TSH受体（TSHR）具有一个大的糖基化胞外域，包括与TSH结合以及Graves病中自身抗体识别有关的分子氨基末端的一半（743个残基中的394个）。在这项研究中，我们采用抗体的氨基末端（Ab 1），中间部分（Ab 2），羧基末端（Ab 3）的TSH-胞外域，以前映射使用重组受体蛋白，检测天然受体存在于洗涤剂溶解的猪甲状腺细胞膜通过免疫印迹。检测到几种形式的受体。在减少样品Abl检测全长holoreceptor存在的非糖基化和糖基化形式的表观分子量分别为80和90 kDa，以及明显的二聚体非糖基化和二聚体糖基化holoreceptor形式的减少。Ab 1还检测到全受体（gly α-亚基）的糖基化氨基末端47- 52-kDa片段，通过Endo F去糖基化还原为42 kDa（α-亚基）。Ab 2检测到所有相同的形式。Ab 3主要检测到35 kDa的羧基末端非糖基化片段（β-亚基）。在未还原的样品中，Ab 1的识别模式没有变化。Ab 2检测切割的和二硫键连接的gly α-β全受体。Ab 3检测到单体和二聚体β-亚基，以及更高级的复合物。未还原制剂中存在的不同TSHR形式通过硫酸铵沉淀进行解析，证实了它们的自主性。数据表明存在多种形式的天然TSHR。它们在TSH作用和TSHR自身免疫中的作用需要进一步探索。
The TSH receptor (TSHR) has a large glycosylated ectodomain comprising the amino-terminal half of the molecule (394 of 743 residues) implicated in TSH binding, as well as autoantibody recognition in Graves' disease. In this study we employed antibodies specific for the amino-terminus (Ab1), midportion (Ab2), and carboxyl-terminus (Ab3) of the TSHR-ectodomain, previously mapped using recombinant receptor proteins, to detect the natural receptor present in detergent-solubilized porcine thyroid cell membranes via immunoblotting. Several forms of the receptor were detected. In reduced samples Abl detected full-length holoreceptors present in both nonglycosylated and glycosylated forms of apparent molecular masses 80 and 90 kDa, respectively, as well as apparent dimeric nonglycosylated and dimeric glycosylated holoreceptor forms resistant to reduction. Also detected by Ab1 were a glycosylated amino-terminal 47- to 52-kDa fragment of the holoreceptor (gly alpha-subunit), reduced to 42 kDa (alpha-subunit by Endo F deglycosylation. Ab2 detected all of the same forms. Ab3 detected primarily a carboxy-terminal, nonglycosylated fragment of 35 kDa (beta-subunit). In unreduced samples, the recognition pattern was unchanged with Ab1. Ab2 detected cleaved and disulfide-linked gly alpha-beta holoreceptors. Ab3 detected monomeric and dimeric beta-subunits, as well as higher order complexes. The different TSHR forms present in unreduced preparations were resolved by ammonium sulfate precipitation, confirming their autonomy. The data demonstrate the presence of multiple forms of the natural TSHR. Their roles in TSH action and TSHR autoimmunity require further exploration.