Latif R, Graves P, Davies T F
Division of Endocrinology, Diabetes and Bone Diseases, Mount Sinai School of Medicine, New York, New York 10029-6574, USA.
J Biol Chem. 2001 Nov 30;276(48):45217-24. doi: 10.1074/jbc.M103727200. Epub 2001 Sep 4.
To examine thyrotropin (TSH) receptor homophilic interactions we fused the human TSH receptor (hTSHR) carboxyl terminus to green fluorescent protein (GFP) and the corresponding chimeric cDNA was expressed in Chinese hamster ovary cells. Fluorescent TSH receptors on the plasma membrane were functional as assessed by TSH-induced cAMP synthesis. The binding of TSH, as well as TSHR autoantibodies, induced time- and dose-dependent receptor capping. Fluorescence resonance energy transfer between receptors differentially tagged with GFP variants (RFP and YFP) provided evidence for the close proximity of individual receptor molecules. This was consistent with previous studies demonstrating the presence of TSHR dimers and oligomers in thyroid tissue. Co-immunoprecipitation of GFP-tagged and Myc-tagged receptor complexes was performed using doubly transfected cells with Myc antibody. Western blotting of the immunoprecipitated complex revealed the absence of noncleaved TSH holoreceptors. This further suggested that cleavage of the holoreceptor into its two-subunit structure, comprising disulfide-linked TSHR-alpha and TSHR-beta subunits, was required for the formation of TSHR dimers and higher order complexes.
为了研究促甲状腺激素（TSH）受体的同源相互作用，我们将人TSH受体（hTSHR）的羧基末端与绿色荧光蛋白（GFP）融合，并将相应的嵌合cDNA在中国仓鼠卵巢细胞中表达。通过TSH诱导的cAMP合成评估，质膜上的荧光TSH受体具有功能。TSH以及TSHR自身抗体的结合诱导了时间和剂量依赖性的受体聚集。用GFP变体（RFP和YFP）进行差异标记的受体之间的荧光共振能量转移为单个受体分子的紧密接近提供了证据。这与先前证明甲状腺组织中存在TSHR二聚体和寡聚体的研究一致。使用Myc抗体对双重转染的细胞进行GFP标记和Myc标记的受体复合物的共免疫沉淀。免疫沉淀复合物的蛋白质印迹显示不存在未切割的TSH全受体。这进一步表明，全受体切割成其二亚基结构（包括二硫键连接的TSHR-α和TSHR-β亚基）是形成TSHR二聚体和高阶复合物所必需的。
