Van Sande J, Massart C, Costagliola S, Allgeier A, Cetani F, Vassart G, Dumont J E
Institute of Interdisciplinary Research, School of Medicine, Free University of Brussels, Belgium.
Mol Cell Endocrinol. 1996 May 31;119(2):161-8. doi: 10.1016/0303-7207(96)03804-x.
The identification of 16 different activating mutations in the TSH receptor, found in patients suffering from toxic autonomous adenomas or congenital hyperthyroidism, leads to the concept that this receptor is in a constrained conformation in its wild-type form. We used mild trypsin treatment of CHO-K1 cells or COS-7 cells, stably or transiently transfected with the human TSH receptor, respectively, and measured its consequences on the TSH receptor coupled cascades, i.e. cyclic AMP and inositol-phosphates accumulation. A 2-min, 0.01% trypsin treatment increased stably cyclic AMP but not inositol-phosphates formation. This was not observed after chymotrypsin, thrombin and endoproteinase glu C treatment. The TSH action on cyclic AMP was decreased by only 25%. The effect was also observed in cells expressing the dog TSH receptor. It was not observed in MSH receptor, LH receptor expressing or mock transfected cells (vector alone). It is therefore specific for the TSH receptor, for its action on the Gs/adenylate cyclase cascade, and for the proteolytic cleavage caused by trypsin. Using monoclonal (A. Johnstone and P. Shepherd, personal communication) and polyclonal antibodies directed against the extracellular domain of the TSH receptor, it was shown that treatment by trypsin removes or destroys a VFFEEQ epitope (residues 354-359) from the receptor. The effect mimics the action of TSH as it activates Gs alpha and enhances the action of forskolin. It is not reversible in 1 h. The results support the concept that activation of the receptor (by hormone, autoantibodies, mutations or mild proteolysis) might involve the relief of a built-in negative constrain. They suggest that the C-terminal portion of the large extracellular domain plays a role in the maintenance of this constrain.
在患有毒性自主性腺瘤或先天性甲状腺功能亢进症的患者中发现，促甲状腺激素（TSH）受体存在16种不同的激活突变，这引发了一种观点，即该受体的野生型处于一种受限构象。我们分别对稳定或瞬时转染人TSH受体的CHO-K1细胞或COS-7细胞进行了温和的胰蛋白酶处理，并测量了其对TSH受体偶联级联反应的影响，即环磷酸腺苷（cAMP）和肌醇磷酸的积累。2分钟的0.01%胰蛋白酶处理增加了cAMP的稳定积累，但未增加肌醇磷酸的形成。在胰凝乳蛋白酶、凝血酶和内肽酶Glu C处理后未观察到这种情况。TSH对cAMP的作用仅降低了25%。在表达犬TSH受体的细胞中也观察到了这种效应。在表达促黑素（MSH）受体、促黄体生成素（LH）受体的细胞或空载体转染细胞（仅载体）中未观察到这种效应。因此，这对于TSH受体、其对Gs/腺苷酸环化酶级联反应的作用以及胰蛋白酶引起的蛋白水解切割具有特异性。使用针对TSH受体细胞外结构域的单克隆抗体（A. Johnstone和P. Shepherd，个人交流）和多克隆抗体，结果表明胰蛋白酶处理会从受体上去除或破坏一个VFFEEQ表位（残基354 - 359）。这种效应模拟了TSH的作用，因为它激活Gsα并增强了福斯可林的作用。在1小时内不可逆。这些结果支持了这样一种观点，即受体的激活（通过激素、自身抗体、突变或温和的蛋白水解）可能涉及解除一种内在的负性限制。它们表明大细胞外结构域的C末端部分在维持这种限制中起作用。
