Sasaki S, Nakamura H, Tagami T, Miyoshi Y, Nakao K
Department of Internal Medicine, Kyoto University School of Medicine, Japan.
Mol Cell Endocrinol. 1995 Aug 30;113(1):109-17. doi: 10.1016/0303-7207(95)03621-d.
Previously, we identified a point mutation of the T3 receptor (TR) beta gene (R338W) in a patient with pituitary resistance to thyroid hormone (PRTH). The mutation existed in one of two hot spot areas in TRbeta gene where clusters of mutations have been found in subjects with generalized resistance to thyroid hormone (GRTH). Interestingly, R338W induces the phenotypical features responsible for PRTH. In the present study, we examined the functional properties of R338W in comparison with those of a GRTH-mutant, K443E. The levels of thyroid hormones and inappropriately elevated TSH (SITSH) were similar between subjects with K443E and R338W. Transcriptional activities and dominant negatives potencies were measured by CAT assay in CV1 cells transfected with each mutant TRbeta1 or along with wild-type TR. When a reporter gene containing T3-responsive elements (TRE), TRE-pal2, DR4 or myosin heavy chain alpha subunit, was used, transcriptional activation induced by R338W was higher than that by K443E. At 50 nM T3, K443E decreased the transcriptional activity of wild-type TRbeta1 on TRE-pal2 by 31.5%, while R338W reduced by 13.6% (n = 15, P < 0.05). Co-expression of retinoid X receptor (RXR) alpha increased transcriptional activity of R338W and K443E, but not of wild-type TRbeta1. Dominant negative activity on TRE-TSHalpha subunit of R338W was milder than that of K443E. When T3-binding activities of mutant TRbeta1s expressed in the cells were assayed under the same cell conditions for CAT assay, both mutant TRbeta1 showed remarkably reduced activity with no difference between the two. Gel mobility shift assay using TRE-DR4 showed poor homodimer formation of R338W. Heterodimerization with RXRalpha was similar between R338W, K443E and wild-type TRbeta1. The result of the present study suggested that R338W had relatively mild transcriptional and dominant negative activities on several TREs including TRE-TSHalpha subunit. We also showed poor homodimerization of R338W, which might be related to its weak dominant negative potency.
此前，我们在一名垂体甲状腺激素抵抗（PRTH）患者中鉴定出T3受体（TR）β基因的一个点突变（R338W）。该突变存在于TRβ基因的两个热点区域之一，在全身性甲状腺激素抵抗（GRTH）患者中已发现该区域存在突变簇。有趣的是，R338W导致了PRTH的表型特征。在本研究中，我们将R338W的功能特性与GRTH突变体K443E的功能特性进行了比较。K443E和R338W患者的甲状腺激素水平和不适当升高的促甲状腺激素（SITSH）相似。通过CAT分析在转染了每个突变体TRβ1或与野生型TR一起的CV1细胞中测量转录活性和显性负性效力。当使用含有T3反应元件（TRE）、TRE-pal2、DR4或肌球蛋白重链α亚基的报告基因时，R338W诱导的转录激活高于K443E。在50 nM T3时，K443E使野生型TRβ1对TRE-pal2的转录活性降低31.5%，而R338W降低13.6%（n = 15，P < 0.05）。视黄酸X受体（RXR）α的共表达增加了R338W和K443E的转录活性，但没有增加野生型TRβ1的转录活性。R338W对TRE-TSHα亚基的显性负性活性比K443E弱。当在与CAT分析相同的细胞条件下测定细胞中表达的突变体TRβ1的T3结合活性时，两个突变体TRβ1的活性均显著降低，两者之间无差异。使用TRE-DR4的凝胶迁移率变动分析显示R338W的同源二聚体形成较差。R338W、K443E和野生型TRβ1与RXRα的异源二聚化相似。本研究结果表明，R338W对包括TRE-TSHα亚基在内的几种TRE具有相对较弱的转录和显性负性活性。我们还表明R338W的同源二聚化较差，这可能与其较弱的显性负性效力有关。
