THE STUDY OF THE FUNCTIONS AND EXPRESSIONS OF WILD-TYPE AND MUTANT THYROID HORMONE RECEPTORS

对野生型（w）和突变型（m）甲状腺激素受体（TR）的功能和表达进行分析，得到以下结果.构建了含有延伸因子基因启动子区、β F451X-1和α F397X-1基因的转基因小鼠（TG），并将其显微注射入小鼠受精卵原核。将卵子转移到假孕的养母输卵管中，并通过PCR和Southern印迹分析来检测后代中转基因的存在。β F451X-TG的产率为17%（9/53），在本所各代TG中处于平均水平，而α F397X-TG的产率很低，仅为3.4%（4/117）。在四个alphaF397X-TG中，一个出生时死亡，另一个在生长过程中死亡。两个alphaF397X-TG，但 关于我们 在β F451-TG中，没有一个显示体重增加减少。TG无明显异常，基础T_4和TSH水平在正常范围内。学习和行为的研究表明，TG，特别是雌性betaF451X-TG与同窝仔相比，有更高的自发运动活动的趋势。未发现学习能力等其他异常，提示甲状腺激素抵抗患者中未发现TR α 1突变的原因可能是由于突变型TR α 1.2的致死作用。用酵母双杂交系统检测TR与维甲酸X受体（RXR）之间的异源二聚体形成能力（HD），在CV1细胞中表达两种酵母DNA结合域GAL 4与RXR配体结合域（LBD）的融合蛋白（GAL-RXR）和TRbetaLBD与VP16的融合蛋白（VP-TR），可反式激活GAL报告基因。由于共表达的TR与VP-TR竞争结合RXR，因此可以定量和灵敏地测定mTR和VP-TR的HD能力。不同mTRs的RXR结合活性与其显性负效应（DNE）一致，提示HD在DNE中的重要性.分析betaF451X和alphaf397X的沉默活性。betaF451X和alphaf397X都具有非常强且相同的沉默活性。与每个野生型TR相比，与RXR和辅阻遏物如SMRT和N-CoR的结合活性在betaF451X中显著更高，但在alphaF397X中不显著更高，这表明两种截短TR的沉默机制不一定相同。对TR/β 1的氨基端A/B区进行功能分析。利用缺失突变体研究表明，TR/β 1的32 - 51区氨基酸具有反式激活功能，51 - 94区氨基酸具有抑制功能。有趣的是，这些区域可能会影响HD能力。TR与维生素D受体（VDR）的转录相互作用：我们发现TR和VDR只有在各自配体存在的情况下才能在转录水平上相互干扰。骨肉瘤细胞内内源性受体之间也存在相互作用。实验结果表明，在某些共激活因子中，TR和VDR之间存在功能竞争。少

The functions and expressions of wild-type (w) and mutant (m) thyroid hormone receptors (TR) were analyzed and the following results were obtained.1. Generation of the transgenic mice (TG) expressing the carboxyl (C)-terminal 11 amino acid-truncated TRbeta (betaF451X) or TR alpha1 which possesses the same C-terminal truncation as betaF451X (alphaF397X).The expression vectors bearing the promotion region of the elongation factor gene and betaF451X-or alphaF397X-gene were constructed and microinjected into the pronucleus of fertilized mouse eggs. The eggs were transferred into the oviducts of pseudopregnant foster mothers and the existence of the transgene was examined in offsprings by PCR and Southern blot analysis. The productivity of betaF451X-TG was 17% (9/53 mice), which was average in various TG generation in our institute, while very low as only 3.4% in alphaF397X-TG (4/117 mice). Among four alphaF397X-TG,one was born dead and another died during growth. The two alphaF397X-TG,but … More none in betaF451-TG,showed decrease in body weight gain. No apparent abnormality was noticed in either TG.The basal T4 and TSH levels were within normal limits. The study of learning and behavior showed the tendency of higher spontaneous motor activity in TG,especially female betaF451X-TG as compared with the litter mates. No other abnormality including learning ability was found. The results suggest that the reason no mutation has been found in TRalpha1 in patients with thyroid hormone resistance may be due to the lethal effect of mutant TRalpha1.2. Measurement of the heterodimer formation (HD) ability between TR and retinoid X receptor (RXR) by a new assay system of yeast two hybrid system.Expression of two fusion proteins of yeast DNA binding domain, GAL4, and ligand binding domain (LBD) of RXR (GAL-RXR), and TRbetaLBD and VP16 (VP-TR) in CV1 cells transactivate the GAL reporter gene. Since TR coexpressed competes with VP-TR in RXR binding, HD ability of w-and mTR can be assayd quantitatively and sensitively. The RXR binding activities of various mTRs were well paralleled with their dominant negative effects (DNE), suggesting the importance of HD in DNE.3. Analysis of the silencing activity of betaF451X and alphaF397X.Both betaF451X and alphaf397x have very strong and identical silencing activities. The binding activities with RXR and corepressors such as SMRT and N-CoR were significantly higher in betaF451X,but not in alphaF397X,compared with each wild-type TR,suggesting that the mechanism of silencing was not necessarily identical in both truncated TRs.4. Functional analysis of amino (N) -terminal A/B region of TR/beta1.Studies using deletion mutants demonstrated the transactivating function in region 32-51 amino acid and suppressing function in region 51-94 amino acid. interestingly, these regions may affect the HD ability.5. Transcriptional interaction between TR and vitamin D receptor (VDR).We found that TR and VDR interfered each other in transcriptional level only when in the presence of each ligand. The interaction was observed between the endogenous native receptors in osteosarcoma cells also. The experimental date strongly suggested the functional competition between TR and VDR in some coactivator (s). Less