PHOTOAFFINITY LABELING OF THYROID HORMONE RECEPTORS

甲状腺激素受体是染色质相关蛋白，可以 用0.4M氯化钾提取，沉淀在3.8M S，并有估计 分子量(MR)为54,000。相反，在低离子强度时 微球菌核酸酶以6.5 S的形式切除受体，并 作为一个数量较少的12.5 S种，似乎代表单核小体 相关受体。S表的M(R)估计为149,000和 密度估计表明，它由85%的蛋白质组成(Mr=127,000) 和15%的DNA(Mr=22,000或35个碱基对)。这表明，6.5岁的S 受体由54,000个MR 3.8的二聚体S结合组分组成 单独或以单体形式与其他结合的独特蛋白质结合 到大约35个碱基对的DNA结构域。为了进一步探索 染色质中激素结合成分的相互作用 合成了一种L的光亲和标记衍生物-三碘甲腺原氨酸 与完整细胞中的核受体结合(N-2-重氮， 3，3，3-三氟丙酰基-L-三碘甲腺原氨酸)。254 nm紫外光照射 随后的SDS-聚丙烯酰胺凝胶电泳法显示了丰富的 47,000种MR受体和数量较少的57,000种MR。两者都有 组分产生与金黄色葡萄球菌相同的125I-肽片段 V8蛋白水解酶或胰酶。57,000到47,000的蛋白质水解性转化 MR Form没有解释这两个已识别的物种。几个 对这两种MR受体提出了解释。这些措施包括： (1)受体结合组分的UV蛋白质-蛋白质交联 到染色质中的10,000个MR成分；(2)非结合亚单位，其可以 通过蛋白质-蛋白质与47,000个MR物种共价连接 交叉链接；(3)一种MR形式到另一种形式的特定转换 通过合成后修饰或细胞核中的特定切割 这导致了受体的激活；(4)两个M(R)成分可以是 衍生自杂二聚体结构，如已报道的 孕激素受体。这项建议中的研究将利用 结合接触部位的光亲和标记衍生物 用于探测染色质成分的交联剂 受体相互作用。这些研究将在培养的GH1大鼠身上进行 生长激素细胞以及正常和甲状腺激素抵抗的细胞 含正常甲状腺激素核的成纤维细胞 感受器。

Thyroid hormone receptors are chromatin associated proteins which can be extracted with 0.4 M KCl, sediment at 3.8 S, and have an estimated molecular weight (Mr) of 54,000. In contrast, at low ionic strength micrococcal nuclease excises the receptors as a precominent 6.5 S form and as a less abundant 12.5 S species which appears to represent mononucleosome associated receptor. The 6.5 S form has an estimated M(r) of 149,000 and density estimates suggest that it is composed of 85% protein (Mr = 127,000) and 15% DNA (Mr = 22,000 or 35 base pairs). This suggests that the 6.5 S receptor is composed of the 54,000 Mr 3.8 S binding component in dimeric form alone or in monomeric form associated with other unique proteins bound to a DNA domain of approximately 35 base pairs. To further explore the interaction of the hormone binding component in chromatin we have synthesized a photoaffinity label derivitive of L-triiodothyronine which binds to nuclear receptors in intact cells (N-2-diazo, 3,3,3-trifluoropropionyl-L-triiodothyronine). UV irradiation at 254 nm followed by SDS-polyacrylamide gel electrophoresis demonstrates an abundant 47,000 Mr receptor form and a less abundant 57,000 Mr species. Both components yield identical 125I-peptide fragments with Staphlacocus aureus V8 protease or trypsin. Proteolytic conversion of the 57,000 to the 47,000 Mr form does not account for the two identified species. Several explanations for the two Mr receptor species are proposed. These include: (1) the UV protein-protein cross-linking of the receptor binding component to a 10,000 Mr component in chromatin; (2) a non-binding subunit which can be covalently linked to the 47,000 Mr species by protein-protein cross-linking; (3) specific conversion of one Mr form to the other either by post-synthetic modification or specific cleavage in the cell nucleus which results in receptor activation; (4) the two M(r) components may be derived from a heterodimer structure as has been reported for the progesterone receptor. Studies in this proposal will utilize the photoaffinity label derivative in conjunction with contact site cross-linking agents to probe the chromatin components with which the receptor interacts. These studies will be performed in cultured GH1 rat somatotroph cells as well as normal and thyroid hormone resistant fibroblasts which contain a normal complement of thyroid hormone nuclear receptors.