Thyroid hormone receptors. Binding characteristics and lack of hormonal dependency for nuclear localization.
Thyroid hormone receptors. Binding characteristics and lack of hormonal dependency for nuclear localization.
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甲状腺激素受体。
DOI：
10.1016/s0021-9258(19)41393-8
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发表时间：
1975
期刊：
影响因子：
--
通讯作者：
J. Baxter
中科院分区：
文献类型：
--
作者：
B. Spindler;K. Macleod;J. Ring;J. Baxter
甲状腺激素对生长和新陈代谢有不同的影响。结合三碘甲状腺原氨酸和其他生物活性类似物并可能参与甲状腺激素作用的特异性“受体”蛋白最近已在反应组织的细胞核中发现。本报告介绍了这些受体在大鼠肝细胞核的研究。结合数据的Scatchard分析与以前的报道一致，表明反应，三碘甲状腺原氨酸+特异性受体与三碘甲状腺原氨酸-受体复合物平衡，在22 ℃下的表观平衡解离常数（Kd）约为190 pM，每mg DNA的三碘甲状腺原氨酸结合位点的容量约为1 pmol。结合的动力学也进行了检查。三碘甲状腺原氨酸受体复合物的形成是二级的，解离是一级的。在22 ℃时，表观缔合（k+1）和解离（k-1）速率常数分别为4.7 × 10-7 m-1 min-1和7.6 × 10- 3 min-1。由速率常数之比（k减1：k+1）估算的表观Kd约为150 pM，与由平衡数据测定的值相似。这些数据支持上述甲状腺激素与其受体相互作用的表达。另外的动力学实验表明，一些三碘甲腺原氨酸结合的无细胞核是由激素在完整的动物先前占据的网站，提供进一步的证据表明，完整的细胞和无细胞的反应是相同的。以前发现，核结合的三碘甲腺原氨酸定位于染色质中。我们发现，分离的染色质保留类似于分离的细胞核的特异性结合活性。因此，结合可能不需要细胞质、核质或核膜因子。这些发现可能意味着受体的染色质定位不依赖于激素。这一想法得到了早期发现的支持，即甲状腺切除动物的细胞核中存在结合活性。然而，许多刺激物如类固醇激素、细菌诱导物和细菌中的环腺苷3 '：5'-单磷酸通过促进蛋白质添加到染色质或从染色质去除而在基因水平上影响调节蛋白。因此，我们在受体稳定性和可逆结合的测定条件下研究了甲状腺激素对受体核含量的影响。甲状腺功能减退动物中的受体水平与甲状腺功能正常动物中的受体水平相同。这些数据表明，激素不影响受体的核定位。因此，甲状腺激素作用的基础可能是调节染色质中受体的活性，而不是促进受体加入染色质或从染色质中去除。
Thyroid hormones have diverse effects on growth and metabolism. Specific "receptor" proteins which bind triiodothyronine and other biologically active analogs and which may be involved in thyroid hormone action have been recently found in nuclei of responsive tissues. This report presents studies of these receptors in rat liver nuclei. Confirming previous reports, a Scatchard analysis of the binding data suggests the reaction, triiodothyronine + specific receptor in equilibrium with triiodothyronine-receptor complex, with an apparent equilibrium dissociation constant (Kd) at 22 degrees of about 190 pM and a capacity of about 1 pmol of triiodothyronine-binding sites per mg of DNA. The kinetics of the binding were also examined. Triiodothyronine-receptor complex formation is second order and dissociation is first order. The apparent association (k+1) and dissociation (k minus 1) rate constants at 22 degrees are, respectively, 4.7 times 10-7 m-minus 1 min-minus 1 and 7.6 times 10-minus 3 min-minus 1. The apparent Kd, estimated from the ratio of the rate constants (k minus 1:k+1), was about 150 pM, similar to that determined from the equilibrium data. These data support the expression written above for the interaction of thyroid hormone with its receptor. Additional kinetic experiments indicate that some of the triiodothyronine binding by cell-free nuclei is to sites previously occupied by hormone in the intact animal, providing further evidence that the intact cell and cell-free reactions are the same. It was previously found that nuclear-bound triiodothyronine is localized in chromatin. We found that isolated chromatin retains specific binding activity similar to that of isolated nuclei. Thus, binding may not require cytoplasmic, nucleoplasmic, or nuclear membrane factors. These findings may imply that chromatin localization of the receptor does not depend on the hormone. This idea is supported by an earlier finding that binding activity is present in nuclei from thyroidectomized animals. However, many stimuli such as steroid hormones, bacterial inducers, and cyclic adenosine 3':5'-monophosphate in bacteria influence regulatory proteins at the gene level by promoting the protein's addition to or removal from chromatin. Thus, we studied the effect of thyroid hormone on the nuclear content of receptors under assay conditions of receptor stability and reversible binding. Receptor levels in hypothyroid animals are identical with those in euthyroid animals. These data suggest that the hormone does not influence the nuclear localization of receptors. Thus, the basis for thyroid hormone action may be to regulate the activity of receptors resident in chromatin rather than to promote receptor addition to or removal from chromatin.