STRUCTURE/FUNCTION RELATIONSHIPS OF HUMAN THYROTROPIN

本建议的目的是阐明结构- 人促甲状腺激素（hTSH）及其受体的功能关系 开发和表征新的类似物的作用， 结合亲和力、体外和体内生物活性（超活性 类似物）。 有广泛的研究功能域的 促性腺激素，但只有有限的研究，这些在TSH，主要是从 我们的实验室，这表明显着差异的结构-功能 关系。此外，在我们最近的观察之前， 已报告任何糖蛋白激素或胱氨酸的任何成员 结生长因子超家族的这种类似物的主要增加， 受体结合亲和力或体外生物活性。我们已经启动了 基于进化考虑的超活性类似物的设计， 不同物种序列的同源性比较，以及同源性 hTSH模型。 第一代超活性hTSH类似物 显示出高达1,000倍的效力增加和2倍增加 最大反应 设计出第二代更有效的 类似地，我们将选择性地引入带电残基的簇， 存在于同源激素中，在hTSH的外周环内， 单独地和以各种组合。 在目前的提案中，我们 将：1）在外周中进行系统的多结构域诱变， 两个hTSH亚基的发夹环，以优化效力和功效 2）表达和纯化大量的最具活性的 类似物并表征其生化和生物学特性; 3)试图确定hTSH受体的区域， hTSH环内带正电荷的残基 超活性类似物; 4）测试是否位点特异性去除唾液酸 酸将增加受体结合和体外活性， 选择的hTSH类似物。 这些研究应该提供新的见解， TSH的结构-功能关系和一种新的方法， 促甲状腺激素受体激活研究。 高活性重组hTSH 类似物在几种体外应用中应该具有很大的价值 与受体结合或激活有关， 刺激131 I摄取和甲状腺球蛋白的治疗益处 甲状腺癌和非毒性甲状腺肿患者的分泌物。 高 亲和hTSH类似物也可能具有独特的能力， 涉及靶向递送的甲状腺癌的治疗 放射性核素、毒素、肿瘤抑制基因或钠/碘 共转运体基因 此外，它们的设计提供了一个合理的战略， 已知胱氨酸的其他成员的类似物的开发 生长因子超家族 最后，公共卫生的重要性 这一建议得到了大量新病例的强调， 在欧洲发现的异常侵袭性的儿童甲状腺癌， 切尔诺贝利事故以及美国预计将出现的新的成人病例。 因为20世纪50年代在内华达州进行的核试验。

The objective of this proposal is the elucidation of the structure- function relationships of human thyrotropin (hTSH) and its receptor by developing and characterizing the action of novel analogs with enhanced binding affinity, in vitro and in vivo biological activity ( superactive analogs ). There are extensive studies of functional domains of gonadotropins but only limited studies of those in TSH, primarily from our laboratory, which show significant differences in structure-function relationships. Moreover, until our recent observations, there had not been reports for any glycoprotein hormone or any member of the cystine knot growth factor superfamily of such analogs with major increases in receptor binding affinity or in vitro bioactivity. We have initiated the design of superactive analogs based on evolutionary considerations, homology comparison of sequences from various species, and homology modeling of hTSH. The first generation of superactive hTSH analogs displayed up to 1,000-fold increase in potency and 2-fold increased maximal response. To design a second generation of even more potent analogs we will selectively introduce clusters of charged residues, present in homologous hormones, within the peripheral loops of hTSH, separately and in various combinations. In the current proposal we will: 1) perform systematic multidomain mutagenesis in the peripheral hairpin loops of both hTSH subunits to optimize the potency and efficacy of the analogs; 2) express and purify large amounts of the most active analogs and characterize their biochemical and biological properties; 3) attempt to determine the areas of the hTSH receptor that interact with the positively charged residues within the loops of hTSH superactive analogs; 4) test whether site-specific removal of sialic acid will increase the receptor binding and in vitro activity of selected hTSH analogs. These studies should provide novel insights into structure-function relationships of TSH and a novel approach to the study of TSH receptor activation. Highly active recombinant hTSH analogs should be of great value in several in vitro applications related to receptor binding or activation and should have improved therapeutic benefit in stimulating 131I uptake and thyroglobulin secretion in patients with thyroid cancer and nontoxic goiter. High affinity hTSH analogs may also have unique capabilities for novel therapies of thyroid cancer involving targeted delivery of radionuclides, toxins, tumor suppressor genes or the sodium/iodide symporter gene. Moreover, their design provides a rational strategy for the development of similar analogs for other members of the cystine know growth factor superfamily. Finally, the public health importance of this proposal is underscored by the large number of new cases of unusually aggressive childhood thyroid cancer detected in Europe because of the Chernobyl accident and of new adult cases expected in the U.S. because of nuclear testing in Nevada in the 1950's.