STRUCTURE/FUNCTION RELATIONSHIPS OF HUMAN THYROTROPIN

这项建议的目的是澄清结构- 人促甲状腺激素(HTSH)及其受体的功能关系 开发和表征增强的新型类似物的作用 结合亲和力、体外和体内生物活性(超活性 类比)。有广泛的研究，功能结构域的 促性腺激素，但对促性腺激素的研究有限，主要来自 我们的实验室，在结构功能上表现出显著的差异 两性关系。此外，在我们最近的观察之前，还没有 关于任何糖蛋白激素或任何胱氨酸成员的报道 结生长因子超家族的这类类似物与主要增加 受体结合亲和力或体外生物活性。我们已经启动了 基于进化考虑的超级活性类似物的设计， 不同物种序列的同源性比较和同源性 HTSH的建模。第一代超活性HTSH类似物 显示出高达1,000倍的效力提升和2倍的提升 最大反应。为了设计出第二代更强大的 类比，我们将有选择地引入带电残基簇， 存在于HTSH外周环内的同源激素中， 分开并以各种组合的形式。在目前的提案中，我们 将：1)在外周进行系统的多结构域突变 两个HTSH亚基的发夹环，以优化效力和疗效 2)表达和纯化大量最具活性的 模拟并表征它们的生化和生物学特性； 3)尝试确定HTSH受体相互作用的区域 带正电的残基在HTSH环内 超活性类似物；4)测试特定部位唾液酸的去除 酸性会增加受体结合和体外活性。 精选的HTSH类似物。这些研究应该为我们提供新的见解 促甲状腺激素的结构-功能关系及其研究新途径 促甲状腺激素受体激活的研究。高效重组hTSH 类似物在几种体外应用中应该有很大的价值 与受体结合或激活有关，应该有所改善 激发~(131)I摄取和甲状腺球蛋白的疗效 甲状腺癌和非毒性甲状腺肿患者的分泌物。高 亲和力HTSH类似物也可能具有独特的能力 甲状腺癌的靶向递送治疗 放射性核素、毒素、肿瘤抑制基因或钠/碘 符号转运蛋白基因。此外，他们的设计为 为胱氨酸的其他成员开发类似的类似物已知 生长因子超家族。最后，公共卫生的重要性 大量的新病例强调了这一建议。 欧洲发现异常侵袭性的儿童甲状腺癌 切尔诺贝利事故和预计美国将出现新的成人病例。 因为20世纪50年代在内华达州进行的核试验，S。

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.