HCG STRUCTURE/FUNCTION RELATIONSHIPS

HCG 结构/功能关系

• 批准号: 2139204
• 负责人: J DAVID PUETT
• 金额: $ 27.08万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-09-01 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2139204
• 关键词: Sf9 cell line; animal genetic material tag; biological signal transduction; chorionic gonadotropin; circular dichroism; conformation; cyclic AMP; electron spin resonance spectroscopy; fluorescence microscopy; hormone receptor; human genetic material tag; luteinizing hormone; microcalorimetry; mutant; protein folding; protein sequence; protein structure function; radioimmunoassay; receptor binding; secretion; site directed mutagenesis; structural biology; thermodynamics; thermostability

The long-range goal of this project is elucidation at the molecular level of the structural and functional aspects of the two glycoprotein hormones, hCG and LH, and their common G protein-coupled receptor, LH/CGr. This research is guided by the hypotheses that (i) discrete regions of the alpha and beta subunits may function both in holoprotein formation and receptor binding, and (ii) conformation changes occur upon heterodimer formation and receptor binding. The work will be a continuation of current studies designed to identify and delineate amino acid residues and discrete regions of the alpha and beta subunits that are important in hormone function. Site-directed mutagenesis will be used to replace or delete certain amino acid residues and putative functional domains that are known or suspected to be involved in secretion, subunit assembly, receptor binding, and perhaps both subunit and receptor binding. A number of the beta replacements will be made in the Keutmann steroidogenic loop (residues 38-57), the Ward determinant loop (residues 93-lOO), and other regions identified as important in function. These mutant forms of hCG will be assayed in vitro via competitive binding and steroidogenesis. Differential trace labeling will be done with [3H]acetic anhydride to map the reactivities of amino groups in ovine LH and equine CG for comparison with hCG. A selected number of residues on LH/CGr will be replaced by site-directed mutagenesis and the mutants assayed for hormone binding and cAMP stimulation. These results will provide information on the roles of specific groups of the extracellular domain and extracellular connecting loops on hormone binding and the function of residues in the transmembrane helices and intercellular connecting loops on signal transduction. Physicochemical studies will be conducted on a few of the more interesting C-terminal deletion mutants, e.g. those where stable tertiary structure is forming, to gather data on their conformational aspects. The gonadotropins hCG and LH, acting through their common receptor, are required for gonadal steroidogenesis, ovulation, and maintenance of early pregnancy. Elucidation of their structure-function relationships may provide new insights into fertility regulation and the etiology of infertility.

该项目的长期目标是在分子水平上阐明 两种糖蛋白激素的结构和功能方面， hCG和LH及其共同的G蛋白偶联受体LH/CGr。这 研究是由以下假设指导的：（一）离散区域的 α和β亚基可以在全蛋白形成中起作用， 受体结合，以及（ii）异二聚体发生构象变化 形成和受体结合。这项工作将是当前 旨在鉴定和描述氨基酸残基的研究， α和β亚基的离散区域， 激素功能 将使用定点诱变来替代或 删除某些氨基酸残基和推定的功能结构域， 已知或怀疑参与分泌，亚单位组装， 受体结合，以及可能亚基和受体结合。一些 的β取代将在Keutmann类固醇生成环中进行 （残基38-57），Ward决定簇环（残基93- 100），和其他 在功能上被确定为重要的区域。hCG的这些突变形式 将通过竞争性结合和类固醇生成进行体外测定。 将使用[3 H]乙酸酐进行差异痕量标记，以绘制图谱 绵羊LH和马CG中氨基反应性的比较 hCG。LH/CGr上选定数量的残基将被替换为 定点诱变，测定突变体的激素结合， cAMP刺激。这些结果将提供有关以下作用的信息： 胞外结构域和胞外连接的特定基团 激素结合的环和跨膜中残基的功能 螺旋和细胞间连接环对信号转导的影响。 物理化学研究将在一些更有趣的 C-末端缺失突变体，例如其中稳定的三级结构被破坏的那些。 形成，以收集关于其构象方面的数据。促性腺激素 hCG和LH通过它们共同的受体起作用，是性腺发育所必需的。 类固醇生成、排卵和维持早期妊娠。 阐明它们的结构-功能关系可能提供新的 了解生育调节和不孕症的病因。