CHEMISTRY AND BIOLOGY OF HUMAN AND OTHER GONADOTROPINS

人类和其他促性腺激素的化学和生物学

• 批准号: 2196629
• 负责人: OM P BAHL
• 金额: $ 19.28万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-12-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2196629
• 关键词: Baculoviridae; G protein; adenylate cyclase; biological signal transduction; carbohydrate structure; chemical structure function; chemical substitution; chorionic gonadotropin; clearance rate; crystallization; glycoprotein structure; glycosylation; hormone receptor; hormone regulation /control mechanism; immunoaffinity chromatography; laboratory mouse; laboratory rabbit; laboratory rat; luteinizing hormone; molecular site; mutant; ovulation; phospholipase C; protein purification; protein reconstitution; protein sequence; recombinant DNA; selenomethionine; tissue /cell culture; transfection; transfection /expression vector; tunicamycin

The proposed studies relating to the structure and function relationships in human choriogonadotropin (hCG) and its receptor are a continuation of our ongoing studies. HCG and its subunits and the rat lutropin receptor have been expressed in an heterologous eukaryotic expression system, baculovirus-insect cell system. The hormone and its subunits were expressed in a high efficiency and could be purified by immunoaffinity chromatography. Since the recombinant hormones are similar to the native hormone and its subunits in immunological and biological properties, they can replace the native urinary hormones in all immunodiagnostic applications. More importantly, it has been possible to substitute selenomethionine for methionine in hCG. The recombinant selenomethionyl hCG has considerable potential in the determination of its three dimensional structure. The recombinant LH/hCG receptor expressed in baculovirus system occurs predominantly as a dimer. The specific aims of the present studies are twofold. I. Studies on the structure and function relationships of gonadotropins will include the preparation and characterization of: (1) Recombinant selenomethionyl hCG on a large scale and to attempt to obtain x-ray diffraction grade crystals for three dimensional structural analysis. (2) Recombinant unglycosylated hCG and its subunits to resolve the question of the carbohydrate role in the function of hCG and selenomethionyl unglycosylated hCG. (3) The polypeptide and carbohydrate chains of recombinant hCG and its subunits. (4) Recombinant hCG, hCGalpha and hCGbeta on a large scale for further evaluation of their in vivo biological properties. (5) Carbohydrate mutants of hCGalpha and hCGbeta in baculovirus expression system to evaluate the contribution of individual N-linked carbohydrates to the function of hCG. II. Studies on the structure and function of rat LH/hCG receptor will include the preparation and characterization of: (1) Recombinant rat LH/hCG receptor. (2)Recombinant 1-294 amino acid residue extracellular polypeptide and its selenomethionyl analog. (3) Various deletion mutants of LH/hCG receptor in insect cells in order to delineate the ligand binding domain of the receptor and (4) Delineation of the hormone and Gs binding sites of the LH/hCG receptor and the possible involvement of extracellular and cytoplasmic loops in the ligand or Gs binding respectively. (5) Homologous reconstitution of LH receptor with GTP binding protein (Gs) and with Gs and adenylate cyclase or phospholipase C from rat ovarian membranes in liposomes to study the role of carbohydrate in the hormone and the receptor in the mechanism of signal transduction. All of the above studies should enhance our understanding of the regulation of human fertility and thus facilitate the development of safer methods of contraception.

关于结构和功能的拟议研究 人绒毛膜促性腺激素（hCG）及其受体之间的关系是一个 继续我们正在进行的研究。 人绒毛膜促性腺激素及其亚单位与大鼠 促黄体激素受体已经在异源真核细胞中表达， 表达系统、杆状病毒-昆虫细胞系统。 激素及其 亚基以高效率表达，并可通过 免疫亲和层析 由于重组激素是 类似于免疫学和免疫学中的天然激素及其亚基 生物学特性，它们可以取代天然的尿激素， 所有免疫诊断应用。 更重要的是， 有可能用硒代蛋氨酸取代hCG中的蛋氨酸。 的 重组硒甲硫酰hCG在免疫治疗中具有相当大的潜力， 确定其三维结构。 重组 在杆状病毒系统中表达的LH/hCG受体主要作为 二聚体。 本研究的具体目标有两个方面。I. 促性腺激素结构与功能关系的研究 将包括制备和表征：（1）重组 硒甲硫酰hCG的大规模生产，并试图获得X射线 用于三维结构分析的衍射级晶体。 (2)重组非糖基化hCG及其亚单位，以解决 碳水化合物在hCG功能中的作用问题， 硒甲硫酰非糖基化hCG。(3)多肽和碳水化合物 重组人绒毛膜促性腺激素及其亚单位的链。(4)重组hCG， 以进一步评价它们在 体内生物学特性(5)hCG α的碳水化合物突变体和 hCG β在杆状病毒表达系统中的作用，以评估 单个N-连接的碳水化合物对hCG的功能。二.研究 大鼠LH/hCG受体的结构和功能将包括 制备和表征：（1）重组大鼠LH/hCG 受体的（2）重组1-294氨基酸残基胞外 多肽及其硒代甲硫酰类似物。 (3)各种删除 LH/hCG受体的突变体，以描绘 受体的配体结合结构域和（4）激素的描绘 LH/hCG受体的Gs结合位点和可能的参与 细胞外和细胞质环的配体或Gs结合 分别 (5)LH受体与GTP的同源重组 结合蛋白（Gs）和Gs与腺苷酸环化酶或磷脂酶 从大鼠卵巢膜中研究C脂质体的作用 糖在激素和受体信号机制中的作用 转导 以上的研究都应该增进我们的理解 调节人类生育力，从而促进发展 更安全的避孕方法。