HORMONE-BINDING DOMAINS OF LH/HCG RECEPTOR

促黄体激素受体（Lutropin receptor，LHR）属于严重跨膜受体超家族 结构域G偶联受体。 其长的胞外氨基末端 341-368个氨基酸残基的多肽具有高亲和力， 激素结合序列。 由于可以准备公平的 量的来自E.杆菌和 CHO细胞，它现在已经成为可行的进行其详细的 理化、免疫学和生物学表征。 的 这些研究的最终目标是尽可能精确地定义 受体的配体结合位点，目的是开发 人绒毛膜促性腺激素（hCG）的拮抗剂。 胞外结构域 将制备大鼠（LHR）（1-294和1-341残留物），单位为mg 数量从E。coli、CHO和SF-9昆虫细胞。 经过初步 大鼠LHR的重组氨基末端肽的表征 氨基酸组成、肽图谱和氨基和羧基末端 测序，（1）确定配体结合位点的研究 将进行。 一些方法，如使用删除和 通过定点诱变的截短突变体，使用蛋白水解 胞外结构域多肽和合成肽的片段 将绘制LHR的高亲和力激素结合位点。（二） LHR将通过研究一些 翻译后修饰，包括二硫键的位置 键和碳水化合物的详细结构， 来自CHO和SF-9昆虫细胞的胞外结构域。 (3)三 空间结构分析和尝试将结晶 氨基末端1-294和1-341残基多肽及其 硒代甲硫氨酸类似物。 加入硒可以解决这个问题 重金属离子同晶置换的x射线衍射 问题研究 (4)抑制细胞增殖的多克隆和单克隆抗体 hCG与受体的结合。 (5)最后，体外和体内 大鼠LHR胞外区的生物学特性 研究hCG对大鼠hCG升高的抑制作用 将进行子宫重量、排卵和孕酮合成。 这些研究将有助于评估 作为生育力的hCG拮抗剂的LHR细胞外多肽 调控 对大鼠LHR的初步研究将扩展到人类LHR。

Lutropin receptor (LHR) belongs to the superfamily of sever transmembrane domain G-coupled receptors. Its long extracellular aminoterminal polypeptide of 341-368 amino acid residues contains the high affinity hormone binding sequences. Since it has been possible to prepare fair amounts of the extracellular aminoterminal polypeptide from E. coli and CHO cells, it has now become feasible to undertake its detailed physicochemical, immunological and biological characterization. The ultimate goal of these studies is to define as precisely as possible the ligand binding site of the receptor with the objective of developing antagonists of human choriogonadotropin (hCG). The extracellular domain of rat (LHR) (1-294 and 1-341 residues) will be prepared in milligram quantities from E. coli, CHO and SF-9 insect cells. After initial characterization of the recombinant aminoterminal peptides of rat LHR by amino acid composition, peptide mapping and amino and carboxyterminal sequencing, (1) studies on the determination of the ligand binding site will be undertaken. Several approaches such as the use of deletion and truncation mutants by site directed mutagenesis, use of proteolytic fragments of the extracellular domain polypeptides and synthetic peptides will be made to map the high affinity hormone binding site of LHR. (2) The LHR will be further characterized by studying some of the posttranslational modifications including the positions of the disulfide bonds and the detailed structures of the carbohydrate unites in the extracellular domains from CHO and SF-9 insect cells. (3) For three dimensional structural analysis and attempt will be made to crystallized the aminoterminal 1-294 and 1-341 residue polypeptides as well as its selenomethionly analogs. Insertion of selenium will obviate the problem of isomorphous replacement by heavy metal ion in x-ray diffraction studies. (4) Polyclonal and monoclonal antibodies which inhibit the binding of hCG to the receptor. (5) Finally, the in vitro and in vivo biological characterization of the extracellular domain of rat LHR by studying the inhibition of hCG action in hCG induced increase in rat uterine weight, ovulation and progesterone synthesis will be carried out. These studies will help in the evaluation of the potential of the extracellular polypeptides of LHR as antagonists of hCG for fertility regulation. Initial studies on rat LHR will be extended to human LHR.