STRUCTURE AND CELLULAR BIOLOGY OF GPH RECEPTORS

GPH 受体的结构和细胞生物学

• 批准号: 2888888
• 负责人: JAMES Alan DIAS
• 金额: $ 20.45万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2888888
• 关键词: RNase protection assay; SDS polyacrylamide gel electrophoresis; chemical models; circular dichroism; confocal scanning microscopy; conformation; enzyme linked immunosorbent assay; epitope mapping; flow cytometry; follicle stimulating hormone; gonadotropins; hormone receptor; laboratory rabbit; mass spectrometry; molecular site; monoclonal antibody; posttranslational modifications; protein structure function; radionuclides; receptor binding; receptor expression; site directed mutagenesis; tissue /cell culture; western blottings; yeast two hybrid system

The pituitary/placental glycoprotein hormone receptor (GPHR) fancy includes follicle stimulating hormone receptor (FSHR), luteinizing hormone receptor, thyroid stimulating hormone receptor and chorionic gonadotropin receptor. Glycoprotein hormones control reproduction, sexual development and thyroid function. Studies conducted during the previous grant period focused on the structure-activity-relationships of hFSH. We have obtained direct structural information about amino acids which constitute the FSH residues involved in FSH/FSH-receptor interaction and hFSHalpha/hFSHbeta association. Together, the solved crystal structure of hCG and the hFSH mutagenesis studies have provided a better understanding of the glycoprotein hormone-receptor interaction. In this application, we turn our attention towards elucidation of the hormone binding site of the receptor and towards a better understanding of the cell biology and physiology of the receptor. A detailed knowledge of liganded receptor structure is needed and will complete understanding of the diverse physiological processes which this family of receptors control. Our major focus is to use site directed mutagenesis of FSHR to provide fine resolution of principal residues critical to stabilization of the hormone-receptor complex. Models of the GPHR are now available and will be used as a conceptual guide to assess experimental outcomes. The tertiary structure of the GPHR will also be studied by immunochemical methods which we have previously used to appraise the assembled epitopes of hFSH .Biophysical approaches will be used to study the hFSHR, identifying postranslational modifications, including disulfide bond assignments. We have previously described the cell surface distribution of hFSHR and this work will be extended using confocal microscopy to directly analyze the cell surface distribution of the receptors during the activation process. Desensitization of the GPHR is correlated with phosphorylation of cytoplasmic residues of the GPHR and the proteins involved in this mechanism of regulation of gonadal cellular responsiveness to ligand will be identified using an interaction trap ("yeast two hybrid system") method.

垂体/胎盘糖蛋白激素受体（GPHR）花哨 包括卵泡刺激激素受体（FSHR），黄体生成 激素受体，甲状腺刺激激素受体和绒毛膜 促性腺激素受体。糖蛋白激素控制繁殖，性 发育和甲状腺功能。 在上一个赠款期间进行的研究重点是 HFSH的结构活动关系。我们已经直接获得了 有关构成FSH的氨基酸的结构信息 涉及FSH/FSH受体相互作用和HFSHALPHA/HFSHBETA的残留物 协会。一起，HCG和HFSH的溶液晶体结构 诱变研究提供了更好的了解 糖蛋白激素受体相互作用。在此应用程序中，我们转身 我们注意阐明激素结合位点的 受体并更好地了解细胞生物学和 受体的生理学。 需要对配体受体结构的详细知识，并将 完全了解这 受体家族控制。我们的主要重点是使用定向网站 FSHR的诱变可很好地解决主要残基 对激素受体复合物的稳定至关重要。模型 GPHR现在可用，将用作评估的概念指南 实验结果。 GPHR的三级结构也将是 通过免疫化学方法研究，我们以前曾经用过 评估HFSH的组装表位。 用于研究HFSHR，识别后倾斜修饰， 包括二硫键分配。我们以前已经描述了 HFSHR的细胞表面分布和这项工作将使用 共聚焦显微镜直接分析细胞表面分布 激活过程中的受体。 GPHR的脱敏 与GPHR的细胞质残基的磷酸化相关 以及这种调节性腺调节机制涉及的蛋白质 使用相互作用确定对配体的细胞反应性 陷阱（“酵母两个混合系统”）方法。