MEMBRANE EVENTS FOLLOWING HORMONE BINDING TO LH RECEPTOR

促黄体生成素/绒毛膜促性腺激素（LH/CG）受体 黄体和Leydig细胞结合黄体生成素（LH）和人 绒毛膜促性腺激素（hCG）调节类固醇的合成和分泌 从这些腺体。 这个项目的目的是为了更好地了解 LH/CG受体结构在生育调节中的作用及其机制 促性腺激素可调节LH/CG受体功能，包括 G蛋白相互作用、受体内化和CAMP激活。 首先，我们将检查促性腺激素结合是否引起大小变化 在功能性LH受体复合物中使用两种激光光学方法， 测量蛋白质分子运动，偏振荧光损耗， 荧光光漂白恢复 单细胞荧光能量 转移测量将用于评估受体-受体 聚集在膜上。 第二，LH/CG结构对 受体分子运动将使用组织培养的细胞进行评估 携带C-末端截短的LH/CG受体。 配体结合 这些截短的受体导致CAMP激活增加， 快速内化，可能是由于更快的LH/CG受体外侧 在质膜中扩散。 三、去糖基化的影响 将在大鼠上评价hCG和天然hCG对LH/CG受体运动影响 Leydig细胞和MA-10细胞。 去糖基化hCG与 LH/CG受体减缓受体下调和内化， 可能是由于受体横向扩散较慢， 受体与内化所需蛋白质的相互作用。 最后， 包括G蛋白亚基在内的非受体蛋白的结合， MHC I类抗原与LH/CG受体将通过 光邻近标记技术，其允许识别和 分离物理上接近已知膜位点的蛋白质。 科罗拉多州立大学有一个不寻常的专业知识融合， 生殖生理学和膜的仪器设备 结构研究。 这种情况为我们提供了一个独特的机会， 研究LH受体的组织以及这种组织如何调节 受体功能

Luteinizing hormone/chorionic gonadotropic (LH/CG) receptors on the corpus luteum and on Leydig cells bind luteinizing hormone (LH) and human chorionic gonadotropin (hCG) to regulate steroid synthesis and secretion from these glands. The goal of this project is to better understand the role of LH/CG receptor structure in regulation of fertility and how gonadotropins may modulate events in LH/CG receptor function including G-protein interactions, receptor internalization and CAMP activation. First, we will examine whether gonadotropin binding causes size changes in functional LH receptor complexes using two laser optical methods for measuring protein molecular motions, polarized fluorescence depletion and fluorescence photobleaching recovery. Single-cell fluorescence energy transfer measurements will be used to assess receptor-receptor aggregation on the membrane. Second, the effect of LH/CG structure on receptor molecular motions will be assessed using tissue-cultured cells bearing LH/CG receptors with C-terminal truncations. Ligand binding to these truncated receptors results in increased CAMP activation and more rapid internalization, perhaps due to more rapid LH/CG receptor lateral diffusion in the plasma membrane. Third, the effects of deglycosylated hCG and native hCG on LH/CG receptor motions will be evaluated on rat Leydig cells and on MA-10 cells. Binding of deglycosylated hCG to the LH/CG receptor slows receptor down-regulation and internalization, effects that may result from slower receptor lateral diffusion and fewer receptor interactions with proteins needed for internalization. Finally, the association of non-receptor proteins including G-protein subunits and MHC class I antigen with LH/CG receptors will be explored by a photoproximity labeling technique that permits identification and isolation of proteins physically proximal to a known membrane site. Colorado State University has an unusual confluence of expertise is reproductive physiology and instrumental facilities for membrane structural studies. This situation provides us a unique opportunity to study both LH receptor organization and how this organization modulates receptor function.