MEMBRANE EVENTS FOLLOWING HORMONE BINDING TO LH RECEPTOR

促黄体生成素/绒毛膜促性腺激素受体 黄体和间质细胞结合黄体激素和人 绒毛膜促性腺激素(HCG)调节类固醇合成和分泌 从这些腺体中。本项目的目标是更好地了解 黄体生成素/卵泡刺激素受体结构在生育调节中的作用及其机制 促性腺激素可能调节黄体生成素/卵泡刺激素受体功能的事件，包括 G蛋白相互作用、受体内化和cAMP激活。 首先，我们将研究促性腺激素结合是否会导致大小改变。 在功能性黄体生成素受体复合体中使用两种激光光学方法 测量蛋白质分子运动、偏振荧光耗尽和 荧光光漂白回收。单细胞荧光能量 转移测量将用于评估受体-受体 膜上的聚集。第二，黄体生成素/卵泡刺激素的结构对 受体分子运动将使用组织培养细胞进行评估 携带带有C端截断的黄体生成素/卵泡刺激素受体。配体结合到 这些被截断的受体导致cAMP激活增加 快速内化，可能是由于更快的黄体生成素/卵泡刺激素受体侧向 在质膜中扩散。第三，脱糖基的影响 人绒毛膜促性腺激素和天然人绒毛膜促性腺激素对大鼠黄体生成素/卵泡刺激素受体运动的影响 间质细胞和MA-10细胞。脱糖人绒毛膜促性腺激素与细胞 黄体生成素/卵泡刺激素受体减缓受体下调和内化， 可能由较慢的受体横向扩散和较少的 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与 具有促黄体生成素/卵泡刺激素受体的MHC I类抗原将通过 光邻近标记技术，允许识别和 分离已知膜位置附近的蛋白质。 科罗拉多州立大学拥有不同寻常的专业知识汇聚 膜的生殖生理和仪器设备 结构研究。这种情况为我们提供了一个独特的机会 研究黄体生成素受体组织以及该组织如何调节 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.