ROLE OF BETA ARRESTIN IN LH/CG RECEPTOR DESENSITIZATION

与七螺旋促黄体激素/绒毛膜促性腺激素结合的激动剂 受体（LH/CG R）激活刺激性鸟嘌呤核苷酸 结合蛋白（Gs）和下游腺苷酸环化酶（AC）。LH/CG R随后表现出降低的腺苷酸依赖性AC活性，或 对饱和激动剂反应的脱敏。与脱敏不同， 许多G蛋白偶联的R，LH/CG R的体内脱敏反应， 在排卵前LH峰的卵巢卵泡中， 无细胞条件因为卵泡膜LH/CG R脱敏 在不添加任何 外源蛋白，研究人员可以研究参与的机制， LH/CG R脱敏在无并发症的未扰动膜中 与R的内化和循环有关。使用该模型， 研究人员已经表明，LH/CG R刺激的AC活性的脱敏 需要GTP，并被阻止G蛋白的GDP类似物逆转 激活，LH/CG R磷酸化不是LH/CG R必需的 脱敏。研究人员证明， 含有β-抑制蛋白的膜， 特别是阻止脱敏的发展。添加重组 纯化的β-arrestin模拟hCG促进LH/CG R脱敏。一 合成肽对应于第三胞内（3 i）环的 LH/CG R特异性地阻止该肽的脱敏和饱和 用外源性β-抑制蛋白恢复脱敏。这些结果导致 研究人员假设β-抑制蛋白参与了 LH/CG R的激动剂依赖性脱敏， 通过与LH/CG R的3 i环结合，以磷酸化非依赖性方式， 防止G的激活。这些目标验证了这一核心假设。目标1将 测试β-抑制蛋白与血浆紧密且独特结合的假设 卵泡膜，可能以FSH依赖的方式。目标2将 测试假设，β-抑制蛋白的结合活性，但 非磷酸化LH/CG R是必需的，足以介导LH/CG R 脱敏目标3将检验以下假设： LH/CG R与β-抑制蛋白依赖性LH/CG R寡聚化相关。目的 4将检验LH/CG R中的GTP依赖性步骤 脱敏包括从细胞膜释放β-抑制蛋白 对接点这些研究的结果将提供关于 LH/CG R的信号传导被淬灭， 生殖周期提前，并可能允许发展 停止生殖周期或减少过度活跃的活性的试剂 R的。

Agonist binding to the heptahelical luteinizing hormone/choriogonadotropin receptor (LH/CG R) activates the stimulatory guanine nucleotide binding protein (Gs) and downstream adenylyl cyclase (AC). The LH/CG R subsequently exhibits reduced hormone-dependent AC activity or desensitization in response to saturating agonist. Unlike desensitization of many G-protein coupled R's, the in vivo desensitization response of the LH/CG R in ovarian follicles to the preovulatory LH surge can be mimicked under cell-free conditions. Because LH/CG R desensitization in follicular membranes exhibits physiologically relevant kinetics without the addition of any exogenous proteins, the investigators can study the mechanisms involved in LH/CG R desensitization in an unperturbed membrane without complications associated with R internalization and recycling. Using this model the investigators have shown that desensitization of LH/CG R-stimulated AC activity requires GTP and is reversed by a GDP analog that prevents G protein activation, and that LH/CG R phosphorylation is not obligatory for LH/CG R desensitization. The investigators demonstrated that purified follicular membranes contain beta-arrestin and that neutralizing anti-arrestin antibodies specifically block development of desensitization. Addition of recombinant purified beta-arrestin mimicked hCG to promote LH/CG R desensitization. A synthetic peptide corresponding to the third intracellular (3i) loop of the LH/CG R specifically prevented desensitization and saturation of this peptide with exogenous beta-arrestin revived desensitization. These results lead the investigators to hypothesize that beta-arrestin participates in agonist-dependent desensitization of the LH/CG R in an apparent phosphorylation-independent manner by binding to the 3i loop of the LH/CG R to prevent activation of Gs. The aims test this central hypothesis. Aim 1 will test the hypothesis that beta-arrestin binds tightly and uniquely to the plasma membrane of ovarian follicles, perhaps in an FSH dependent manner. Aim 2 will test the hypothesis that the binding of beta-arrestin to the active but unphosphorylated LH/CG R is necessary and sufficient to mediate LH/CG R desensitization. Aim 3 will test the hypothesis that desensitization of the LH/CG R is associated with beta-arrestin-dependent LH/CG R oligomerization. Aim 4 will test the hypothesis that the GTP-dependent step in LH/CG R desensitization consists of the release of beta-arrestin from a membrane docking site. Results from these studies will provide precise knowledge of the cellular mechanisms by which signaling from the LH/CG R is quenched to allow the reproductive cycle to advance and will likely permit development of reagents to halt the reproductive cycle or to reduce the activity of overactive R's.