ROLE OF BETA ARRESTIN IN LH/CG RECEPTOR DESENSITIZATION

激动剂与七螺旋黄体生成素/绒毛膜促性腺激素结合 黄体生成素/胆碱能受体激活刺激性鸟嘌呤核苷酸 结合蛋白(Gs)和下游的腺酰环化酶(AC)。黄体生成素/卵泡刺激素 R随后表现出激素依赖性AC活性降低或 对饱和激动剂的反应是脱敏。与脱敏不同 多个G蛋白偶联受体--黄体生成素/促性腺激素受体的体内脱敏反应 在卵泡到排卵前的黄体生成素高峰下可以模拟 无细胞条件。因为卵泡膜中的黄体生成素/促性腺激素受体脱敏 显示出与生理相关的动力学，而不添加任何 外源蛋白，研究人员可以研究其参与的机制 无干扰膜中的黄体生成素/促性腺激素受体脱敏反应 与R内化和循环有关。使用此模型， 研究表明，黄体生成素/促性腺激素受体刺激的AC活性的脱敏 需要GTP，并被一种防止G蛋白的GDP类似物逆转 激活，并且黄体生成素/卵泡刺激素受体的磷酸化不是促黄体生成素/卵泡刺激素受体必需的。 脱敏。研究人员证明，纯化的卵泡 含有β-arrestin和中和抗arrestin抗体的膜 具体地说，阻断脱敏作用的发展。重组人的添加 纯化的β-arrestin模拟hCG促进Lh/CG R脱敏。一个 与细胞内(3I)环相对应的合成肽 L H/CG R特异性地阻止了该多肽的脱敏和饱和 用外源性β-arrestin复苏性脱敏。这些结果领先于 调查人员假设β-arrestin参与了 激动剂依赖的黄体生成素/卵泡刺激素受体脱敏 通过与LH/CG R的3I环结合而不依赖于磷酸化的方式 防止激活Gs。AIMS对这一核心假设进行了检验。目标1将 验证β-arrestin与血浆紧密且唯一结合的假设 卵泡膜，可能是以FSH依赖的方式。目标2将 检验β-arrestin与活性BUT结合的假设 非磷酸化的黄体生成素/卵泡刺激素受体是介导黄体生成素/卵黄素受体的必要条件和充分条件 脱敏。目标3将检验这样一种假设，即 促黄体生成素/促性腺激素受体与依赖β-精氨酸的促黄体生成素/促性腺激素受体寡聚有关。目标 4将检验以下假设：促黄体生成素/cGR中依赖GTP的步骤 脱敏作用包括膜上β-arrestin的释放。 对接地点。这些研究的结果将提供关于 来自黄体生成素/黄体生成素受体的信号被猝灭的细胞机制 生殖周期将提前，并可能允许发展 停止生殖周期或减少过度活动的试剂 R‘s。

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.