BIOACTIVE FSH AND REPRODUCTION

黄体生成素和卵泡刺激素受体属于G蛋白偶联亚家族 具有七个跨膜区和一个大的胞外受体 含有富含亮氨酸重复序列的结构域。我们对人类的分析 促性腺激素受体允许表征-获得和失去- 家族性男性性早熟和家族性早熟患者的功能突变 间质细胞发育不良。另外，两种受体的共表达 配基结合或信号转导缺陷可恢复配基 发信号。与啮齿动物的受体不同，人类的受体。有 独特的物种特定配基识别特性。较早的表达式 促性腺激素受体胞外区的研究表明 结构域保留配体结合能力，但仍被困在内部 转基因细胞。这一现象阻碍了对它们在 信号转导，并防止它们的WE作为激素特异性的 结合蛋白。我们最近证明了细胞外区 黄体生成素受体和卵泡刺激素受体可与质膜结合 保留配基结合能力。我们现在建议将其描述为 锚定的促性腺激素受体及其作用机制 锚定受体与突变体共转染后的激活 含有信号转导结构域的受体。使用定位 受体，我们将确定最小的多肽序列和N-连接 配基识别所需的碳水化合物。我们进一步插入了一个 锚定受体中的凝血酶裂解部位允许特定的酶 可溶性受体片段的切割和产生 放射配基受体分析中的竞争。我们将测试它的能力 这些受体的可溶性配体结合域的特异性 在体外中和促黄体生成素或促卵泡刺激素的作用。使用杆状病毒表达 系统中，我们建议获得大量的可溶性黄体生成素和卵泡刺激素受体。 用于检测为受体拮抗剂或结合蛋白的片段 活着。这项建议将进一步加深我们对 促性腺激素激活其受体的机制 产生能够选择性拮抗这些作用的截断受体 动物和患者循环中的促黄体生成素和促卵泡刺激素。

LH and FSH receptors belong to a sub-family of G protein-coupled receptors with seven transmembrane regions and a large extracellular domain containing leucine-rich repeats. Our analysis of human gonadotropin receptors has allowed characterization of gain- and loss-of- function mutations in patients with familial male precocious puberty and Leydig cell hypoplasia, respectively. Also, coexpression of two receptors defective in either ligand binding or signal transduction restores ligand signaling. The human receptors, unlike their rodent counterparts. have unique species-specific ligand recognition properties. Earlier expression of extracellular regions of gonadotropin receptors indicated that this domain retains ligand binding ability, but remains trapped inside transfected cells. This phenomenon hampers analysis of their role in signal transduction and prevents the we of them as hormone-specific binding proteins. We recently demonstrated that the extracellular region of LH and FSH receptors can be anchored to the plasma membrane with retention of ligand binding capacity. We now propose to characterize the anchored gonadotropin receptors and the mechanisms underlying receptor activation following co-transfection of anchored receptors with mutant receptors containing the signal transduction domain. Using anchored receptors, we will determine minimal peptide sequences and N-linked carbohydrates needed for ligand recognition. We further inserted a thrombin cleavage site in anchored receptors to allow specific enzyme cleavage and generation of soluble receptor fragments capable of competition in the radioligand receptor assay. We will test the ability of the soluble, ligand binding domain of these receptors to specifically neutralize LH or FSH actions in vitro. Using a Baculovirus expression system, we propose to obtain large amounts-of soluble LH and FSH receptor fragments for testing as receptor antagonists or binding proteins in vivo. The present proposal will further our understanding of the mechanism by which gonadotropins activate their receptors and should yield truncated receptors capable of selectively antagonizing the actions of circulating LH and FSH in animals and patients.