BIOACTIVE FSH AND REPRODUCTION

LH和FSH受体属于G蛋白偶联受体亚家族， 受体具有七个跨膜区和一个大的细胞外 富含亮氨酸的重复序列。我们对人类的分析 促性腺激素受体允许表征的增益和损失的， 家族性男性性早熟患者的功能突变， Leydig细胞发育不全。同时，两种受体的共表达 配体结合或信号转导缺陷恢复配体 信号人类的受体，不像啮齿类动物。有 独特的物种特异性配体识别特性。早期表达 促性腺激素受体的细胞外区域表明， 结构域保留了配体结合能力，但仍被困在 转染细胞这一现象阻碍了对它们在以下方面的作用的分析： 信号转导，并阻止它们的wet作为细胞特异性 结合蛋白我们最近证明细胞外区域 LH和FSH受体可以锚定在质膜上， 保留配体结合能力。我们现在建议将 锚定促性腺激素受体及其机制 锚定受体与突变体共转染后的活化 含有信号转导结构域的受体。使用锚定 受体，我们将确定最小的肽序列和N-连接 配体识别所需的碳水化合物。我们还插入了一个 锚定受体中的凝血酶切割位点， 切割和产生可溶性受体片段， 放射性配体受体测定中的竞争。我们将测试 这些受体的可溶性配体结合域， 在体外中和LH或FSH的作用。使用杆状病毒表达 系统，我们建议获得大量的可溶性LH和FSH受体 用于作为受体拮抗剂或结合蛋白测试的片段 vivo.本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作用的截短受体 循环LH和FSH在动物和患者。

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.