MOLECULAR CHARACTERIZATION OF GNRH RECEPTORS

促性腺激素释放激素(GnRH)是一种下丘脑十肽类物质。 生殖过程的关键神经调节器。它的受体位于 在垂体前叶促性腺激素的细胞膜中 黄体生成素和卵泡刺激素的释放 (FSH)。除垂体前叶外，GnRH受体还被报道为 存在于卵巢、睾丸、胎盘和脑中。近年来， 许多研究人员还在不同的人类体内发现了GnRH受体 肿瘤(乳腺、结肠、前列腺、卵巢、子宫和胰腺)，而 在正常情况下，这些组织中不会检测到GnRH受体。 然而，促性腺激素释放激素受体在癌症中的作用尚不清楚。宽阔的，长的- 拟议研究的长期目标是探讨 促性腺激素释放黄体生成素以及促性腺激素释放激素受体在癌症中的作用。 更具体地说，对于这次拨款申请，我们的努力将集中在 克隆的促性腺激素释放激素受体(S)的分子特征 人前叶垂体腺及其在正常和 恶性组织。我们从一个人类前部分离出一个克隆 该文库由1557个核苷酸组成。它将编码 一种与其他G蛋白偶联的7-G蛋白具有相似拓扑结构的蛋白质 跨膜受体。克隆受体的结合研究 显示出高亲和力和类似于的药理特性 天然的人类脑垂体促性腺激素释放激素受体。人的Northern印迹分析 垂体RNA显示3个hGnRH受体mRNAs的存在 对人类基因组DNA的印迹分析表明，存在不止一个 GnRH受体基因。逆转录酶/聚合酶链式反应分析揭示 受体基因在脑下垂体、卵巢、睾丸、肾上腺、 乳腺、乳腺肿瘤、前列腺、前列腺肿瘤和乳腺肿瘤细胞系 (MCF-7)。该提案的具体目标是：克隆、测序、 表达和鉴定所有三种mRNAs的GnRH受体cDNA； 克隆、测序并鉴定编码GnRH-1的所有基因 受体；测定细胞中各受体mRNA的存在和数量 正常和恶性组织；制备抗该基因的单抗 受体(S)用于检测和测量受体数量 乳腺癌和前列腺癌中的蛋白质；研究单抗的作用 乳腺和前列腺肿瘤细胞在培养和培养中生长的抗体 定位染色体上每个受体(S)的基因。这些研究 应提供有关GnRH潜在作用的广泛新信息- 激素依赖性癌症中的受体。

Gonadotropin-releasing hormone (GnRH), a hypothalamic decapeptide, is the key neural regulator of the reproductive process. Its receptor is located in the cell membrane of anterior pituitary gonadotropes where it mediates the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH). In addition to anterior pituitary, GnRH receptors are reported to be present in the ovary, testis, placenta and brain. In recent years, numerous investigators also have found GnRH receptors in various human tumors (breast, colon, prostate, ovarian, uterine and pancreatic), whereas under normal conditions GnRH receptors are not detected in these tissues. However, the role of GnRH receptors in cancer is unclear. The broad, long- term objective of the proposed studies is to investigate the mechanism of LH release from gonadotropes, and the role of GnRH receptors in cancer. More specifically, for this grant application our efforts will be directed toward the molecular characterization of GnRH receptors(s) cloned from the human anterior pituitary gland and their regulation in normal and malignant tissues. We have isolated a clone from a human anterior pituitary cDNA library which is composed of 1557 nucleotides. It encodes a protein with topology similar to that of other G-protein coupled 7- transmembrane receptors. Binding studies of the cloned receptor demonstrate a high affinity and pharmacological properties similar with the native human pituitary GnRH receptor. Northern blot analysis of human pituitary RNA reveals the presence of 3 hGnRH-receptor mRNAs and Southern blot analysis of human genomic DNA suggests the existence of more than one GnRH receptor gene. Reverse transcriptase/PCR analysis of RNA revealed that the receptor mRNA is expressed in pituitary, ovary, testis, adrenal, breast, breast tumor, prostate, prostate tumor, and breast tumor cell line (MCF-7). The specific aims of the proposal are to: clone, sequence, express and characterize GnRH receptor cDNAs for all three mRNAs; to clone, sequence, and characterize all the genes encoding the GnRH- receptors; to determine the presence and amount of each receptor mRNA in normal and malignant tissues; to prepare monoclonal antibodies to the receptor(s) for detection and measurement of the amount of receptor protein in breast and prostate cancers; to study the effects of monoclonal antibodies on growth of breast and prostate tumor cells in culture and; to localize the gene for each receptor(s) on the chromosome. These studies should provide extensive new information about the potential role of GnRH- receptors in hormonally-dependent cancers.