RESPONSIVENESS OF GNRH RECEPTOR SIGNALING

GNRH 受体信号传导的反应性

• 批准号: 2740071
• 负责人: JIMMY D NEILL
• 金额: $ 22.2万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-12-15 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2740071
• 关键词: G protein; RNase protection assay; antisense nucleic acid; arrestins; enzyme inhibitors; enzyme linked immunosorbent assay; estradiol; gene expression; gonadotropin releasing factor; hormone receptor; hormone regulation /control mechanism; hypothalamic pituitary axis; inositol phosphates; laboratory rat; luteinizing hormone; phospholipase C; phosphorylation; pituitary gonadal axis; protein kinase; receptor coupling; secretion; tissue /cell culture

The long-term aim of the proposed studies is to investigate regulation of the responsiveness of GnRH receptor signaling. The health importance of these studies is that the mechanism of GnRH's use as a treatment of prostate cancer is based on its propensity to desensitize LH secretion which, in turn, reduced testosterone secretion. Intracellular proteins that have come under scrutiny has potential regulators of the responsiveness of GnRH receptor signaling are members of the GRK family (G protein coupled receptor kinases) and beta arrestins 1 and 2. GRKs 2, 3, and 5 and beta arrestin 1 and 2 are expressed naturally in the rat anterior pituitary gland, and their experimental expression in GnRH receptor expressing COS-1 cells suppresses GnRH-stimulated production of IP3, a member of the second messenger cascade evoke by GnRH. Development of an innovative adenoviral approach for expression of GRKs in rat anterior pituitary cells has shown that they are strongly inhibitory. Therefore, in the proposed studies, this innovative adenoviral expression approach will be used to probe the extent to which the GRKs and beta arrestins participate in setting the responsiveness level of GnRH receptor signaling. The Specific Aims of the proposal are: Specific Aim 1: Determine the effects of GRK2, GRK3, beta arrestin 1 and 2 alone and in combination on GnRH-stimulated LH secretion using adenoviral-mediated gene expression in rat pituitary cells. Specific Aim 2: Investigate ablation or neutralization of endogenous GRK2/3 on responsiveness of GnRH receptor signaling using adenoviral-mediated gene transfer in pituitary gonadotropes of: a) a peptide GRK inhibitor that competitively inhibits GRKs), and; b) a GRK 2/3 antisense construct that suppresses synthesis of GRKs. Specific Aim 3: Determine if the external factors (estradiol, GnRH, etc.) that set responsiveness of GnRH receptor signaling do so by regulating the level of expression of GRK 2, 3, and 6 and beta arrestins 1 and 2. Specific Aim 4: Investigate GRKs' site and mode of action and inhibiting GnRH receptor signaling: a) Measure IP3-responsiveness of gonadotropes during sensitized and desensitized states to determine if GRK's site of action occurs before or after phospholipase C. b) Determine if LH secretion in gonadotropes infected with adeno-GRK2 and beta arrestin 2 remains stimulable to NaF treatment, a reagent that directly activated G proteins; 3) Look for phosphorylation of the epitope-tagged GnRH receptor in GRK2/beta arrestin 2 expressing COS-1 cells using epitope- tagged angiotensin II receptor as a positive control.

拟议研究的长期目的是调查调节 GnRH受体信号传导的响应能力。健康的重要性 这些研究是GnRH用作治疗的机制 前列腺癌基于其脱敏LH分泌的倾向 反过来，这减少了睾丸激素的分泌。细胞内蛋白 受到审查的可能是 GNRH受体信号的响应能力是GRK家族的成员（G 蛋白偶联受体激酶）和β抑制蛋白1和2。grks2，3， 5和β抑制素1和2自然在大鼠中表达 前垂体及其在GnRH中的实验表达 表达COS-1细胞的受体抑制GNRH刺激的产生 ip3，第二使者级联的成员GNRH唤起了GNRH。发展 一种创新的腺病毒方法，以表达大鼠的GRK 前垂体细胞表明它们是强烈抑制性的。 因此，在拟议的研究中，这种创新的腺病毒表达 方法将用于探测GRK和Beta的程度 逮捕蛋白参与设定GNRH受体的响应水平 信号。该提案的具体目的是：特定目标1： 单独确定GRK2，GRK3，Beta抑制蛋白1和2的影响 使用腺病毒介导的基因组合GnRH刺激的LH分泌 大鼠垂体细胞中的表达。特定目标2：调查消融或 内源性GRK2/3的中和GnRH受体反应性 使用垂体中腺病毒介导的基因转移的信号传导 促性腺激素：a）竞争性抑制的肽GRK抑制剂 grks），; b）抑制合成的GRK 2/3反义构建体 Grks。特定目标3：确定外部因素（雌二醇，gnrh， 等等）设定GNRH受体信号的响应能力是这样做的 调节GRK 2、3和6的表达水平以及β抑制蛋白 1和2。特定目标4：调查GRKS的站点和行动方式， 抑制GNRH受体信号：a）测量IP3的反应性 在敏化和脱敏状态期间的促性腺激素，以确定是否是否 GRK的作用部位发生在磷脂酶C之前或之后。B）确定 如果LH分泌在感染Adeno-Grk2和Beta逮捕的促性腺激素中 2仍然可以刺激NAF治疗，该试剂直接激活 G蛋白； 3）寻找表位标记的GnRH的磷酸化 GRK2/beta抑制蛋白2中的受体使用表位 - 表达COS-1细胞 标记血管紧张素II受体作为阳性对照。