RESPONSIVENESS OF GNRH RECEPTOR SIGNALING

拟议研究的长期目标是调查对 促性腺激素释放激素受体信号的反应性。健康的重要性 这些研究表明，促性腺激素释放激素用于治疗糖尿病的机制 前列腺癌是基于其对黄体生成素分泌不敏感的倾向。 这反过来又减少了睾丸素的分泌。胞内蛋白质 已经受到审查的潜在监管机构 GnRH受体信号的反应性是GRK家族的成员(G 蛋白偶联受体激酶)和β拦阻蛋白1和2。GRKs 2，3， 和5以及β-arrestin 1和2在大鼠体内自然表达 垂体腺及其在促性腺激素释放激素中的实验表达 表达受体的COS-1细胞抑制促性腺激素释放激素的产生 IP3，是GnRH引起的第二信使级联中的一个成员。发展 一种新型的腺病毒方法在大鼠体内表达GRKs 脑垂体前叶细胞表现出强烈的抑制性。 因此，在拟议的研究中，这种创新性的腺病毒表达 将使用方法来调查GRK和Beta的程度 抑制素参与调节促性腺激素释放激素受体的反应水平 发信号。该提案的具体目标是：具体目标1： 确定GRK2、GRK3、β-arrestin 1和2单独和在 腺病毒介导基因联合应用于促性腺激素释放激素促黄体生成素分泌 在大鼠垂体细胞中的表达。具体目标2：调查消融或 内源性GRK2/3对GnRH受体反应性的中和作用 腺病毒介导的基因转移在垂体中的信号转导 促性腺激素：a)竞争性抑制GRK的多肽 GRKs)，以及；b)抑制GRK2/3合成的反义构建物 GRKS。具体目标3：确定外部因素(雌二醇、促性腺激素释放激素、 等)设置GnRH受体信号的反应性是通过 调节GRK 2、3、6和β拦阻蛋白的表达水平 1和2.具体目标4：调查GRK的地点和行动方式 抑制GnRH受体信号转导：A)测定IP3反应性 致敏和脱敏状态下的促性腺激素 GRK的作用部位发生在磷脂酶C.b)确定之前或之后 Adeno-GRK2和β-arrestin感染的促性腺激素分泌 2仍然对NaF处理有刺激作用，NaF是一种直接激活的试剂 G蛋白；3)寻找表位标记的GnRH的磷酸化 GRK2/β-arrestin 2受体在COS-1细胞中的表达 标记血管紧张素II受体作为阳性对照。

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.