FSH REGULATION OF A-KINASE ANCHORING PROTEINS

FSH 对 A 激酶锚定蛋白的调节

• 批准号: 2205631
• 负责人: DANIEL W CARR
• 金额: $ 11.53万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-02-01 至 2000-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2205631
• 关键词: SDS polyacrylamide gel electrophoresis; antisense nucleic acid; binding proteins; biological signal transduction; cell adhesion molecules; cell differentiation; chemical binding; cytoskeletal proteins; enzyme substrate; follicle stimulating hormone; gene induction /repression; genetic library; granulosa cell; hormone regulation /control mechanism; immunocytochemistry; laboratory rat; membrane proteins; molecular cloning; phosphorylation; protein isoforms; protein kinase A; protein purification; protein sequence; protein structure function; western blottings

cAMP is a second messenger which regulates responses to many different hormones and neurotransmitters. Elevated levels of cAMP results in the activation of the cAMP-dependent protein kinase (PKA) which produces a variety of distinct physiological responses. One hypothesis to explain how one enzyme can perform many different functions is that the subcellular localization of PKA is tightly controlled in each different cell type. Accordingly, PKA would be targeted or anchored adjacent to its physiological substrate. PKA consists of four subunits, two regulatory and two catalytic. The anchoring of PKA is believed to be accomplished through the interaction of the regulatory subunit (R) of PKA with specific cellular proteins, referred to as A-Kinase Anchoring Proteins (AKAPs). Although characterization of the protein-protein interaction between PKA and anchoring proteins has been extensive, the physiological function of most AKAPs remains unknown. Granulosa cells provide an ideal system for studying the functional role of AKAPs. It is well recognized that FSH and LH sequentially promote granulosa cell differentiation via a mechanism dependent upon the generation of cAMP and the subsequent activation of PKA. However, the mechanism by which PKA regulates this biological process remains elusive. Two AKAPs have recently been identified in primary granulosa cell cultures whose expression is differentially regulated by follicle stimulating hormone (FSH). The expression of one protein, AKAP 80, is stimulated by FSH, while the induction of the other protein, AKAP 140, is blocked by FSH. FSH also causes the induction of one isoform of PKA, regulatory subunit (RIIbeta) and the translocation of another isoform of PKA (RIIalpha). All of this data is consistent with the hypothesis that FSH promotes a redistribution of PKA, possibly to enable the cells to respond to a second hormone. LH, which also causes an increase in cAMP. To test this hypothesis we propose: l) to isolate and characterize both AKAP 80 and 140; 2) to determine the subcellular location of the AKAPs and RIIalpha and RIIbeta; and 3) to determine the functions of AKAPs 80 and 140 and isoforms of PKA in granulosa cells by either blocking the interaction of PKA with anchoring proteins through the use of anchoring inhibitor peptides or blocking the induction of these proteins through the use of antisense oligonucleotides. These studies will provide insights into the physiologic role of specific AKAPs and also new information on the cellular signaling pathway utilized by FSH and LH in granulosa cells.

营地是第二个使者，可调节对许多不同的回应 激素和神经递质。营地水平升高导致 依赖CAMP的蛋白激酶（PKA）的激活，该蛋白激酶（PKA）产生A 各种不同的生理反应。一个假设来解释如何 一种酶可以执行许多不同的功能是亚细胞 PKA的定位在每种不同的细胞类型中都受到严格控制。 因此，PKA将被靶向或锚定 生理基板。 PKA由四个亚基组成，两个监管和 两个催化。据信PKA的锚定是通过 PKA的调节亚基（R）与特定的相互作用 细胞蛋白，称为A-激酶锚定蛋白（AKAP）。 尽管PKA之间的蛋白质蛋白质相互作用的表征 锚定蛋白一直广泛，是 大多数AKAP仍然未知。颗粒细胞为 研究AKAP的功能作用。 众所周知，FSH和 LH通过机制顺序促进颗粒细胞分化 取决于营地的产生和随后的激活 PKA。 但是，PKA调节该生物学的机制 过程仍然难以捉摸。最近在 原代颗粒细胞培养物的表达差异 由卵泡刺激激素（FSH）调节。一个人的表达 蛋白质AKAP 80被FSH刺激，而另一个蛋白质的诱导 蛋白质AKAP 140被FSH阻塞。 FSH还导致一个 PKA的同工型，监管亚基（Riibeta）和易位 PKA（riialpha）的另一种同工型。所有这些数据都与 FSH促进PKA的重新分布的假设，可能是为了启用 细胞对第二激素反应。 LH，也导致 营地增加。为了检验这一假设，我们提出：l）分离和 表征AKAP 80和140； 2）确定亚细胞 Akaps和Riialpha和Riibeta的位置； 3）确定 Akaps 80和140的功能以及颗粒细胞中PKA的同工型的功能 要么阻止PKA与锚定蛋白的相互作用 使用锚定抑制剂肽或阻止这些诱导 通过使用反义寡核苷酸的蛋白质。这些研究会 提供有关特定AKAP和新的生理作用的见解 有关FSH和LH使用的细胞信号传导途径的信息 颗粒细胞。