STEROID ACTION AT THE OOCYTE PLASMA MEMBRANE

类固醇对卵母细胞质膜的作用

• 批准号: 2196723
• 负责人: GENE A MORRILL
• 金额: $ 14.87万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-02-01 至 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2196723
• 关键词: G protein; Rana; adenylate cyclase; affinity labeling; alternatives to animals in research; biological signal transduction; cell membrane; cyclic AMP; diacylglycerols; egg /ovum; electron spin resonance spectroscopy; enzyme activity; fluidity; high performance liquid chromatography; hormone regulation /control mechanism; hydrolysis; intracellular membranes; lipid metabolism; meiosis; membrane activity; methylation; mitogens; phosphatidylcholines; phosphatidylinositols; phospholipase C; phosphorylation; plasmalogens; progesterone; progesterone receptors; protease inhibitor; protein kinase C

Progesterone binding at the amphibian oocyte plasma membrane induces meiosis via synthesis of the maturation promoting factor, now recognized to be a universal mitogen. This proposal is concerned with the mechanism of steroid action at the plasma membrane which leads to mitogen synthesis. We have found that the following reactions are triggered by progesterone in isolated oocyte plasma membranes: phosphatidylethanolamine (PE) N-methylation (within seconds), the release of protease and free Ca2+, a rise and fall in 1,2-diacylglycerol (DAG) derived from phosphatidycholine (PC) by phospholipase C (PLC) (within two minutes), activation of protein kinase C (PKC) and increased inositol phosphate turnover. Phospholipid N-methylation, leading to PC synthesis, occurs only within plasma membranes, but 80-90% of the DAG, which activates PKC and is now recognized as a second messenger, is generated from PC. This represents the transduction of a plasma membrane receptor signal to second messenger activation at intracellular membranes. The mechanism by which steroid binding to the plasma membrane receptor activates an enzyme may be G protein-mediated, since our experiments indicate that G proteins directly regulate plasma membrane N- methyltransferase and/or phospholipase C. The first specific aim of this proposal is to use photoaffinity labeling to identify G proteins which are functionally associated with the progesterone receptor; the ultimate goal being to reconstitute the hormone-receptor-G protein system in membrane vesicles. The second aim is to determine the molecular species and phospholipid source of the DAGs generated both from plasma membrane and intracellular membranes. About 30% of the total [3H]-glycerol- labeled 1,2--DAG in the prophase oocyte is derived from plasmalogen, a choline phospholipid containing an alpha, beta-unsaturated ether linkage in the 1-position, high in sperm and excitable tissues (brain, heart). The third aim is to investigate transient changes in diacylglycerol kinase activity after hormone stimulation; this enzyme controls both DAG levels and the branch point promoting resynthesis of phosphatidylinositols at the expense of resynthesis of the major membrane phospholipids such as PC. The fourth aim is to examine the possible role of PKC-dependent protein phosphorylation and/or decreased membrane fluidity in the down-regulation of adenylate cyclase. The proposed research should contribute to the understanding of mitogenic signaling, steroid hormone action at the plasma membrane, and role of phospholipids in generating a second messenger, and the spread of that second messenger signal from plasma to intracellular membranes.

两栖动物卵母细胞质膜处的孕酮结合可诱导 通过合成成熟促进因子的减数分裂，现在已被认可 成为通用有丝分裂原。 该提议与机制有关 在质膜上的类固醇作用 合成。 我们发现以下反应是由 分离的卵母细胞质膜中的孕酮： 磷脂酰乙醇胺（PE）N-甲基化（几秒钟内），释放 蛋白酶和游离Ca2+，在1,2-二酰基甘油（DAG）中升高和下降 由磷脂酶C（PLC）源自磷脂酰胆碱（PC）（在两个 分钟），激活蛋白激酶C（PKC）和肌醇增加 磷酸盐周转率。 磷脂N-甲基化，导致PC合成， 仅发生在质膜内，但DAG的80-90％ 激活PKC，现在被认为是第二个使者 来自PC。 这表示质膜受体的转导 在细胞内膜上向第二信使激活发出信号。 这 类固醇与质膜受体结合的机制 激活酶可能是G蛋白介导的，因为我们的实验 表明G蛋白直接调节质膜N- 甲基转移酶和/或磷脂酶C. 建议是使用光性标签来识别G蛋白 在功能上与孕酮受体有关；最终 目的是重新构建激素受体 - G蛋白系统 膜囊泡。 第二个目的是确定分子物种 以及从质膜产生的DAG的磷脂来源 和细胞内膜。 大约30％的总[3H] - 甘油 - 预言卵母细胞中标记为1,2--DAG是源自质子原的，A 胆碱磷脂，含有α-β-未饱和醚键 在1位中，精子和兴奋的组织（大脑，心脏）高。 第三个目的是研究二酰基甘油的短暂变化 激素刺激后的激酶活性；这种酶控制两个DAG 水平和分支点促进了重新合成的 磷脂酰肌醇以主要膜重新合成为代价 磷脂，例如PC。 第四个目的是检查可能的角色 依赖PKC的蛋白质磷酸化和/或膜降低 腺苷酸环化酶下调中的流动性。 提议 研究应有助于理解有丝分裂信号， 类固醇激素在质膜上的作用和磷脂的作用 在产生第二个使者以及第二个使者的传播时 从血浆到细胞内膜的信号。