Second Messengers in the Retina

视网膜中的第二信使

• 批准号: 6621248
• 负责人: ROBERT E ANDERSON
• 金额: $ 36.63万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 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 2005-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6621248
• 关键词: SDS polyacrylamide gel electrophoresis; biotechnology; enzyme activity; immunocytochemistry; inositol phosphates; insulin receptor; insulinlike growth factor; laboratory rat; mass spectrometry; molecular cloning; phosphatidylinositols; phospholipase C; phosphorylation; photoactivation; protein kinase; protein kinase C; protein sequence; retina; rod cell; second messengers; visual phototransduction; western blottings

DESCRIPTION (From the Applicant's Abstract): Studies from our laboratory over the past two decades have shown that the retina and rod outer segments (ROS) have an active phosphoinositide (P1) metabolism (called the P1 Cycle) that generates several lipid second messengers. During the last year, we discovered that the insulin receptor -subunit (IR) is tyrosine phosphorylated in vivo and in vitro in response to light and directly activates PT 3-kinase, which forms PI-3,4,5-P3 and other D3-phosphorylated PIs in ROS. In other tissues, these second messengers have been shown to affect a large number of intracellular events, including secretion, cell survival, membrane trafficking, and actin polymerization, all of which are important in the retina. It is our hypothesis that light-stimulation of this reaction in photoreceptor cell outer segments affects important photoreceptor cell function(s) not related to visual transduction. The long-term goal of our research is to elucidate the physiological role of these lipid second messengers in photoreceptor cells. To this end, we will determine the mechanism of activation of the enzymes that synthesize these messengers and identify their downstream effectors. Expression of GST-fusion proteins containing the JR motif will be used in pull-down assays to obtain proteins that can be identified on Western Blots, by 2D gel electrophoresis followed by in-gel digestion and mass spectrometry of the peptide fragments, or by protein sequencing followed by cloning and sequencing the gene. Once these proteins have been identified, we will attempt to determine their role in the light-driven reactions involving the insulin receptor and the P1 Cycle. We will use new gene knockout technology now available in our laboratory to selectively eliminate the gene products of several enzymes of the P1 Cycle in photoreceptor cells and determine the effect on photoreceptor structure and function.

描述（摘自申请人的摘要）：我们实验室的研究 过去的二十年表明，视网膜和杆外段（ROS） 具有活性的磷酸肌醇（P1）代谢（称为P1周期） 生成几个脂质第二使者。在去年，我们发现了 胰岛素受体 - 亚基（IR）在体内被酪氨酸磷酸化， 体外响应光并直接激活Pt 3-激酶，形成形成的Pt 3-激酶 ROS中的PI-3,4,5-P3和其他D3磷酸化的PI。在其他组织中，这些 已显示第二使者会影响大量细胞内 包括分泌，细胞生存，膜贩运和肌动蛋白在内的事件 聚合，所有这些在视网膜中都很重要。这是我们的假设 光感受器细胞外部段中这种反应的光刺激 影响与视觉无关的重要光感受器细胞功能 转导。我们研究的长期目标是阐明 这些脂质第二信使在感光细胞中的生理作用。到 这个目的，我们将确定激活酶的机理 合成这些使者并确定其下游效应子。表达 含有JR图案的GST融合蛋白的蛋白质将用于下拉测定 获得可以在蛋白质印迹上鉴定的蛋白质，用2D凝胶 电泳，然后是凝胶内消化和质谱法 肽片段，或通过蛋白质测序，然后进行克隆和测序 基因。一旦确定了这些蛋白质，我们将尝试 确定它们在涉及胰岛素的轻驱动反应中的作用 受体和P1周期。我们现在将使用新的基因淘汰技术 在我们的实验室中可用以选择性地消除 P1循环在感光细胞中的几种酶，并确定效果 在光感受器的结构和功能上。