REGULATION OF RETINAL MUSCARINIC RECEPTOR EXPRESSION

视网膜毒蕈碱受体表达的调节

• 批准号: 6525093
• 负责人: Neil Marc Nathanson
• 金额: $ 26.19万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2004-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6525093
• 关键词: chickens; complementary DNA; confocal scanning microscopy; expression cloning; genetic mapping; genetic transcription; growth /development; high performance liquid chromatography; immunocytochemistry; mass spectrometry; molecular cloning; muscarinic receptor; protein purification; protein sequence; protein structure function; receptor expression; reporter genes; retina; tissue /cell culture; transcription factor

DESCRIPTION (provided by applicant): The long-term goal of this research is to determine the molecular and cellular mechanisms responsible for the regulation of muscarinic acetylcholine receptor (mAChR) gene expression in the retina during development. Muscarinic receptors have an important role in the processing of visual information in the retina, and have also been implicated in the development of the embryonic retina. Our laboratory has found that there is receptor subtype- and cell-specific regulation of mAChR in the chick retina during embryonic development. The developmental regulation of the M2 receptor-in vivo can be mimicked in retina cell culture by an apparently novel developmentally-regulated secreted factor. This application will identify and determine the mechanism of action of the M2-inducing factor. The specific aims are: (1) To purify the M2 receptor-inducing factor. We have developed a serum-free culture system that supports secretion of this factor, and have used a rapid reporter gene assay to obtain highly enriched preparations of this factor. We will refine this purification scheme to purify the factor to sufficient homogeneity to allow biochemical and molecular biological analyses. (2) To isolate cDNA clones encoding the M2 receptor-inducing factor. We will use both a protein sequence determination approach and expression cloning to isolate cDNA clones encoding the factor. (3) To test the hypothesis that the M2 receptor-inducing factor regulates M2 receptor expression in vivo. We will determine if the factor identified on the basis of its ability to induce M2 receptor expression in retinal cells in culture is indeed responsible for the developmental induction of retinal M2 receptor expression in vivo. (4) To determine the molecular and cellular mechanisms responsible for the regulation of the M2 receptor by the M2 receptor-inducing factor. These experiments should provide valuable new information on the mechanisms responsible for the developmental regulation of neurotransmitter receptor gene expression in the retina.

描述（由申请人提供）：本研究的长期目标是 确定负责调节的分子和细胞机制 视网膜中毒蕈碱乙酰胆碱受体 (mAChR) 基因表达的影响 在开发过程中。毒蕈碱受体在 视网膜中视觉信息的处理，也与 在胚胎视网膜的发育过程中。我们实验室发现有 是小鸡视网膜中 mAChR 的受体亚型和细胞特异性调节 在胚胎发育过程中。 M2的发育调节 体内受体可以通过一种明显新颖的方法在视网膜细胞培养物中进行模拟 发育调节的分泌因子。该应用程序将识别并 确定 M2 诱导因子的作用机制。具体目标 是： (1)纯化M2受体诱导因子。我们开发了一种无血清 支持该因子分泌的培养系统，并使用了快速 报告基因测定以获得该因子的高富集制剂。我们 将完善这个纯化方案，将因子纯化到足够的程度 均质性以允许生化和分子生物学分析。 (2)分离编码M2受体诱导因子的cDNA克隆。我们将 同时使用蛋白质序列测定方法和表达克隆 分离编码该因子的 cDNA 克隆。 (3)检验M2受体诱导因子调节M2的假设 体内受体表达。我们将确定该因素是否在 其在视网膜细胞中诱导 M2 受体表达的能力的基础 文化确实负责视网膜 M2 的发育诱导 体内受体表达。 (4) 确定造成该现象的分子和细胞机制 M2受体诱导因子对M2受体的调节。 这些实验应该提供有关机制的有价值的新信息 负责神经递质受体基因的发育调节 视网膜上的表达。