CONTROL OF CALCIUM AND PHOSPHORYLATION-REGULATED SIGNALLING PATHWAYS

钙和磷酸化调节信号通路的控制

• 批准号: 3817420
• 负责人: R L KINCAID
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3817420
• 关键词: 3'5' cyclic nucleotide phosphodiesterase; afferent nerve; brain metabolism; calcium; calcium binding protein; calmodulin; central neural pathway /tract; cerebellar Purkinje cell; enzyme mechanism; gene expression; histochemistry /cytochemistry; immunochemistry; messenger RNA; molecular cloning; neural transmission; phosphopeptides; phosphoproteins; synapses

Investigations on the physiology and molecular biology of calcium and phosphoprotein-regulated signalling pathways have been initiated, with emphasis on the calmodulin (CaM)-dependent phosphatase, calcineurin (CN), and cyclic nucleotide phosphodiesterase (PDE). Immunocytochemical studies indicate that the calmodulin-dependent isoform of brain PDE is expressed predominantly in regional output neurons, consistent with a role in integration of synaptic input. Further, chemical destruction of climbing fiber afferents to cerebellar Purkinje cells causes loss of PDE immunoreactivity suggesting a "transsynaptic" model for regulation of gene expression. Current studies on the cloning of this enzyme are designed to determine the control mechanism(s) for expression. Recent studies have shown that CN is the major cytosolic CaM-binding protein (BP) in lymphocytes and that it is differentially expressed in subpopulations of lymphoid cells. To examine its regulation in the immune system (and brain), cDNAs for the catalytic subunit of this phosphatase have been cloned using expression vector immunoscreening and a novel plaque hybridization method employing biotinylated restriction fragments. Tissue- specific mRNA species are now being characterized and in vitro mutagenesis will be carried out to examine important allosteric and catalytic domains on the enzyme, some of which are suggested by clear similarities to other recently cloned phosphatases. Ongoing studies with model phosphopeptides will be used to investigate immunologically the phosphorylation of receptors and proteins involved in control of intermediary metabolism and cytoskeletal organization.

钙的生理学和分子生物学研究 和磷蛋白调节的信号通路 发起，重点是钙调蛋白（CaM）依赖性 磷酸酶、钙调神经磷酸酶 (CN) 和环核苷酸 磷酸二酯酶（PDE）。 免疫细胞化学研究表明 表达大脑 PDE 的钙调蛋白依赖性亚型 主要存在于区域输出神经元，与角色一致 突触输入的整合。 此外，化学破坏 攀爬纤维传入小脑浦肯野细胞的原因 PDE 免疫反应性的丧失表明存在“跨突触”模型 基因表达的调控。 克隆研究现状 该酶旨在确定控制机制 表达。 最近的研究表明 CN 是主要的 淋巴细胞中的胞质 CaM 结合蛋白 (BP) 在淋巴细胞亚群中差异表达。 到 检查其在免疫系统（和大脑）中的调节，cDNA 该磷酸酶的催化​​亚基已使用克隆 表达载体免疫筛选和新型噬菌斑杂交 使用生物素化限制性片段的方法。 组织- 目前正在对特定 mRNA 种类进行表征并在体外进行 将进行诱变以检查重要的变构和 酶上的催化结构域，其中一些是由 与其他最近克隆的磷酸酶有明显的相似之处。 进行中 磷酸肽模型研究将用于调查 免疫学上受体和蛋白质的磷酸化 参与中间代谢和细胞骨架的控制 组织。