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: 3808680
• 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/3808680
• 关键词: 3'5' cyclic nucleotide phosphodiesterase; afferent nerve; biochemical evolution; biological signal transduction; brain; brain metabolism; calcium; calcium binding protein; calmodulin; calmodulin dependent protein kinase; central neural pathway /tract; cerebellar Purkinje cell; developmental genetics; electron microscopy; enzyme mechanism; enzyme structure; gene expression; genetic promoter element; genetic regulation; histochemistry /cytochemistry; immunochemistry; messenger RNA; molecular cloning; molecular genetics; neural transmission; neurons; phosphodiesterases; phosphopeptides; phosphorylation; protein structure; synapses

Analysis of mammalian cDNA and genomic clones for the catalytic subunit of the calmodulin (CaM)-dependent protein phosphatase, calcineurin (CN) indicate multiple molecular isoforms that result from alternative splicing of distinct genes. The evolutionary relationship between these genes has been characterized, as have their localizations on human chromosomes and differences in mRNA expression. To study the origins of this genetic complexity and its regulatory controls, we are comparing the corresponding genes in lower eukaryotes. In these simpler organisms primary structure is highly conserved in catalytic and regulatory domains, while regions of unrelated sequence near the carboxyl terminus suggest areas that may impart unique substrate specificity. In Neurospora intron/exon boundaries corresponded to those we have found in the mammalian gene, suggesting a step-wise evolution of DNA control features. In both Neurospora and Dictyostelium, mRNA expression appears to be developmentally controlled, and studies to examine promoter regulation are being pursued. A bacterially-expressed fungal catalytic subunit having high enzyme activity was produced and a set of deletion mutants of the mammalian protein delineated the minimum structural domain needed for CaM binding; a synthetic peptide based on this region exhibited an inhibitory constant of one-fourth 5 nM. Developmental studies on phosphodiesterase (PDE) showed a pattern of expression that coincided with synaptogenesis in most brain areas; the e.m. localization of PDE in neurons is now being studied to suggest its possible functional roles. A novel brain-specific CaM binding protein of 140 kDa (native MW) has been characterized that is localized primarily in limbic structures.

哺乳动物 cDNA 和基因组克隆的催化亚基分析 钙调蛋白 (CaM) 依赖性蛋白磷酸酶、钙调神经磷酸酶 (CN) 表示由选择性剪接产生的多种分子亚型 不同的基因。这些基因之间的进化关系 已被表征，以及它们在人类染色体上的定位和 mRNA 表达的差异。研究这种基因的起源 复杂性及其监管控制，我们正在比较相应的 低等真核生物中的基因。在这些更简单的生物体中，一级结构是 在催化和调控领域高度保守，而区域 羧基末端附近不相关的序列表明可能赋予 独特的底物特异性。脉孢菌内含子/外显子边界 与我们在哺乳动物基因中发现的一致，表明 DNA控制特征的逐步进化。在脉孢菌属和 盘基网柄菌，mRNA 表达似乎受发育控制， 正在开展检查启动子监管的研究。一个 细菌表达的具有高酶活性的真菌催化亚基 产生了一组哺乳动物蛋白的缺失突变体 描绘了 CaM 结合所需的最小结构域；一个 基于该区域的合成肽表现出抑制常数 四分之一 5 nM。磷酸二酯酶 (PDE) 的发育研究表明 大多数大脑中与突触发生一致的表达模式 地区；电磁波目前正在研究 PDE 在神经元中的定位 提出其可能的功能作用。一种新型的大脑特异性 CaM 结合 140 kDa（天然分子量）的蛋白质已被表征为局部化的 主要存在于边缘结构。