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Histological study on the gene expression of several proteins related to the intracellular Ca-signals.

与细胞内 Ca 信号相关的几种蛋白质的基因表达的组织学研究。

基本信息

批准号：
04404020
负责人：
KONDO Hisatake
金额：
$ 16.64万
依托单位：
TOHOKU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-04404020/
关键词：
Ca-signal gene expression rat in situ hybridization gene cloning protein kinase プロテインキナーゼ in situ hybridization カルシウム結合蛋白質プロティンキナーゼ免疫組織化学 in situ hybidization

项目摘要

The gene cloning, sequencing and expression localization of several proteins initimately related to the intracellular Ca-signal mechanism were undertaken in the present study. These proteins included calbindin calreticulin, Ca/calmodulin dependent protein kinase(CaMK) II and IV, diacylglycerol kinase(DGK), calpain, calpastatin, 14-3-3 protein (presumptive regulator for Ca-dependent protein kinase C) ; and further protein phosphatases (PP-2A, 2B, 2C), -adrenergic receptor kinase and neuronal and non-neuronal enolases. By in situ hybridization histochemistry using specific cDNA probes for each protein which were obtained by PCR amplification, spatial and temporal heterogeneity in the expression intensity was remarkable in developing and mature rat brain. For example, cerebellar granule cells expressed intensely CaMKII and IV and PP subunits and subtypes, while no significant expression for calbindin and calreticulin was detected in these cells. This heterogeneity suggests the functional heterogeneity of these Ca-signal-related proteins in different neuronal populations, and further suggests the existence of multiple isoforms for some of these proteins. The latter possibility was pursued by using DGK and CaMKIV as targets. As a result, 80 kDa- and 90 kDa-DGKs were identified by the gene cloning. They showed 58% identity to each other and contained zinc finger-like sequences, E-F hand motifs and ATP-binding sites. The gene expression for 80 kDa-DGK was confined to oligodendrocytes, suggesting the involvement of this isoform in Ca-signals related to the myelin formation. The gene of 90 kDa-DGK was expressed predominantly in neurons of the caudate putamen, suggesting its involvement in Ca-signals related the dopaminergic transmission. In addition, a cDNA encoding CaMKIV -subtype was isolated and sequenced from rat brain. Although addition of 28 amino acids in its amino terminal region was the only difference of the -subtype from the *, the diffirential gene expression fo

本研究对几种与细胞内钙信号机制密切相关的蛋白质进行了基因克隆、测序和表达定位。这些蛋白包括钙结合蛋白网织蛋白、钙/钙调蛋白依赖性蛋白激酶(CaMK)II和IV、二酰甘油蛋白激酶(DGK)、钙蛋白、钙蛋白抑素、14-3-3蛋白(钙依赖性蛋白激酶C的假定调节因子)，以及其他蛋白磷酸酶(PP-2A、2B、2C)、肾上腺素能受体激酶以及神经元和非神经元烯醇化酶。采用原位杂交组织化学方法，对各蛋白进行原位杂交组织化学检测，发现发育成熟大鼠脑内各蛋白表达强度的时空异质性显著。例如，小脑颗粒细胞强烈表达CaMKII和IV以及PP亚基和亚型，而在这些细胞中未检测到明显的钙结合蛋白和钙网织蛋白表达。这种异质性表明这些钙信号相关蛋白在不同神经元群体中的功能异质性，并进一步表明其中一些蛋白存在多种异构体。后一种可能性是通过使用DGK和CaMKIV作为目标来实现的。通过基因克隆鉴定出80 kDa和90 kDa的DGKs。它们之间有58%的同源性，并含有锌指类序列、E-F手序和ATP结合位点。80 kDa-DGK的基因表达仅限于少突胶质细胞，提示该亚型参与了与髓鞘形成相关的钙信号。90 kDa-DGK基因主要在尾壳核神经元中表达，提示其参与了与多巴胺能传递相关的钙信号。此外，还从大鼠脑组织中分离并测序了编码CaMKIV亚型的基因。虽然在其氨基末端增加了28个氨基酸是-亚型与*亚型的唯一不同之处，但其基因表达的差异