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

本研究中进行了与细胞内CA信号机制有关的几种蛋白质的基因克隆，测序和表达定位。这些蛋白质包括钙蛋白钙网蛋白，CA/钙调蛋白依赖蛋白激酶（CAMK）II和IV，二酰基甘油激酶（DGK），Calpain，Calpastatin，14-3-3-3蛋白（用于CA依赖性蛋白激酶C）的推定性调节剂；以及进一步的蛋白质磷酸酶（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酶。通过原位杂交组织化学，使用每种蛋白质的特定cDNA探针通过PCR扩增获得，在表达强度中获得的空间和时间异质性在发育和成熟的大鼠脑中非常明显。例如，小脑颗粒细胞在这些细胞中检测到Calbindin和Calbindin和Calreticulin的强烈表达CAMKII和IV和PP亚型和亚型。这种异质性表明这些CA信号相关蛋白在不同神经元种群中的功能异质性，进一步表明其中某些蛋白质的多种同工型存在。通过使用DGK和CAMKIV作为目标来追求后一种可能性。结果，通过基因克隆确定了80 kDa和90 kDa-dgk。他们彼此表现出58％的身份，并包含锌指的序列，E-F手提基序和ATP结合位点。 80 kDa-DGK的基因表达仅限于少突胶质细胞，这表明该同工型参与与髓磷脂形成相关的CA信号。 90 kDa-DGK的基因主要在尾状壳核的神经元中表达，这表明它参与了与多巴胺能传播有关的Ca-Signals。另外，分离了编码CAMKIV -SUBTYPE的cDNA并从大鼠脑中测序。尽管在其氨基末端区域中添加了28个氨基酸是 *与 *的subtype的唯一差异，但差异基因表达fo