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Moleculap biological and Pustochemical study on phospholipid me tabolic enzymes inviolved in signal transduction

参与信号转导的磷脂代谢酶的分子生物学和化学研究

基本信息

批准号：
07457001
负责人：
KONDO Hisatake
金额：
$ 4.93万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

Phosphoinositides (PI) turnover produces second messengers such as ciacylglycerol (DG) and inositol triphosphate in response to external stimuli. In addition, its intermediate products such as PI 4-monophosphate (PI4-P), PI4,5-bisphosphate (PI4,5-P) play roles in the membrane transport. It is thus important to clarify the molecular identity and characteristics of enzymes involved in the PI turnover for understanding the signal transduction and membrane transport. The present study addressed this point and revealed the molecular feature of DG kinase and PI 4-kinase, and localized them at mRNA levels in the brain of developing and adult rats by in situ hybridization histochemistry. DG kinase converts DG to phosphatidic acid (PA), and PI 4-kinase phosphorylates phosphatidyl-inositol to produce PI 4,5-P.Four subtypes of DG kinase (termed type I-IV) were identified by gene cloning from rat brain. The DG kinase type I is of soluble form and localized in the oligodendrocyte, but not neurons, … More and the absence of its expression is seen in the brain of myelin-deficient (shiverer) mice, suggesting the role in formation and maintenance of the myelin. The type II DG kinase is of membrane-associated form and localized in the medium-spiny neurons, neurons in the nucleus accumbens and olfactory tubercle, and in the endocrine cells of the pituitary inermediate lobe, suggesting the involvement in the dopaminergic transmission. The type III is localized dominantly in the Purkinije neurons and substantially in the granule cells of the cerebellum, suggesting its role in the cerebellar motor-control. The type IV is localized in the cerebral and cerebellar cortical neurons, but peculiar in the molecular structure : while the former three contain two EF-hand and zinc-finger motifs in addition to the ATP-binding domain, the type IV contains no EF-hand motifs, but possesses ankyrin-like repeats. This structure has a high homology to rdgA (a gene of Drosophila inducing the retinal degeneration), but this rat molecule is localized in the bipolar cell, but not the photoreceptor cell of the retina, suggesting some different roles for this homologous molecule between different animal species.Two subtypes of PI 4-kinase were idenfified with the molecular weight of 230kDa and 92kDa. The former has a high homology to Pikl and the latter to Stt4, both of which are yeast PI 4-kinases and has previously been cloned by other authors. Both PI 4-kinases are localized at mRNA levels widely in almost all neurons with much higher expression in immature cells. When overexpressed in COS-cells, these molecules are localized in the Golgi apparatus. It is thus suggested that these PI 4 kinases are involved in the vesicle-transport within cells. Less

磷脂酰肌醇(PI)代谢产生第二信使，如甘油三酯(DG)和肌醇三磷酸，以响应外界刺激。此外，其中间产物PI4-单磷酸(PI4-P)、PI4，5-二磷酸(PI4，5-P)在膜转运中发挥作用。因此，弄清参与PI周转的酶的分子特性和特性对于理解PI的信号转导和膜转运是非常重要的。本研究针对这一点，揭示了DG和PI4-激酶的分子特征，并用原位杂交组织化学方法对其在发育和成年大鼠脑内的mRNA水平进行了定位。DG激酶将DG转化为磷脂酸(PA)，PI 4-激酶使磷脂酰肌醇磷酸化生成PI 4，5-P。通过基因克隆从大鼠脑中鉴定了4种亚型DG激酶(称为I-IV型)。DG激酶I型是可溶性的，定位于少突胶质细胞，而不是神经元，…在髓鞘缺陷(颤抖)小鼠的大脑中可以看到更多和缺乏它的表达，这表明它在髓鞘的形成和维持中发挥了作用。II型DG激酶为膜相关形式，定位于中棘神经元、伏隔核和嗅结节的神经元，以及垂体中叶的内分泌细胞，提示其参与了多巴胺能传递。III型主要定位于Purkinije神经元，大量定位于小脑的颗粒细胞，提示其在小脑运动控制中的作用。IV型定位于大脑和小脑皮质神经元，但在分子结构上是独特的：前三种除了三磷酸腺苷结合区外，还含有两个EF-手和锌指基序，而IV型不含EF-手基序，但具有Ankyrin样重复序列。该结构与果蝇视网膜变性基因rdgA有很高的同源性，但这种大鼠分子定位于视网膜的双极细胞，而不是光感受器细胞，这表明这种同源分子在不同的动物物种中具有不同的作用。前者与Pik1有很高的同源性，后者与Stt4有很高的同源性，这两个基因都是酵母PI 4-激酶，以前已经被其他作者克隆过。这两种PI4-激酶在几乎所有神经元中都广泛地定位于mRNA水平，在未成熟细胞中的表达要高得多。当在COS细胞中过表达时，这些分子定位于高尔基体。因此，这些PI-4激酶参与了细胞内的囊泡运输。较少