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

在外界刺激下，磷酸肌苷（PI）的转化产生第二信使如甘酰甘油（DG）和三磷酸肌醇。此外，其中间产物PI4-单磷酸（PI4- p）、pi4,5 -二磷酸（pi4,5 - p）也参与了膜转运。因此，阐明参与PI转换的酶的分子特性和特征对于理解信号转导和膜转运具有重要意义。本研究针对这一问题，通过原位杂交组织化学方法，揭示了DG激酶和PI 4激酶的分子特征，并在发育和成年大鼠脑中的mRNA水平上进行了定位。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产生PI 4,5- p。从大鼠脑中克隆了DG激酶的4个亚型（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，在髓磷脂缺乏（震颤）小鼠的大脑中可以看到它的表达缺失，这表明它在髓磷脂的形成和维持中起作用。II型DG激酶为膜相关形式，定位于中棘神经元、伏隔核和嗅结节神经元以及垂体中叶的内分泌细胞，提示其参与多巴胺能传递。III型主要定位于浦肯野神经元，大量定位于小脑颗粒细胞，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，但在分子结构上很特殊：前三种除了atp结合结构域外，还包含两个EF-hand和锌指基元，而IV型不包含EF-hand基元，但具有锚蛋白样重复序列。该结构与rdgA（果蝇诱导视网膜变性的基因）具有高度的同源性，但该大鼠分子定位于双极细胞，而不是视网膜的光感受器细胞，这表明该同源分子在不同动物物种之间具有不同的作用。鉴定出PI 4激酶的两个亚型，分子量分别为230kDa和92kDa。前者与Pikl具有高度同源性，后者与Stt4具有高度同源性，两者都是酵母PI 4激酶，此前已被其他作者克隆。这两种PI - 4激酶在几乎所有神经元中都以mRNA水平广泛定位，在未成熟细胞中表达量更高。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。少

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