| Budget Amount *help |
¥7,700,000 (Direct Cost: ¥7,700,000)
Fiscal Year 1996: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1995: ¥5,000,000 (Direct Cost: ¥5,000,000)
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| Research Abstract |
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,
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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
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