| Co-Investigator(Kenkyū-buntansha) |
UMEDA Masato The University of Tokyo, Faculty of Pharmaceutical Sciences, 薬学部, 助手 (10185069)
BENNETT Vann Duke University Medical Center, 医療センター・生化学部, 教授
VANN Bennett デューク大学医療センター, 生化学部, 教授
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| Budget Amount *help |
¥4,400,000 (Direct Cost: ¥4,400,000)
Fiscal Year 1993: ¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1992: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
Ankyrins are a family of spectrin-binding proteins that link the spectrin/actin network to cytoplasmic domains of integral membrane proteins which include ion channels and cell adhesion molecules. Three different ankyrins are currently known to be expressed in brain tissue : ankyrin_R, ankyrin_B, and ankyrin_<node>. Ankyrin_B includes two isoforms of 220kD and 440kD which are generated from a single gene by alternative splicing of pre-mRNA. 220kD ankyrin_B is the major ankyrin isoform in adult rat brain, while 440kD ankyrin_B, in contrast, is maximally expressed in developing neonatal rat brain. 440kD ankyrin_B shares the same NH_2-terminal and COOH-terminal regions as 220kD ankyrin_B and contains, in addition, an inserted domain of 220kD. Both Isoforms are expressed in primary cerebellar cells in a manner similar to that in vivo ; the larger isoform appears first when axogenesis is actively taking place, and the smaller isoform arises later. 440kD ankyrin_B is localized to axons using
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an antibody raised against the insert region, while 220kD isoform can be localized to the cell bodies and dendrites of neurons by an antibody raised against a synthetic peptide corresponding to the splice site. Astroglial cells also express 220kD ankyrin_B. These results indicate that 440kD ankyrin_B is a neuron-specific isoform and that the inserted domain of 440kD ankyrin_B is essential for the targeting of this isoform to the axon.
Brain specific isoforms of ankyrin, 440kD and 220kD ankyrin_B, are both expressed in human neuroblastoma NB-1 cells and rat pheochromocytoma PC12h cells. Expression of the larger isoform was increased upon induction of neurite outgrowth by dibutyryl cAMP or nerve growth factor, while that of smaller isoform remained unchanged. Messenger RNA encoding 440kD ankyrin_B was also increased in differentiating NB-1 cells, whereas mRNA encoding 220kD ankyrin_B stayed almost unchanged. In methylmercury-treated NB-1 cells, polypeptide and mRNA of 440kD ankyrin_B were selectively attenuated in association with the retraction of neurites, while those of 220kD isoform were not affected. The expressed 440kD ankyrin_B is concentrated at the tip of growing neurites and some of them are colocalized with GAP(growth-associated protein)-43. These results indicate that the expression of 440kD ankyrin_B is intimately associated with the neurite outgrowth and retraction in neuronal cell lines, and is regulated at mRNA level. Less
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