Versatile roles of R-Ras GAP in neurite formation of PC12 cells and embryonic vascular development
Versatile roles of R-Ras GAP in neurite formation of PC12 cells and embryonic vascular development
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DOI：
10.1074/jbc.c600293200
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发表时间：
2007-02-09
影响因子：
4.8
通讯作者：
Song, Si-Young
中科院分区：
文献类型：
--
作者：
Iwashita, Shintaro;Kobayashi, Mariko;Song, Si-Young
Ras GTP酶激活蛋白（GAP）是Ras的负调节因子，其将活性Ras-GTP转化为非活性Ras-GDP。R-Ras GAP是一种膜结合分子，对R-Ras（整合素的激活剂）的GAP活性比H-Ras强。我们发现，在大鼠嗜铬细胞瘤PC 12细胞突起形成过程中，神经生长因子（NGF）下调R-Ras GAP，R-Ras GAP的瞬时表达或显性负性R-ras cDNA可阻断NGF下调R-Ras GAP。通过建立稳定表达外源R-Ras GAP的PC 12亚克隆，发现NGF降低内源R-Ras GAP，但不降低外源R-Ras GAP，表明R-Ras GAP的下调发生在转录水平。为了阐明R-Ras GAP的生理作用，我们产生了表达具有敲低活性的突变型Ras GAP的小鼠。虽然杂合子是正常的，但纯合子小鼠在E12.5-13.5死于大量皮下和实质内出血，可能是由于毛细血管内皮细胞之间的粘附连接发育不全。这些结果表明R-Ras GAP在发育和分化中的重要作用：其表达是胚胎发育血管屏障所需的，而其下调通过维持激活的R-Ras促进NGF诱导的PC 12细胞神经突形成。
Ras GTPase-activating proteins (GAP) are negative regulators of Ras that convert active Ras-GTP to inactive Ras-GDP. R-Ras GAP is a membrane-associated molecule with stronger GAP activity for R-Ras, an activator of integrin, than H-Ras. We found that R-Ras GAP is down-regulated during neurite formation in rat pheochromocytoma PC12 cells by nerve growth factor (NGF), which is blocked by the transient expression of R-Ras gap or dominant negative R-ras cDNA. By establishing a PC12 subclone that stably expresses exogenous R-Ras GAP, it was found that NGF reduced endogenous R-Ras GAP but not exogenous R-Ras GAP, suggesting that down-regulation of R-Ras GAP occurs at the transcription level. To clarify the physiological role of R-Ras GAP, we generated mice that express mutant Ras GAP with knocked down activity. While heterozygotes are normal, homozygous mice die at E12.5-13.5 of massive subcutaneous and intraparenchymal bleeding, probably due to underdeveloped adherens junctions between capillary endothelial cells. These results show essential roles of R-Ras GAP in development and differentiation: its expression is needed for embryonic development of blood vessel barriers, whereas its down-regulation facilitates NGF-induced neurite formation of PC12 cells via maintaining activated R-Ras.