Versatile Roles of R-Ras GAP in Neurite Formation of PC12 Cells and Embryonic Vascular Development

Shintaro Iwashita,Mariko Kobayashi,Yuya Kubo,Yoshimi Hinohara,Mariko Sezaki,Kenji Nakamura,Rika Suzuki-Migishima,Minesuke Yokoyama,Showbu Sato,Mitsunori Fukuda,Masayuki Ohba,Chieko Kato,Eijiro Adachi,Si-Young Song

doi:10.1074/jbc.c600293200

Abstract

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.

Highlights

Biochemical reactions mediated by receptors in the plasma membrane produce signaling for cell growth and differentiation and down-regulate this signaling for the maintenance of cellular homeostasis [1]
We show a positive correlation between the down-regulation of R-Ras GTPase-activating proteins (GAP) and neurite outgrowth of PC12 cells and the embryonic lethality of R-Ras gap mutant mice accompanied with massive hemorrhage
To address the biological significance of R-Ras GAP down-regulation in neurite outgrowth, we employed transient transfection experiments to examine the effects of Ras or Ras GAP on nerve growth factor (NGF)-mediated neurite outgrowth of PC12 cells

Summary

ACCELERATED PUBLICATION

Versatile Roles of R-Ras GAP in Neurite Formation of PC12 Cells and Embryonic Vascular Development*□S. 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. Biochemical reactions mediated by receptors in the plasma membrane produce signaling for cell growth and differentiation and down-regulate this signaling for the maintenance of cellular homeostasis [1]. An arginine situated in the GRD loop, the “arginine finger,” has been identified as essential for all Ras GAP activity [16] Based on this result, we generated mice mutant for R-Ras gap, which lacks the region including the arginine finger, to clarify its physiological significance in vivo. We show a positive correlation between the down-regulation of R-Ras GAP and neurite outgrowth of PC12 cells and the embryonic lethality of R-Ras gap mutant mice accompanied with massive hemorrhage

RESULTS

Extracellular ligand

DISCUSSION