Studies on the Roles of Ras femily GTPases in the Signal Transduction in Hyppocampal Neuronal Cells.

丝裂原活化蛋白(MAP)激酶在体外和活体动物体内建立长时程增强过程中起着重要作用。MAP激酶在多种刺激下被激活，包括通过N-甲基-D-天冬氨酸受体和L型钙通道、cAMP和神经营养素的钙内流。为了研究RAS在海马神经元MAP激酶和CREB(cAMP反应元件结合蛋白)激活中的作用，我们通过腺病毒载体过表达RAS GTP酶激活蛋白Gap1m或显性阴性RAS来抑制RAS功能。Gap1m的表达几乎完全抑制了NMDA、钙离子载体、膜去极化、Forsklin和脑源性神经营养因子(BDNF)对MAP激酶的激活。显性负值RAS也表现出类似的效应。另一方面，Rap1 GAP对Forsklin诱导的MAP激活没有明显的抑制作用。这些结果表明，RAS信号转导信号在大范围的刺激下映射到海马神经元中的激酶。我们还分析了培养细胞中RAP1的功能。在引入激活RAP1的因素后，观察到细胞粘附性的增强。显性阴性的RAP1阻断了这种增强，显性活性的RAP1强烈激活了细胞黏附，暗示了RAP1在细胞黏附中的作用。此外，我们还建立了Rin的基因靶向小鼠，Rin是一种仅在神经元组织中特异表达的Ras样GTP酶。该Rin基因敲除小鼠可能对Rin在体内的功能研究非常有用。

Mitogen-activated protein (MAP) kinase plays important roles in the establishment of long term potentiation both in vitro and in living animals. MAP kinase is activated in response to a broad range of stimuli including calcium influx through N-methyl-D-aspartate (NMDA) receptor and L-type calcium channel, cAMP, and neurotrophins. To investigate the role of Ras in the activation of MAP kinase and CREB (cAMP response element-binding protein) in hippocampal neurons, we inhibited Ras function by overexpressing a Ras GTPase- activating protein, Gap1m, or dominant negative Ras by means of adenovirus vectors. Gap1m expression almost completely suppressed MAP kinase activation in response to NMDA, calcium ionophore, membrane depolarization, forskolin, and brain-derived neurotrophic factor (BDNF). Dominant negative Ras also showed the similar effects. On the other hand, Rap1 GAP did not significantly inhibit the forskolin-induced activation of MAP kinase. These results demonstrate that Ras transduces signals elicited by a broad range of stimuli to MAP kinase in hippocampal neurons.We also analyzed Rap1 function in cultured cells. Upon introduction of factors that activated Rap1, enhancement of cell adhesion property was observed. Dominant negative Rap1 blocked this enhancement and dominat active Rap1 strongly activated the cell adhesion, suggenting the role of Rap1 in cell adhesion.Moreover, we made a gene targeting mouse for Rin, a Ras-like GTPase expressed secifically only in neuronal tissues. This Rin knockout mouse maybe very useful for the functional study of Rin in vivo.