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

有丝分裂原活化蛋白激酶（MAP）在体外和活体动物长时程增强的建立中起重要作用。MAP激酶响应于广泛的刺激而被激活，包括通过N-甲基-D-天冬氨酸（NMDA）受体和L-型钙通道、cAMP和神经营养因子的钙内流。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 GT3激活蛋白Gap 1 m或显性负性Ras来抑制Ras功能。Gap 1 m的表达几乎完全抑制了MAP激酶对NMDA、钙离子载体、膜去极化、毛喉素和脑源性神经营养因子（BDNF）的激活。显性负性Ras也表现出类似的效应。另一方面，Rap 1 GAP没有显著抑制毛喉素诱导的MAP激酶的激活。这些结果表明Ras可以将多种刺激引起的信号转导至海马神经元的MAP激酶，并分析了Rap 1在培养细胞中的功能。通过导入激活Rap 1的因子，观察到细胞粘附性的增强。显性负性Rap 1阻断了这种增强作用，显性活性Rap 1则强烈激活细胞粘附，提示Rap 1在细胞粘附中的作用。Rin基因敲除小鼠的建立为Rin基因的体内功能研究提供了重要的实验依据。

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.