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

裂丝原活化蛋白激酶（MAP）在体外和活体动物体内长期增强的建立中起着重要的作用。MAP激酶在广泛的刺激下被激活，包括通过n -甲基- d -天冬氨酸（NMDA）受体和l型钙通道、cAMP和神经营养素流入的钙。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 GTPase激活蛋白Gap1m或显性阴性Ras来抑制Ras的功能。Gap1m表达几乎完全抑制MAP激酶对NMDA、钙离子载体、膜去极化、forskolin和脑源性神经营养因子（BDNF）的激活。显性负Ras也表现出类似的效果。另一方面，Rap1 GAP对福斯克林诱导的MAP激酶活化无明显抑制作用。这些结果表明，Ras转导信号在广泛的刺激下引起海马神经元的MAP激酶。我们还分析了Rap1在培养细胞中的功能。在引入激活Rap1的因子后，观察到细胞粘附性能增强。显性阴性Rap1阻断了这种增强，而显性活性Rap1则强烈激活细胞粘附，提示Rap1在细胞粘附中起作用。此外，我们制作了一种针对小鼠的基因，用于Rin，一种仅在神经元组织中特异性表达的ras样GTPase。该基因敲除小鼠可能对体内Rin的功能研究具有重要意义。

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.