NOVEL SECOND MESSENGER SIGNALING IN THE STRIATUM

Ras蛋白是生长和分化的关键调节因子，但最近也被认为与大脑的学习和记忆功能有关。Rap蛋白通过MAP激酶途径抑制Ras信号，或通过B-Raf激活MAP激酶。因此，Rap和Ras蛋白在细胞中可能具有拮抗或互补的功能。鸟嘌呤核苷酸交换因子（GEFs）可以激活Rap和Ras蛋白。许多Ras GEF，但直到工作描述，只有Rap GEF，已经确定。我们已经发现了新的富含大脑的基因家族，它们编码ras -超家族gef和第二信使结合基序。我们建议集中研究钙和二酰基甘油调控的gef， caldag - gef，它们在纹状体中高度富集。我们发现CalDAG-GEFI激活Rap1，而CalDAG-GEFII激活Ras， Ebinu等人也在1998年报道过。Ca2+和DAG都增强了这种激活。CalDAG-GEFI和CalDAG-GEFII都在纹状体的投射神经元中强烈表达，它们共存于纹状体的许多神经元中。这种共表达可能提供了一种机制，通过这种机制，钙和DAG信号可以协调或从Rap1切换到Ras激活，这取决于CalDAG-GEF参与。Ras参与了学习和记忆的神经可塑性，我们假设Rap1也可能参与其中。为了验证这一广泛的假设，我们建议在细胞和系统水平的实验中使用结合分析、突变遗传方法、分布和细胞定位研究、CalDAG-GEFI和II与谷氨酸和多巴胺神经递质系统的偶联分析、缺乏CalDAG-GEF功能的转基因和敲除小鼠的学习、记忆和其他神经功能测试来检验CalDAG-GEF。这项工作与健康相关的意义在于，这种新蛋白具有结合基序的第二信使分子对正常的大脑功能、可塑性和记忆至关重要。因此，了解这些新的第二信使信号分子将有助于理解重要的大脑功能，并有助于阐明大脑疾病。

Ras proteins are key regulators of growth and differentiation, but recently have also been implicated in learning and memory functions in the brain. Rap proteins inhibit Ras signaling through the MAP kinase pathway or, via B-Raf, also can activate MAP kinase. Thus Rap and Ras proteins may have antagonistic or complementary functions in cells. Guanine nucleotide exchange factors (GEFs) can activate Rap and Ras proteins. Many Ras GEFs, but until the work described, only Rap GEF, have been identified. We have discovered new families of brain-enriched genes that code for Ras-superfamily GEFs and second messenger binding motifs. We proposed to concentrate on the calcium and diacylglycerol- regulated GEFs, CalDAG-GEFs, which are highly enriched in the striatum. We have found that CalDAG-GEFI activates Rap1, whereas CalDAG-GEFII activates Ras, as also reported by Ebinu et al., 1998. Ca2+ and DAG both augment this activation. CalDAG-GEFI and CalDAG-GEFII are both strong expressed in the projection neurons of the striatum and they co-exist in many if not most of these neurons. This co-expression may provide a mechanism by which calcium and DAG signaling can be coordinated or switched from Rap1 to Ras activation, depending on the CalDAG-GEF engaged. Ras is involved in the neuroplasticity underlying learning and memory, and we hypothesize that Rap1 may be as well. To test this broad hypothesis, we propose to examine the CalDAG-GEFs in cellular and systems-level experiments using binding assays, mutational genetic approaches, distribution and cellular localization studies, assays for CalDAG-GEFI and II coupling to glutamate and dopamine neurotransmitter systems, and tests of learning and memory and other neurologic function in transgenic and knockout mice lacking CalDAG-GEF function. The health- related significance of this work rests in the fact that the second messenger molecules for which the novel proteins have binding motifs are essential for normal brain function and for plasticity and memory. Understanding these novel second messenger signaling molecules will therefore bring insights to understanding fundamentally important brain functions and will help in elucidating brain disorders.