ANALYSIS OF NEUROFIBROMATOSIS TYPE 1 GENE FUNCTION

这项建议旨在了解人类神经纤维瘤病1型突变基因的功能。该病的特点是各种病理表现(如肿瘤、色素沉着改变、行为异常、生长缺陷)，其中许多基础尚不清楚。对NF1功能的主流观点认为，NF1主要作为RAS的GTP酶激活蛋白(GAP)对RAS GTP酶起负性调节作用。为了研究NF1在生物体内的功能，我们克隆并鉴定了一个高度保守的果蝇同源基因。对果蝇NF1同源物的研究没有显示出在NF1突变体中Ras1介导的信号改变的证据，这表明NF1作为Ras1调节因子的假设作用在体内可能是多余的或不那么重要。相反，在NF1和编码cAMP/PKA信号通路组件的基因之间观察到了强烈的遗传交互作用。NF1突变体在生长和突触传递方面存在缺陷。这种生长缺陷被一种缺乏GAP活性的突变蛋白所拯救，这种突变蛋白似乎需要NF1的非细胞自主功能。本文描述的实验旨在阐明NF1基因表面上不依赖于Ras1的功能及其通过非细胞自主机制在调节生长中的作用。具体目标1是提供NF1如何调节体内生长的详细描述。包括对NF1与cAMP/PKA通路相互作用的分析。具体目的2是通过识别NF1蛋白中与其他途径相互作用的结构域来确定在体内介导NF1非RAS功能的信号通路。具体目标3是确定在体内与NF1相互作用的基因。这些基因将通过对亚型NF1等位基因的蛹大小表型的增强子和抑制子的遗传筛选，或者通过使用基因克隆微阵列并寻找NF1突变体中基因表达的显著变化来识别。这些研究将有助于我们了解NF1基因在体内的正常功能，并有助于我们了解疾病过程的各种表现。

This proposal is aimed at understanding the function of the gene that is mutated in the human disease Neurofibromatosis type1. The disease is characterized by a wide variety of pathological manifestations (e.g. tumors, pigmentation changes, behavioral abnormalities, growth defect) and the basis of many of these is not known. The prevailing view of NF1 function is that it is primarily a negative regulator of the Ras GTPase by acting as a GTPase activating protein (GAP) for Ras. To study the function of NF1 in vivo in an organism amenable to genetic manipulations, a highly conserved Drosophila homologue was cloned and characterized. Studies with the Drosophila NF1 homologue have failed to show evidence of altered Ras1-mediated signaling in NF1 mutants suggesting that the postulated role of NF1 as a Ras1 regulator ma either be redundant or less important in vivo. In contrast strong genetic interactions were observed between NF1 and genes encoding components of the cAMP/PKA signaling pathway. NF1 mutants show defects in growth and in synaptic transmission. The growth defect is rescued by a mutant protein that lacks GAP activity and appears to require a non cell autonomous function of NF1. The experiments described here are aimed at elucidating the apparently Ras1- independent functions of the NF1 gene and its role in regulating growth via a non cell autonomous mechanism. Specific Aim 1 is to provide a detailed description of how NF1 regulates growth in vivo. Included is an analysis of the interaction between NF1 and the cAMP/PKA pathway. Specific Aim 2 is to define the signaling pathways that mediate Ras-independent functions of NF1 in vivo by identifying domains in the NF1 protein that interact with other pathways. Specific Aim 3 is to identify genes that interact with NF1 in vivo. These will be identified either by a genetic screen for enhancers and suppressors of the pupal size phenotype of a hypomorphic NF1 allele or by using a microarray of cDNA clones and looking for significant alteration in gene expression in NF1 mutants. These studies will help us understand the normal function of the NF1 gene in vivo and will contribute to our understanding of the diverse manifestations of the disease process.