ANALYSIS OF NEUROFIBROMATOSIS TYPE 1 GENE FUNCTION

该建议旨在了解在人类疾病1型神经纤维瘤病中发生突变的基因的功能。该疾病的特点是多种多样的病理表现（如肿瘤、色素变化、行为异常、生长缺陷），其中许多病理表现的基础尚不清楚。关于NF1功能的主流观点是，它主要是Ras GTPase的负调节因子，作为Ras的GTPase激活蛋白（GAP）。为了研究NF1在可遗传操作生物体内的功能，克隆了一个高度保守的果蝇同源物并对其进行了表征。对果蝇NF1同系物的研究未能显示出NF1突变体中Ras1介导的信号传导改变的证据，这表明NF1作为Ras1调节剂的假设作用在体内可能是多余的或不那么重要。相反，NF1与编码cAMP/PKA信号通路组分的基因之间存在强烈的遗传相互作用。NF1突变体在生长和突触传递方面表现出缺陷。生长缺陷由缺乏GAP活性的突变蛋白拯救，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基因的明显不依赖于Ras1的功能及其通过非细胞自主机制调节生长的作用。具体目的1是提供NF1如何调节体内生长的详细描述。包括NF1与cAMP/PKA通路之间相互作用的分析。具体目标2是通过鉴定NF1蛋白中与其他途径相互作用的结构域，确定介导NF1 ras独立功能的体内信号通路。特异性目的3是鉴定体内与NF1相互作用的基因。这些突变体可以通过对NF1等位基因的蛹大小表型增强子和抑制子的基因筛选来鉴定，也可以使用cDNA克隆微阵列来寻找NF1突变体中基因表达的显著改变。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多种表现。

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.