FUNCTIONAL ANALYSIS OF NF2 GENE MUTATIONS

描述（改编自申请人的摘要）：神经纤维瘤病（NF 2）是 一种遗传性疾病，其中受影响的患者发展为神经鞘瘤， 脑膜瘤和神经胶质瘤。 NF 2肿瘤抑制基因产物梅林， 与一个蛋白质家族有序列相似性， 具有肌动蛋白细胞骨架的膜糖蛋白（ERM蛋白家族）。 这就提出了merlin通过转导 通过细胞表面蛋白和肌动蛋白的细胞外信号 细胞骨架 这项资助旨在研究NF 2患者的突变 导致Merlin负生长调节缺陷。 具体来说，我们希望 为了检验Merlin函数中的缺陷是以下结果的假设， NF-2突变是由于产生（1）不稳定的梅林蛋白， (2)亚细胞分布改变的merlin蛋白，（3）merlin 形成分子内或分子间复合物的能力降低的蛋白质， 和/或（4）与Merlin结合能力降低的Merlin蛋白 效应蛋白 预测一些NF 2突变产生截短的 并因此导致体内不稳定的Merlin蛋白。 相反， 突变可导致产生稳定的Merlin蛋白， 抑制细胞生长的能力。 我们建议确定NF 2是否 患者突变或选择性剪接的梅林同种型导致 产生不稳定的梅林蛋白。 merlin的正常周转率 将在许旺细胞中建立， 分析NF 2患者突变和可变剪接对 merlin蛋白稳定性。 NF 2患者突变和merlin亚型将在 分析以确定选择性剪接和NF 2基因的作用 梅林作为负生长调节剂的能力的突变 无论是在体外还是在体内。 Merlin生长抑制剂活性将是 通过生长速率、FACS分析、锚定非依赖性生长和 在无胸腺（裸）小鼠中形成肿瘤的能力。 merlin亚型失效 或突变的梅林蛋白抑制细胞生长可能是由于受损的 与关键的梅林效应蛋白的相互作用。 既然梅林是个 作为ERM蛋白家族的一员，设计实验以确定 merlin的哪个区域对于与细胞膜的相互作用是必需的 蛋白质和肌动蛋白细胞骨架，通过分析NF 2患者的影响， 突变和选择性剪接对这些相互作用。 接下来 分子决定因素所需的梅林形成内和 Merlin生长所必需的分子间复合物 将分析抑制剂。 最后，潜在的梅林效应蛋白 将使用包括生物化学在内的多种方法进行鉴定， 和遗传相互作用系统。 上述战略旨在确定 梅林如何通过改变蛋白质相互作用来抑制生长 旨在了解这种新型肿瘤抑制基因的功能， 着眼于设计更有效的治疗方法， 哪个梅林表达式被改变了。

DESCRIPTION (Adapted from Applicant's Abstract): Neurofibromatosis (NF2) is an inherited disorder in which affected patients develop schwannomas, meningiomas, and gliomas. The NF2 tumor suppressor gene product, merlin, shares sequence similarity with a family of proteins that link integral membrane glycoproteins with the actin cytoskeleton (ERM protein family). This raises the possibility that merlin regulates cell growth by transducing an extracellular signal through cell surface-proteins and the actin cytoskeleton. This grant proposes to investigate how NF2 patient mutations lead to defects in merlin negative growth regulation. Specifically, we wish to test the hypothesis that defects in merlin function as a consequence of NF2 mutations result from the generation of (1) unstable merlin proteins, (2) merlin proteins with altered subcellular distributions, (3) merlin proteins with reduced abilities to form intra- or inter-molecular complexes, and/or (4) merlin proteins with reduced abilities to associate with merlin effector proteins. Some NF2 mutations are predicted to produce truncated and, therefore, unstable merlin proteins in vivo. In contrast, missense mutations may lead to the production of a stable merlin protein with reduced ability to suppress cell growth. We propose to determine whether NF2 patient mutations or alternatively spliced merlin isoforms result in the production of unstable merlin proteins. The normal rate of merlin turnover will be established in Schwann cells to provide the foundations for analyzing the effect of NF2 patient mutations and alternative splicing on merlin protein stability. NF2 patient mutations and merlin isoforms will be analyzed to determine the effect of alternative splicing and NF2 gene mutations on merlin's ability to function as a negative growth regulator both in vitro and in vivo. Merlin growth suppressor activity will be determined by growth rates, FACS analysis, anchorage-independent growth and ability to form tumors in athymic (nude) mice. Failure of merlin isoforms or mutant merlin proteins to suppress cell growth may result from impaired interactions with critical merlin effector proteins. Since merlin is a member of the ERM protein family, experiments are designed to determine which region of merlin are essential for interactions with cell membrane proteins and the actin cytoskeleton by analyzing the effect of NF2 patient mutations and alternative splicing on these interactions. Next, the molecular determinants required for merlin to form intra- and inter-molecular complexes necessary for merlin to function a growth suppressor will be analyzed. Finally, potential merlin effector proteins will be identified using a combination of approaches including biochemical and genetic interaction systems. The strategies outlined above to define how merlin functions to suppress growth through altered protein interactions are aimed at understanding the function of this novel tumor suppressor gene with an eye towards the design of more effective therapies for the tumors in which merlin expression is altered.