FUNCTIONAL ANALYSIS OF NF2 GENE MUTATIONS

描述(摘自申请者摘要)：神经纤维瘤病(NF2)是 一种遗传性疾病，受影响的患者会患上神经鞘瘤， 脑膜瘤和神经胶质瘤。NF2肿瘤抑制基因产物梅林， 与连接整体的一组蛋白质具有序列相似性 带有肌动蛋白细胞骨架的膜糖蛋白(ERM蛋白家族)。 这增加了Merlin通过转导调节细胞生长的可能性 通过细胞表面蛋白和肌动蛋白的胞外信号 细胞骨架。这项拨款建议调查NF2患者的突变 导致梅林负增长调控的缺陷。具体来说，我们希望 来检验这样的假设，即梅林的缺陷是由 NF2突变源于(1)不稳定的Merlin蛋白的产生， (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体能力降低的蛋白质， 和/或(4)与Merlin结合能力降低的Merlin蛋白 效应器蛋白。一些NF2突变被预测会导致截短 因此，体内不稳定的Merlin蛋白。相比之下，误解 突变可能导致稳定的Merlin蛋白的产生 抑制细胞生长的能力。我们建议确定NF2是否 患者突变或选择性剪接的Merlin亚型导致 产生不稳定的梅林蛋白。梅林的正常周转率 将在雪旺细胞中建立，为 分析NF2患者突变和选择性剪接对基因表达的影响 梅林蛋白的稳定性。NF2患者的突变和Merlin亚型将是 分析以确定选择性剪接和NF2基因的作用 梅林作为负生长调节因子功能的突变 在体外和体内都有。梅林增长抑制活性将是 由增长率、FACS分析、独立于锚定的增长和 在裸鼠体内形成肿瘤的能力。Merlin异构体的失效 或突变的Merlin蛋白抑制细胞生长可能是由于 与关键的Merlin效应蛋白的相互作用。因为梅林是一个 ERM蛋白家族的成员，实验旨在确定 Merlin的哪些区域是与细胞膜相互作用所必需的 蛋白与肌动蛋白细胞骨架分析对NF2患者的影响 这些相互作用上的突变和选择性剪接。接下来， 梅林形成Intra-And所需的分子决定因素 Merlin发挥生长功能所需的分子间络合物 抑制者将被分析。最后，潜在的Merlin效应蛋白 将使用包括生化方法在内的组合方法进行鉴定 和遗传相互作用系统。上面概述的战略将定义 Merlin如何通过改变蛋白质相互作用来抑制生长 旨在了解这种新的肿瘤抑制基因的功能 着眼于设计更有效的治疗肿瘤的方法 哪个梅林的表达被改变了。

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.