Analysis of cellular function of Neurofibromatosis Type 2 tumor suppressor gene product (Merlin)

2型神经纤维瘤病（NF2）是一种常染色体显性遗传疾病，与包括双侧前庭神经鞘瘤和脑膜瘤在内的良性颅内肿瘤的发展密切相关。NF2基因最近被克隆，其编码的蛋白（称为merlin/schwannomin）被发现与细胞骨架相关蛋白moesin-ezrin-radixin （MER）家族惊人地相似。为了阐明NF2蛋白（merlin）的生物学功能，我们分析了merlin的突变模式、细胞定位以及通过merlin传递的细胞信号。1)采用蛋白截断法分析NF2患者的NF2基因突变。大多数NF2突变是无意义的，帧移位或外显子缺失突变。超过70%的突变集中在编码merlin的n端一半的区域。此外，在一些神经鞘瘤和脑膜瘤中发现了普遍存在的蛋白酶calpain对n -末端的蛋白水解裂解。这些结果表明，n -末端区域对肿瘤抑制功能至关重要。2)建立了突变体和野生NF2的细胞表达系统，并利用共聚焦激光扫描显微镜（CLSM）分析了它们的细胞定位。野生墨林蛋白表现出细胞质和膜下定位，这是由其核输出信号（NES）依赖的转运机制指导的。n端突变导致merlin的核积累。3)从牛脑提取物中纯化了merlin的细胞结合蛋白，通过对其内部氨基酸序列的分析，鉴定其为聚adp -核糖聚合酶、DNA-PK亚基ku -抗原85和70。merlin的n端（19-339）区域对它们的相互作用至关重要。免疫沉淀、western blotting和CLMS研究证实了它们的细胞共定位。NF2突变破坏梅林蛋白相关复合物的形成及其细胞信号传递。这种梅林细胞功能的丧失可能与颅内肿瘤的产生有关。少

The neurofibromatosis type 2 (NF2) is an autosomal dominantly inherited disorder, and strongly associated with development of benign intracranial tumors including bilateral vestibular schwannomas and meningiomas. NF2 gene was recently cloned, and the protein it encodes (termed as merlin/schwannomin) was found to be striking similarity to the moesin-ezrin-radixin (MER) family of cytoskeleton-associated proteins. To elucidate the biological function of NF2 protein (merlin), we analyzed the mutation pattern of merlin, its cellular localization, and the cellular signals via merlin. 1) NF2 gene mutations in NF2 patients were analyzed by protein truncation test. The majority of NF2 mutations are nonsense, frame shift, or exon-missing mutations. More than 70%of the mutations are clustered in the region encoding the N-terminal half of merlin. In addition, post-transrational proteolytic cleavage of N-terminal merlin by the ubiquitous protease calpain was found in some schwannomas and meningioma … More s. These results suggest that the N-terminal region of merlin is the most important for the tumor suppressive function, 2) Cellular expression systems of mutants or wild NF2 were established, and their cellular localization was analyzed by confocal lazer scanning Microscopy (CLSM). The wild merlin showed cytoplasmic and submembranous localization that was found to be directed by its nuclear export signal (NES) dependent transport mechanism. The N-terminal mutation resulted in nuclear accumulation of merlin. 3) Cellular binding proteins of merlin were purified from bovine brain extracts and identified as poly ADP-ribose polymerase, DNA-PK subunits Ku-antigen 85 and 70 by the analysis of their internal amino-acid sequences. The N-terminal(19-339) region of merlin is essential for their interaction. The immuno-precipitation, western blotting, and CLMS study confirmed their cellular co-localization. NF2 mutations impair the merlin-related complex formation and their cellular signal transport. This loss of cellular function of merlin may link to the intracranial tumor generation. Less