Chao Chuck C-K, Chang P-Y, Lu Henry H-P
Tumor Biology Laboratory, Department of Biochemistry, Chang Gung University, Taoyuan, Taiwan, Republic of China.
J Neurosci Res. 2005 Jul 15;81(2):153-62. doi: 10.1002/jnr.20552.
Gas7, a growth-arrest-specific protein, is expressed preferentially in the brain and is required for neurite outgrowth in cultured cerebellar and peripheral murine neurons. Gas7 interacts with F-actin and colocalizes with the terminal part of actin microfilament in cells in which membrane outgrowth is present. Gas7 isoforms were discovered in murine brain by alternative splicing. This work reports the identification of two human Gas7 cDNA: hGas7-a with 2,427 nucleotides, which encodes 330 amino acids, and hGas7-b with 2,610 nucleotides, which encodes 412 amino acids according to predicted open-reading-frames. The predicted hGas7-b protein is 97% homologous to murine homologues, whereas the hGas7-a is homologous to the mouse Gas7-cb form that is expressed preferentially in cerebellum. Alignment analysis of the Gas7 protein sequences revealed a high homology to that in humans: 99% for the monkey, 97% in murine, and around 75% for the puffer fish and chicken. The hGas7-b protein comprises a WW domain, which often associates with other domains that are typically present in proteins in signal transduction processes, and an FCH domain, which participates in rearranging the cytoskeleton. The hGas7-a comprises only the FCH domain. Analysis of the human Gas7 sequences using the DNA database revealed that the two forms resulted from the canonical alternative splicing of a Gas7 genomic sequence. The abundance of both hGas7 mRNA levels, determined by quantitative PCR in tissues including brain, breast cancer, placenta, and head-neck cancer, revealed that the level of hGas7-a was 14 times that of hGas7-b in these tissues. Transfection of cells with hGas7-a or hGas7-b cDNA yielded the predicted 38-kDa or 50-kDa protein, respectively. The ectopic expression of hGas7 caused neurite-like cell processes in both mouse Neuro-2a and human SH-SY5Y neuroblastoma cells. Interestingly, the hGas7-a preferentially elicited the small lamellipodia, whereas the hGas7-b elicited the small filopodia phenotype. These findings reveal the evolutionary conservation of the structure and function of Gas7. They also suggest that the FCH domain in Gas7 may participate in the development of lamellipodia, and the WW domain may participate in the fine-tuning of the filopodia.
Gas7是一种生长停滞特异性蛋白，在大脑中优先表达，是培养的小脑和外周鼠神经元神经突生长所必需的。Gas7与F-肌动蛋白相互作用，并与存在膜生长的细胞中肌动蛋白微丝的末端部分共定位。通过可变剪接在鼠脑中发现了Gas7亚型。这项工作报道了两种人类Gas7 cDNA的鉴定：hGas7-a有2427个核苷酸，根据预测的开放阅读框编码330个氨基酸；hGas7-b有2610个核苷酸，编码412个氨基酸。预测的hGas7-b蛋白与鼠类同源物有97%的同源性，而hGas7-a与优先在小脑中表达的小鼠Gas7-cb形式同源。Gas7蛋白序列的比对分析显示与人类的序列有高度同源性：与猴子的同源性为99%，与鼠类的同源性为97%，与河豚和鸡的同源性约为75%。hGas7-b蛋白包含一个WW结构域，该结构域通常与信号转导过程中蛋白质中典型存在的其他结构域相关联，以及一个FCH结构域，该结构域参与细胞骨架的重排。hGas7-a仅包含FCH结构域。使用DNA数据库对人类Gas7序列进行分析发现，这两种形式是由Gas7基因组序列的典型可变剪接产生的。通过定量PCR测定包括脑、乳腺癌、胎盘和头颈癌在内的组织中两种hGas7 mRNA水平的丰度，结果显示在这些组织中hGas7-a的水平是hGas7-b的14倍。用hGas7-a或hGas7-b cDNA转染细胞分别产生了预测的38 kDa或50 kDa蛋白。hGas7的异位表达在小鼠Neuro-2a和人SH-SY5Y神经母细胞瘤细胞中均引起了神经突样细胞突起。有趣的是，hGas7-a优先引发小片状伪足，而hGas7-b引发小丝状伪足表型。这些发现揭示了Gas7结构和功能的进化保守性。它们还表明Gas7中的FCH结构域可能参与片状伪足的发育，而WW结构域可能参与丝状伪足的微调。
