Zhang J S, Longo F M
Department of Neurology, University of California at San Francisco/Veterans Administration Medical Center 94121.
J Cell Biol. 1995 Feb;128(3):415-31. doi: 10.1083/jcb.128.3.415.
Receptor-linked tyrosine phosphatases regulate cell growth by dephosphorylating proteins involved in tyrosine kinase signal transduction. The leukocyte common antigen-related (LAR) tyrosine phosphatase receptor has sequence similarity to the neural cell adhesion molecule N-CAM and is located in a chromosomal region (1p32-33) frequently altered in neuroectodermal tumors. To understand the function of receptor-linked tyrosine phosphatases in neural development, we sought to identify LAR isoforms preferentially expressed in the nervous system and cellular processes regulating LAR alternative splicing. We report here the isolation of a series of rat LAR cDNA clones arising from complex combinatorial alternative splicing, not previously demonstrated for the tyrosine phosphatase-receptor gene family in general. Isoforms included: (a) deletions of the fourth, sixth and seventh fibronectin type III-like domains; (b) an alternatively spliced novel cassette exon in the fifth fibronectin type III-like domain; (c) two alternatively spliced novel cassette exons in the juxtamembrane region; (d) a retained intron in the extracellular region with in-frame stop codons predicting a secreted LAR isoform; and (e) an LAR transcript including an alternative 3' untranslated region containing multiple stretches of tandem CAG repeats up to 21 repeats in length. This number of repeats was in the range found in normal alleles of genes in which expansions of repeats are associated with neurodegenerative disease and the genetic phenomenon of anticipation. RT-PCR and Northern analysis demonstrated that LAR alternative splicing occurred preferentially in neuromuscular tissue in vivo and in neurons compared to astrocytes in vitro and was developmentally regulated. Alternative splicing was also regulated in PC12 cells by NGF, in 3T3 fibroblasts by cell confluence and in sciatic nerve and muscle subsequent to nerve transection. Western blot analysis demonstrated that alternatively spliced cassette exons result in the presence of corresponding amino acid segments of LAR protein in vivo. These studies suggest specialized functions of LAR isoforms in the nervous system and support our hypothesis that LAR-like tyrosine phosphatase receptors play a role in neural development and regeneration.
受体连接的酪氨酸磷酸酶通过使参与酪氨酸激酶信号转导的蛋白质去磷酸化来调节细胞生长。白细胞共同抗原相关(LAR)酪氨酸磷酸酶受体与神经细胞粘附分子N-CAM具有序列相似性,且位于神经外胚层肿瘤中经常发生改变的染色体区域(1p32 - 33)。为了解受体连接的酪氨酸磷酸酶在神经发育中的功能,我们试图鉴定在神经系统中优先表达的LAR异构体以及调节LAR可变剪接的细胞过程。我们在此报告了一系列源自复杂组合可变剪接的大鼠LAR cDNA克隆的分离,这在酪氨酸磷酸酶受体基因家族中一般未曾被证实。异构体包括:(a)第四、第六和第七个III型纤连蛋白样结构域的缺失;(b)在第五个III型纤连蛋白样结构域中的一个可变剪接的新型盒式外显子;(c)在近膜区域的两个可变剪接的新型盒式外显子;(d)细胞外区域中保留的内含子,带有框内终止密码子,预测一种分泌型LAR异构体;以及(e)一个LAR转录本,包括一个可变的3'非翻译区,含有多段串联的CAG重复序列,长度可达21个重复。这种重复次数处于与神经退行性疾病和遗传早现现象相关的基因正常等位基因中发现的范围内。逆转录聚合酶链反应(RT-PCR)和Northern分析表明,LAR可变剪接在体内的神经肌肉组织以及与体外星形胶质细胞相比的神经元中优先发生,并且受到发育调控。在PC12细胞中,可变剪接受神经生长因子(NGF)调控;在3T3成纤维细胞中,受细胞汇合调控;在坐骨神经横断后的坐骨神经和肌肉中也受到调控。蛋白质免疫印迹分析表明,可变剪接的盒式外显子导致体内LAR蛋白相应氨基酸片段的存在。这些研究表明LAR异构体在神经系统中具有特殊功能,并支持我们的假设,即LAR样酪氨酸磷酸酶受体在神经发育和再生中发挥作用。