Cloning of the human glycine transporter type 1: molecular and pharmacological characterization of novel isoform variants and chromosomal localization of the gene in the human and mouse genomes.
Cloning of the human glycine transporter type 1: molecular and pharmacological characterization of novel isoform variants and chromosomal localization of the gene in the human and mouse genomes.
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发表时间：
1994-04
影响因子：
3.6
通讯作者：
Kyeong Man Kim;Stephen F. Kingsmore;H. Han;Teresa L. Yang-Feng;N. Godinot;Michael F. Seldin;M. G. Caron;Bruno Giros
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作者：
Kyeong Man Kim;Stephen F. Kingsmore;H. Han;Teresa L. Yang-Feng;N. Godinot;Michael F. Seldin;M. G. Caron;Bruno Giros
我们报告的分子克隆的cDNA编码的高亲和力的人甘氨酸转运蛋白。一个1914个核苷酸的开放阅读框编码一个638个氨基酸的蛋白质，该蛋白质以Na+/Cl（-）依赖的方式转运甘氨酸。与其他Na+/Cl（-）依赖性转运蛋白一样，根据其亲水性特征，它具有12个推定的跨膜结构域。该蛋白是先前从大鼠中分离的甘氨酸转运蛋白[甘氨酸转运蛋白1b型（GlyT-1b）]的人类同源物。除了人GlyT-1b，我们还表征了一种通过选择性剪接产生的新型功能同种型。这种亚型GlyT-1c与最近在大鼠中鉴定的GlyT-2不同，在GlyT-1b的氨基末端部分含有编码54个氨基酸的额外外显子，并且主要在脑中表达。这两种亚型是同一基因的产物，位于人类染色体1p31.3和小鼠染色体4上，靠近自发小鼠神经肌肉突变clasper的位点。当在COS-7细胞中表达时，人GlyT-1b和GlyT-1c都显示甘氨酸的时间和剂量依赖性摄取，当Na+或Cl-被其他离子取代时，甘氨酸的摄取被取消。对于GlyT-1b和GlyT-1c，对甘氨酸的亲和力相似，Km值为70-90 μ M，并且这种摄取被具有相似效力的肌氨酸抑制。除了存在于人类基因组中的三种转运蛋白同种型，即，GlyT-1a、GlyT-1b和GlyT-1c是点突变的变体，在COS-7细胞中表达时似乎完全没有甘氨酸摄取活性，通过聚合酶链反应扩增人黑质的mRNA获得。这些变体指向甘氨酸转运蛋白的区域，这些区域可能在该蛋白的加工或转运功能中很重要。
We report the molecular cloning of a cDNA encoding a high affinity human glycine transporter. An open reading frame of 1914 nucleotides encodes a 638-amino acid protein that transports glycine in a Na+/Cl(-)-dependent manner. In common with other Na+/Cl(-)-dependent transporters, it possesses 12 putative transmembrane domains, according to its hydropathicity profile. This protein is the human homologue of a glycine transporter previously isolated from rat [glycine transporter type 1b (GlyT-1b)]. In addition to the human GlyT-1b, we also characterized a novel functional isoform produced by alternative splicing. This isoform, GlyT-1c, which is distinct from GlyT-2 recently characterized in rat, contains an additional exon encoding 54 amino acids in the amino-terminal part of GlyT-1b and is mainly expressed in brain. These two isoforms are products of the same gene and are localized on human chromosome 1p31.3, as well as on mouse chromosome 4, close to the locus for the spontaneous mouse neuromuscular mutation clasper. When expressed in COS-7 cells, both the human GlyT-1b and GlyT-1c display a time- and dose-dependent uptake of glycine, which is abolished when either Na+ or Cl- is substituted with other ions. For both GlyT-1b and GlyT-1c the affinities for glycine are similar, with Km values of 70-90 microM, and this uptake is inhibited by sarcosine with similar potencies. In addition to the three transporter isoforms present in the human genome, i.e., GlyT-1a, GlyT-1b, and GlyT-1c, point-mutated variants, which appear to be totally devoid of glycine uptake activity when expressed in COS-7 cells, were obtained by polymerase chain reaction amplification of mRNA from human substantia nigra. These variants point to regions of the glycine transporter that might be important in the processing or transport function of this protein.