Kim K M, Kingsmore S F, Han H, Yang-Feng T L, Godinot N, Seldin M F, Caron M G, Giros B
Howard Hughes Medical Institute Research Laboratories, Department of Cell Biology and Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Mol Pharmacol. 1994 Apr;45(4):608-17.
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.
我们报道了编码一种高亲和力人甘氨酸转运体的cDNA的分子克隆。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⁻依赖的转运体一样，根据其亲水性图谱，它具有12个假定的跨膜结构域。该蛋白质是先前从大鼠中分离出的甘氨酸转运体[甘氨酸转运体1b型（GlyT-1b）]的人类同源物。除了人GlyT-1b之外，我们还鉴定了一种由可变剪接产生的新型功能异构体。这种异构体GlyT-1c与最近在大鼠中鉴定的GlyT-2不同，在GlyT-1b的氨基末端部分包含一个额外的编码54个氨基酸的外显子，并且主要在脑中表达。这两种异构体是同一基因的产物，定位于人类染色体1p31.3以及小鼠染色体4上，靠近自发小鼠神经肌肉突变 claspers的基因座。当在COS-7细胞中表达时，人GlyT-1b和GlyT-1c都表现出对甘氨酸的时间和剂量依赖性摄取，当Na⁺或Cl⁻被其他离子替代时，这种摄取被消除。对于GlyT-1b和GlyT-1c两者，对甘氨酸的亲和力相似，Km值为70 - 90μM，并且这种摄取被肌氨酸以相似的效力抑制。除了人类基因组中存在的三种转运体异构体，即GlyT-1a、GlyT-1b和GlyT-1c之外，通过从人黑质的mRNA进行聚合酶链反应扩增获得了点突变变体，当在COS-7细胞中表达时，这些变体似乎完全缺乏甘氨酸摄取活性。这些变体指向甘氨酸转运体中可能在该蛋白质的加工或转运功能中起重要作用的区域。
