Farmer M K, Robbins M J, Medhurst A D, Campbell D A, Ellington K, Duckworth M, Brown A M, Middlemiss D N, Price G W, Pangalos M N
Department of Neuroscience Research, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex, CM19 5AW, United Kingdom.
Genomics. 2000 Dec 1;70(2):241-52. doi: 10.1006/geno.2000.6387.
Orphan transporters form a growing subfamily of genes related by sequence similarity to the Na+/Cl- -dependent neurotransmitter superfamily. Using a combination of database similarity searching and cloning methods, we have identified and characterized two novel human orphan transporter genes, v7-3 and NTT5. Similar to other known orphan transporters, v7-3 and NTT5 contain 12 predicted transmembrane domains, intracellular N- and C-terminal domains, and large extracellular loops between transmembrane (TM) domains 3 and 4 and between TM domains 7 and 8. Residues within the extracellular loops are also predicted to contain sites for N-linked glycosylation. Human v7-3, the species orthologue of rat v7-3, contains an open reading frame (ORF) of 730 amino acids. Human NTT5 is a new member of the orphan transporter family and has an ORF of 736 amino acids. The amino acid sequences of human v7-3 and NTT5 are greater than 50% similar to other known orphan neurotransmitter transporters and also show sequence similarity to the human serotonin and dopamine transporters. Radiation hybrid mapping located the human v7-3 and NTT5 genes on chromosomes 12q21.3-q21.4 and 19q13.1-q13.4, respectively. Human mRNA distribution analysis by TaqMan reverse transcription-polymerase chain reaction showed that v7-3 mRNA is predominantly expressed in neuronal tissues, particularly amygdala, putamen, and corpus callosum, with low-level expression in peripheral tissues. In contrast, NTT5 mRNA was highly expressed in peripheral tissues, particularly in testis, pancreas, and prostate. Transient transfection with epitope-tagged transporter constructs demonstrated v7-3 to be expressed at the cell surface, whereas NTT5 was predominantly intracellular, suggestive of a vesicular location. Although the substrates transported by these transporters remain unknown, their specific but widespread distribution suggests that they may mediate distinct and important functions within the brain and the periphery.
孤儿转运体构成了一个不断扩大的基因亚家族，其通过序列相似性与Na⁺/Cl⁻依赖性神经递质超家族相关。通过结合数据库相似性搜索和克隆方法，我们鉴定并表征了两个新的人类孤儿转运体基因，v7-3和NTT5。与其他已知的孤儿转运体相似，v7-3和NTT5包含12个预测的跨膜结构域、细胞内的N端和C端结构域，以及跨膜（TM）结构域3和4之间以及TM结构域7和8之间的大的细胞外环。细胞外环内的残基也预计含有N-连接糖基化位点。人类v7-3是大鼠v7-3的物种直系同源物，包含一个730个氨基酸的开放阅读框（ORF）。人类NTT5是孤儿转运体家族的新成员，有一个736个氨基酸的ORF。人类v7-3和NTT5的氨基酸序列与其他已知的孤儿神经递质转运体的相似性大于50%，并且也与人类血清素和多巴胺转运体显示出序列相似性。辐射杂种图谱分析将人类v7-3和NTT5基因分别定位在染色体12q21.3-q21.4和19q13.1-q13.4上。通过TaqMan逆转录-聚合酶链反应进行的人类mRNA分布分析表明，v7-3 mRNA主要在神经组织中表达，特别是杏仁核、壳核和胼胝体，在周围组织中表达水平较低。相比之下，NTT5 mRNA在周围组织中高度表达，特别是在睾丸、胰腺和前列腺中。用表位标记的转运体构建体进行瞬时转染表明v7-3在细胞表面表达，而NTT5主要位于细胞内，提示其位于囊泡中。尽管这些转运体转运的底物仍然未知，但它们特异但广泛的分布表明它们可能在大脑和外周介导不同且重要的功能。
