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Molecular Cloning and Functional Expression of the Human Glycine Transporter Glyt2 and Chromosomal Localisation of the Gene in the Human Genome

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Molecular Cloning and Functional Expression of the Human Glycine Transporter Glyt2 and Chromosomal Localisation of the Gene in the Human Genome FEBS Letters 439 (1998) 334^340 FEBS 21144 Molecular cloning and functional expression of the human glycine transporter GlyT2 and chromosomal localisation of the gene in the human genome J.A. Morrow*, I.T. Collie, D.R. Dunbar, G.B. Walker, M. Shahid, D.R. Hill Target Discovery Section, Organon Laboratories Limited, Newhouse, Lanarkshire ML1 5SH, UK Received 16 October 1998 Na/Cl3-dependent family includes transporters for Q-amino- Abstract Neurotransmitter transport systems are major targets for therapeutic alterations in synaptic function. We have cloned butyric acid (GABA) [11^15], norepinephrine/epinephrine and sequenced a cDNA encoding the human type 2 glycine [16], dopamine [17^22], serotonin [23,24], proline [25], choline transporter GlyT2 from human brain and spinal cord. An open [26], betaine [27], taurine [28^30], creatine [31] and glycine reading frame of 2391 nucleotides encodes a 797 amino acid [32^37]. protein that transports glycine in a Na+/Cl^-dependent manner. High-a¤nity glycine transporters have so far been shown to When stably expressed in CHO cells, human GlyT2 displays a be encoded by two separate genes. GlyT1 [32^34,36,37] occurs dose-dependent uptake of glycine with an apparent Km of 108 in three separate isoforms (GlyT1a, -1b and -1c) encoded by a WM. This uptake is not affected by sarcosine at concentrations up single gene which di¡er only in their amino-terminal region to 1 mM. Radiation hybrid analysis mapped the GlyT2 gene to [32]. GlyT1a and -1b originate from transcription directed D11S1308 (LOD = 8.988) on human chromosome 11p15.1^15.2. from alternate promoters whereas GlyT1c is a splice variant z 1998 Federation of European Biochemical Societies. of the -1b transcript [38]. GlyT1 is expressed predominantly in Key words: Glycine transporter; cDNA cloning; glial cells [38,39] of brain areas such as hippocampus, cortex Genomic localization; Neurotransmitter; Human spinal cord; and cerebellum, and also in the brain stem and spinal cord Brain stem; Cerebellum [33,37^41]. Based on its tissue distribution, GlyT1 has been suggested to co-localise with the NMDA receptor [37] where it may modulate the concentration of glycine at its co-agonist binding site [3]. Indeed, a recent study has demonstrated that 1. Introduction GlyT1b has the ability to control the response of the NMDA receptor when co-expressed in Xenopus oocytes [42]. The amino acid glycine is thought to have two major roles More recently, a second glycine transporter (GlyT2) was in mammalian central neurotransmission. Firstly, glycine acts reported [35] and shown to be encoded by a separate gene. as the major inhibitory neurotransmitter in the caudal brain GlyT2 is distinguishable from GlyT1 in terms of primary and spinal cord [1] where its action is mediated by the strych- structure (48% amino acid sequence identity), tissue distribu- nine-sensitive glycine receptor to produce inhibitory post-syn- tion and pharmacological properties [35]. In contrast to aptic potentials [2]. In addition to this inhibitory role, glycine GlyT1, GlyT2 is predominantly neuronal in nature [39], and also modulates excitatory neurotransmission by potentiating restricted to the spinal cord, brain stem, and to a lesser extent the action of glutamate at N-methyl-D-aspartate (NMDA) re- the cerebellum [39,41,43,44] where it co-localises with strych- ceptors [3,4]. nine binding sites [44]. This correlation with the distribution The termination of action of most neurotransmitters is of inhibitory, strychnine-sensitive glycine receptors suggests mediated by rapid re-uptake into the pre-synaptic terminal that GlyT2 is involved in the termination of inhibitory neuro- or surrounding glial cells [5,6]. The active transport of neuro- transmission at strychnine-sensitive glycinergic synapses. transmitters across the plasma membrane is driven by the Abnormalities of neurotransmitter transport can contribute transmembrane Na gradient generated by the plasma mem- to neuropathological processes. For example, blockage of brane Na/K-ATPase [6]. To date, molecular cloning studies and/or reversal of the glutamate transporters during ischaemia have lead to the identi¢cation of two distinctive gene families or anoxia elevates the extracellular concentration of L-gluta- encoding neurotransmitter transporter proteins. These include mate to neurotoxic levels resulting in nerve cell damage the Na/K-dependent (but Cl3-independent) excitatory ami- [45,46]. Neurotransmitter transporters are also major targets no acid transporter family GLAST [7], EAAC-1 [8], GLT1 [9], for therapeutic and pathological alterations in synaptic func- and the more recently discovered EAAT4 [10]. The second, tion. This is particularly apparent by the ability of mono- amine reuptake inhibitors such as tricyclic antidepressants, *Corresponding author. Fax: (44) (1698) 732878. amphetamines and cocaine to produce dramatic behavioural E-mail: [email protected] changes [47]. Also, selective GABA reuptake inhibitors (e.g. tiagabine) are being developed as anticonvulsant and anxio- Abbreviations: GlyT, glycine transporter; NMDA, N-methyl-D-aspar- tate; PCR, polymerase chain reaction; RACE, rapid amplification of lytic agents [48]. Similarly, the development of selective GlyT2 cDNA ends; dNTP, deoxyribonucleotide triphosphate; DMEM, inhibitors could prove to be useful agents for modulating Dulbecco's modified Eagle's medium; FBS, foetal bovine serum; glycinergic neurotransmission in the caudal brain and spinal HBSS, Hanks' balanced salts solution cord. The nucleotide sequence presented here has been submitted to the In this study, we report the isolation of a cDNA from GenBank database under accession number AF085412. human brain and spinal cord which is homologous to the 0014-5793/98/$19.00 ß 1998 Federation of European Biochemical Societies. All rights reserved. PII: S0014-5793(98)01390-8 FEBS 21144 20-11-98 J.A. Morrow et al./FEBS Letters 439 (1998) 334^340 335 rat GlyT2 sequence. We show that it encodes a Na- and Cl3- pected (Fig. 4). In an attempt to increase expression from the dependent type 2 glycine transporter with similar pharmacol- pcDNA3/hGlyT2 construct, a genomic/cDNA fusion was created by including this intron in the expression construct. The intron was ogy reported for rat GlyT2, and that this transcript is simi- cloned by PCR using primers directed against exon sequence on either larly located in spinal cord, brain stem and cerebellum. side of the intron (sense: 5P-CCAGTCTTGTCAATAGCGGGTTT- CAC-3P; antisense: 5P-TGCCCTTGCCCACACTCACGTTCGCAT- 2. Materials and methods 3P) using human genomic DNA as a template. PCR was performed using the Clontech Advantage Genomic PCR kit (95³C 1 min, then 35 cycles of: 95³C, 15 s; 68³C, 12 min). This gave rise to a single frag- 2.1. Cloning of human GlyT2 ment of approximately 1800 bp that was puri¢ed and cloned into A human GlyT2 probe was prepared by performing PCR on pCR2.1 (Invitrogen). Dideoxy sequencing con¢rmed that this frag- a human whole brain cDNA library (Vgt11, Clontech) using a ment contained the intron £anked by exon sequence. The genomic/ pair of primers (sense: 5P-CAGTGGGGCTGGGTAATGTTTGG-3P cDNA fusion construct was then generated by purifying the EcoRI/ and antisense: 5P-AAAGATGATGGCCCAGAATGGAGA-3P) cor- Eco72I fragment from the pCR2.1/genomic sequence and ligating this responding to conserved regions in the ¢rst and eighth transmembrane with the Eco72I/XhoI fragment from pcDNA3/hGlyT2 into EcoRI/ domains of rat GlyT2. Reactions (100 Wl) contained 30 pmol of each XhoI-digested pcDNA3. primer, 2.5 U Taq DNA polymerase (Gibco BRL) in 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2 mM MgCl , and subjected to 40 cycles 2 2.3. Northern blot analysis (1 min, 94³C; 1 min, 60³C; 1 min 72³C). This gave rise a product of The cDNA insert from clone 10 was radiolabeled using the Phar- expected size (1045 bp) which was puri¢ed using the Geneclean II kit macia Ready-To-Go DNA labeling kit. Human multiple tissue North- (Bio 101) and ligated into the TA plasmid pCRII (Invitrogen). All plasmids were transformed into competent Escherichia coli TOP10FP ern blots (Clontech) were hybridised for 2 h at 68³C in ExpressHyb hybridisation solution (Clontech) and washed in 2USSC, 0.05% SDS cells (Invitrogen) and propagated using standard conditions [49]. The at room temperature, twice for 40 min with 0.1USSC, 0.05% SDS at DNA sequence of the PCR product was con¢rmed by dideoxy se- 50³C, then exposed to Hyper¢lm MP (Amersham) with one intensify- quencing using the Pharmacia autoread sequencing kit and ALF au- tomated DNA sequencer. This product was then used to screen hu- ing screen at 380³C for up to 6 weeks. man whole brain, spinal cord (both Vgt11) and brainstem-medulla (Vgt10) cDNA libraries (Clontech). Phage (40 000 per 150 mm diam- 2.4. Genomic localisation eter plate) were transferred to nitrocellulose ¢lters (Costar) as de- The genetic locus of hGlyT2, was determined by PCR assay of the scribed in Sambrook et al. [49]. Filters were prehybridised at 42³C Genebridge-4 radiation-reduced somatic cell DNA panel consisting of for 2 h with 50% formamide, 6USSC (0.9 M NaCl, 0.09 M sodium 84 hybrids each containing a previously characterised complement of citrate, pH 7.0), 5UDenhardt's (0.1% w/v polyvinylpyrrolidone, 0.1% human DNA [50] (UK MRC HGMP Resource Centre, Cambridge). w/v Ficoll type 400, 0.1% bovine serum albumin), and 100 Wg/ml This was performed using primers (sense: 5P-GGCTATTTCCA- sheared, denatured salmon sperm DNA. Hybridisation was performed TTCCCCCTTTACCCTTAC-3P and antisense: 5P-TGTTGGCTGG- overnight at 42³C in the same solution containing [32P]dCTP-labeled CGTTTATTCATTTCCTT-3P) directed against hGlyT2 genomic probe (DNA probes were prepared using the Pharmacia Ready-To- sequence (unpublished data) encompassing an intron/exon boundary Go DNA labeling kit). The ¢lters were washed twice with 2USSC, with sub-chromosomal mapping of the 334 bp PCR product being 0.1% SDS (room temperature), then twice with 0.5USSC, 0.1% SDS analysed via the Whitehead Institute radiation hybrid map- (50³C) and exposed to Hyper¢lm MP (Amersham).
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