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DNA Sequence-]. DNA Sequencing and Mapping, Vol. 2, pp. 21 1-2 18 Reprints available directly from the publisher Photocopying permitted by license only
0 1992 Harwood Academic Publishers CmbH Printed in the United Kingdom
The human glutamate receptor cDNA GluR1: cloning, sequencing, expression and localization to chromosome 5 M.-C. POTIER*, M.G. SPILLANTINI and N.P. CARTERt Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England and t Department ofPathology, University of Cambridge, Cambridge CB2 1 QP, England EMBL Data Library Accession No. X58633
The rat glutamate receptor i s a 907 amino acid transmembrane protein. Using the rat GluRl cDNA as a probe, we have isolated cDNA clones from a human hippocampal cDNA library. Sequence of a full length cDNA clone revealed 98,2% and 89.4% identity to the rat sequence at the amino acid and nucleotide levels respectively. The human cDNA clone detected an RNA transcript in human cerebral cortex, hippocampus and cerebellum, similar to that seen in rat. In situ hybridization experiments showed that human CluRl mRNA i s present in granule and pyramidal cells in the hippocampal formation and that there i s no apparent difference of distribution between control patient and patient with Alzheimer's disease. Dot blot analysis of flow-sorted human chromosomes showed that the CluRl gene maps to chromosome 5. KEY WORDS: glutamate receptor, human chromosome 5, in 5 i f U hybridization, human brain
INTRODUCTION L-glutamate i s the major excitatory neurotransmitter in the central nervous system of mammals. It interacts with two types of membrane receptors: the ionotropic receptors which form transmembrane channels permeant to cations and the metabotropic receptors which are coupled to G protein and stimulate inositol phosphate/Ca2+ intracellular pathways. lonotropic receptors can be classified according
*Corresponding author, present address: Centre National de la Recherche Scientifique, Laboratoire de Physiologie Nerveuse, 91 198 Gif-sur-Yvette Cedex, France.
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to their pharmacological properties. NMDA (Nmethyl-D-aspartate) receptors are coupled to large conductance channels permeant to monovalent cations and Ca" whereas AMPA/kainate (AMPA: a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors are permeant to monovalent cations only, mainly Na' under physiological conditions. The amino acid sequences of some non-NMDA glutamate receptors from rodent brain have been obtained through expression cloning. A rat metabotropic receptor consists of 1199 amino acids containing 7 putative transmembrane domains with no sequence similarities to conventional G protein-coupled receptors (Masu et a/., 19911. AMPA/kainate receptors are heterooligomers of at least three classes of related subunits of about 900 amino acids each with 4 transmembrane domains (Hollmann et a/.,l989; Keinanen et a/., 1990; Boulter et a/.,1990; Sommer et a/., 1990; Nakanishi et a/., 1990; Bettler et a/., 1990; Sakimura et a/., 1990; Egebjerg et a/., 1991; Werner e t a / . , 1991). The four known members of the first class (GIuRI to GkuR4) share 56 to 73% amino acid identity. The three known members of the second class (GIuR5-1, GluR5-2 and GluR6) share 80% amino acid identity with each other. These two classes of subunits are 40% identical. Finally, the only member of the third class i s a high affinity kainate receptor (KA-1) sharing 30% amino acid identity with other classes. Giutamate receptors are involved in experimental neurotoxicity (Choi, 1988) and have been
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M.-C. POTIER ET AL.
implicated in pathological processes. After electrolytic seizure in rat, the GIuR1 mRNA i s downregulated in specific brain regions (Gall et a/., 1990). A 7'0% decrease of GluRl mRNA has recently been reported in hippocampus of schizophrenic patients, although an increase in specific kainate binding sites had been demonstrated previously (Harrison el a / . , 1991; Kerwin et a/., 1988). Finally, glutamate receptors have been shown to be modified in Alzheimer's disease (Maragos et a / . , 1987'; Harrison et a/., 1990). In the present report, we describe the cloning, sequencing, expression and chromosomal localization of the human GluRl receptor cDNA.
RESULTS Isolation of human kainate receptor cDNA clones Screening of 100,000 plaques from a human hippocampal cDNA library with a radiolabelled PCR product derived from the nucleotide sequence encoding the C terminal half of the rat glutamate receptor GluRl gave 18 positive clones (KR1 to KR18). Twelve clones were plaque purified after three rounds of screening with a radiolabelled PCR product derived from the nucleotide sequence encoding the N terminal half of the rat GluRl. Six clones had an insert of more than 2 kb and two 'KR4 and KR9) had an insert of about 3 kb. Sequencing of human GluRl cDNA clone K R 4 The 3 kb EcoRl fragment of KR4 was subcloned into M13 mp18 and sequenced on both strands. It consisted of an open reading frame of 2722 nucleotides following with 197 nucleotides of 5 ' untranslated sequence and ending with 74 nucleotides of 3 ' untranslated sequence after the inframe stop codon (Fig. 1 ). The open reading frame codes for a protein of 906 amino acids which shows 98.2% identity with the rat GluRl receptor and 98.4% identity with the mouse GluRl receptor (Fig. 2). Comparison of the human and rat nucleotide sequences shows 89.4% conservation . The deduced protein sequence contains a putat i v e signal peptide o i 18 amino acids at its N terminus. There are 6 putative glycosylation sites, and 4 potential phosphorylation sites: one for Ca'+/calmodul ine-dependent protein kinase I 1 and three for protein kinase C. 4nalvsis ot the deduced amino acid sequence
for hydrophobicity suggests the presence ot four putative transmembrane segments (M, to M4). These sequence data will appear in the EMB L/G e n Ba n k/D DBJ N uc Ieot id e Sequence Da t abases under the accession number X58633 H U M A N GluRl cDNA. Expression of GluRl in normal and Alzheimer brains The distribution of GluRl transcripts was studied by in situ hybridization in the human hippocampal formation and by Northern blot analysis using single stranded D N A probes in the anti-mRNA sense orientation. Northern blot analysis of poly(A+)RNAfrom cortex, hippocampus and cerebellum of two control patients, cortex of two patients who died of Alzheimer's disease and rat brain showed the same major band of 5.2 kb as determined by Hollman et a / . (19891, stronger in rat than i n human (data not shown). Distribution of hybridization positive cells was high in the dentate gyrus and lower in the cornu ammonis with no apparent difference between the fields (Fig. 3a). Hybridization was specific, since only background labelling was detected when a probe in the mRNA sense orientation was used (Fig. 3b). The same qualitative distribution of hybridization positive cells was observed in both control and Alzheimer brains. There was a slrong labelling of granule celli-in the dentate gyrus (Fig. 3d) and pyramidal cells throughout the cornu ammonis (Fig. 3c and e) and enthorinal cortex (Fig. 3f) although not all granule or pyramidal cells were positive. Chromosomal localization The chromosonal localization of GluRl was studied by hybridization to flow sorted human chromosomes. All human chromosomes could be sorted separately, except for chromosomes 9 to 12. Dot blot analysis of flow sorted chromosomes showed that the single stranded radiolabelled Sac1 fragment of KR4 hybridized only to the left dot of the filter containing chromosomes 5 and 6. indicating localisation to chromosome 5 (Fig. 4).
DISCUSSION A cDNA clone encoding the human equivalent ot one subunit of the rat glutamate receptor (CIuR1)
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HUMAN GLUTAMATE RECEPTOR cDNA
CGAAAACAACACCCAGAACAGCGACAAGAATAAAGGGAAAGGGGGGGAAACACCAAATCTATGAITGGACCTGGGCTTCTTTTTCGCCAATCCAAAAAGGAAT M Q E I F A F F C T G F L C A V V G A N F P N N I Q I G G L F P N Q Q S Q E E A ATGCAGCACATTTITCCCTTCTTCIGCACCGGIITCCTAGGCGCGGTAGTAGGTGCCAATTTCCCCAACAAIAICCAGATCGGGGGATTATTTCCAAACCAGCAGTCACAGGAACAIGCT 10
20
30
40
SO
60
70
80
90
110
100
120
A F R F A L S Q L T E P P K L L P Q I D I V N I S D S F E ~ I Y R F C S Q F S X GCTTTTACATIICCITTGTCGCAACTCACAGAGCCCCCGAAGCTGCTCCCCCAGATIGATATIGTGAACATCAGCCACAGCTTTGAGAIGACCIATAGATTCTGTTCCCAGTTCTCCAAA 130
150
lU0
160
170
180
190
200
210
220
2 30
240
G V W A I F G F Y E R R T V N H L T S F C G A L E V C F I T P S F P V D T S N Q CCAGTCTATGCCAICTITGCGTTTIATGAACGIAGGACTGTCAACATGCTGACCTCCTTTTGTGGCGCCCTCCACGTCTGCTICATTACGCCGAGCTTICCGGTTGAIACATCAAATCAG 250
260
270
280
290
300
310
320
330
350
340
360
F V L Q L R P E L Q D A L I S I I D E Y K U Q X F V Y I Y D A D R G L S V L Q X TTTCTCCTTCAGCTGCGCCCTGAACTGCAGGATGCCCTCATCAGCATCATTGACCATTACAAGTGGCAGAAATTTGTCTACATTTATGATGCCGACCGGGGCTTAICCGTCCIGCAGAAA 370
V
L
D
390
380
T
A
A
E
K
N
U
410
400
Q
V
T
A
V
N
(20
I
L
T
T
E
E
450
UP0
130
T
G
Y
R
M
L
F
Q
4 60
D
L
E
410
K
K
K
480
E
R
L
V
CTCCTCCATACAGCTGCTGAGAAGAACTGGCAGGTGACAGCAGTCAACATTTTGACAACCACAGAGCAACGATACCGGATGCTCTTTCAGGACCTGGAGAAGAAAAAGGAGCGGCTGGTG
Mitochondrial DNA Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/21/14 For personal use only.
490
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V V O C E S E R L N A I L G Q I I X L E K N G I G Y ~ Y I L A N L G F M D I D L GTGGTCGACIGTGAATCAGAACGCCTCAATGCTATCTTGGGCCAGATTATAAAGCTACAGAAGAATGGCATCGGCIACCACTACATTCTTGCAAATCTGGGCTTCA~GGACATIGACTTA 620
610
650
640
630
660
67 0
680
690
710
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720
N K F X E S G A N V T C F Q L V N Y T D T I P A K I H Q Q U K N S D A R D E T R AACAAATTCAACGAGACTGCCGCCAAIGTGACAGGTTTCCAGCTGGTGAACTACACAGACACTATTCCGGCCAAGATCATCCACCAGTCGAAGAATAGTGATGCTCCAGACCACACACGG 730
740
750
760
770
780
790
800
810
820
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V D U K R P K Y T S A L T Y D C V X V H A E A F Q S L R R Q R I D I S R R G N A CTCCACICGAACACACCCAAGTACACCTCTGCGCTCACCTACGAIGGGCIGAAGGTGATGGCTGAGCCTTTCCACAGCCTGCCGAGCCACACAA~TGATATATCTCGCCGCGGGAATGCT 850
860
870
880
890
900
910
920
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960
C D C L A N P A V P U G Q G I D I Q R A L Q Q V R F E G L T C N V Q F N E K G R CCCCATTCTCTCCCTAACCCAGCTGTTCCCTGGGGCCAAGGGATCGACATCCAGAGAGCTCTGCAGCAGGTCGCGTTTGAAGCTTTAACACGAAACCTGCAGTT~AATGAGAAAGGACGC 97 0
980
990
1000
1010
1020
1030
1040
1060
1050
1070
1080
R T N Y T L B V I E H K E D C I R K I G Y W N E D D K ~ V P A A T D A Q A G G D CCCACCAACTACACCCTCCACGTCATTCAAATGAAACATCACGGCATCCCAAACATTGCTTACTCCAATCAACArGATAACTTTCTCCCTGCACCCACCCATGCCCAACCTCCCGCCCAT 1090
1100
1110
1120
1130
1140
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1160
1170
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N S S V Q N R T Y I V T T I L E D P Y V U L K X N A N Q ~ C C N D R X E C Y C AATTCAACTCTTCACAACACAACATACATCGTCACAACAATCCTAGAAGATCCTTATCTGATGCTCAACAACAACCCCAATCACTTTGACCCCAATCACCCTTACCACGGCTACTCTC~A 1210
1220
1230
1240
1250
1260
1270
1290
1300
1290
1310
V
1320
E L A A E I A K E V G Y S Y R L E I V S D G X Y G A R D P D T K A U N C M V G E GACCTGCCGCCAGAGATTGCCAAGCACGTGGGCTAC~CCTACCGTCTGGAGATIGTCAGTGATGGAAAATACGGAGCCCGAGACCCTCACACGAAGGCCTGGAATGCCATGGTGGCACAG 1330
1340
1350
1360
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1390
1400
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L V Y G R A D V A V A P L T I T L V R E C V I D F S K P F U $ L G I S I H I K K CTCGTCTATCCAAGAGCACATGTGGCTGTGGCTCCCCTTACTATCACTTTGGICCGGGAAGAAGTTATAGATTTCTCCAAACCATTTATCAGTTTGGGCATCTCCATCATGATTAAAAAA 1460
1450
1470
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P Q K S K P C V F S F L D P L A Y E I U H C I V F A Y I G V S V V L F L V S R F CCACACAAATCCAAGCCCGGIGTCITCTCCTTCCTTGATCCTTTGGCTTAIGAGATTTGGAIGTGCATTGTTTTTGCCTACATTGGAGTGAGTGTTGTCCTCTTCCTGGTCAGCCGCTTC 1510
1580
1590
1600
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1620
1630
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1640
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S P Y E W B S E E F E E C R D Q T T S D Q S N E F G I F N S L U F S L G A F M Q AGTCCCIATGAATCGCACAGTGAAGAGTTTGAGGAAGGACGGGACCAGACAACCAGTGACCAGTCCAATGAGTTTGGGATATTCAACAGTTIGTGGTTCTCCCTGCGACCCTTCATGCAG 1690
1700
1710
1120
1730
1740
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Q G C D I S P R S L S G R I V C C V W U F F T L I I I S S Y T A N L A A F L T V CAACGATCTGACATITCTCCCAGGTCCCTGTCTGGTCGCATCGICGGTGGCGTCTCGTGGT~CTTCACCTTAATCATCATCTCCTCATATACACCCAATCTCGCCGCCTTCCTGACCGTG 1810
1820
1830
1850
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lP00
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E R H V S P I E S A E D L A K Q T E I A Y G T ~ E A G S T K E F F R R S K I A GAGAGGATGGTGTCICCCATTGAGAGTGCAGAGCACCTAGCGAAGCAGACAGAAATTCCCTACGGGACGCTGGAAGCAGGATCTACTAAGGAGTTCTTCACGAGGTCTAAAATTGCAGTG 1930
1940
1950
1960
1910
1980
1990
2000
2010
2020
2030
V
2040
F E K H W T Y M K S A E P S V F V R T T E E G U I R V R K S K G X Y A Y L L E S TITCAGAAGATCTCCACATACATGAAGICAGCAGAGCCATCAGTTITTGTGCGGACAACAGAGGAGGGGATCATTCGAGIGAGGAAATCCAAAGGCAAATATGCCTACCTCCTGGAGTCC 2050
2060
2070
2080
2090
2100
2110
2120
2130
2140
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? H N E Y I E Q R K P C D T M K V C C N L D S K G Y G I ~ T P K G S A L R N P V ACCAIGAATGACTACATICAGCAGCGGAAACCCTGTGACACCATGAAGGTGGGAGGTAACTTGGATTCCAAAGGCTATGGCATTGCAACACCCAAGCGGTCTGCCCTGAGAAATCCAGTA 2170
2180
2190
2200
2210
2220
2230
2240
2250
2260
2270
2280
N L A V L K L N E Q C L L D K L K N X U U Y D X G E C G S G G G D S K D K I S A AACCTCGCAGTGITAAAACTGAACGAGCAGGGGCTTTTGGACAAATTGAAAAACAAATGGTGGTACGACAAGGGCGAGTGCGGCAGCGGGGGAGGTGATTCCAAGGACAAGACAAGCGCI 2290
L
S
L
2300
S
N
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A
2310
G
V
F
2320
Y
I
2330
L
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2340
G
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A
2350
H
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2360
A
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2370
E
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C
Y
2380
K
S
R
2390
S
Z
S
2400
K
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H
K
CICAGCCTCAGCAATGICCCACGCGTGTTCTACATCCTGATCGGAGGACTTGGACTAGCCATGCTGGTTGCCTTAATCGAGTTCTGCTACAAATCCCGTAGTGAATCCAAGCGGATGAAG 2420
2410
G
F
C
L
I
P
Q
2U30
Q
S
I
2440
N
E
A
2450
I
R
T
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2460
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2470
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2480
G
A
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2490
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2500
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2510
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2520
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CCITITICTTIGATCCCACAGCAATCCATCAACGAAGCCATACGGACATCGACCCTCCCCCGCAACAGCGGGGCAGGAGCCAGCAGCGGCGGCAGTGGAGAGAATCGTCGCGIGGTCAGC 2530
2540
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~ D F P K S M Q S I P C H S E S S G H P L G A T ~ ~ ~ C A T C A C T T C C C C A A C I C C A T ~ ~ A A I ~ ~ A I I ~ ~ T T ~ ~ A T ~ A ~ ~ ~ A ~ A ~ T T C A G ~ ~ A T ~ C C C T T G G G A ~ C C A C 2~1~3 0~ A T T G T A 2 1A4C0 T G G A G C2 A 7 5G0A T G C A G2A7 6C0C C C I T G G G G A G C A 2680 2690 2700 2110 2120 2650 2660 2670 TCCCCAGCCCCAICCCAAACCCIICAGTGCCAAAAACA~CAACAAAATGAAACGCAACCGCAAI~C 2770
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Figure 1 Nucleotide and deduced amino acid sequence of cDNA clone KR4 encoding the human glutamate receptor subunit GluRl. The amino acid sequence i s shown above the nucleotide sequence. Nucleotides are numbered in the 5 ' to 3 ' direction beginning with the first residue of the initiation codon. The in-frame stop codon i s indicated with an asterisk.
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M.-C. POTIER ET AL.
Rat Human
Mouse
b
MPYIFAFFCT GFLGAW AN FPNNIQIGGL FPNQQSQEHA AFRFALSQLT EPPKLLPQID I V ~ S C T F E M MQHIFAFFCT GFLGAWGAN FPNNIQIGGL FPNQQSQEHA AFRFALSQLT EPPKLLPQID IVNISDSFEK MPYIFAFFCT GFLGAWGAN FPNNIQIGGL FPNQQSQEHA AFRFALSQLT EPPKLLPQID IVNISDTFEK TYRFCSQFSK GWAIFGFYE RRTVNMLTSF CGALHVCFIT PSFPVDTSNQ FVLQLRPELQ EALISIIDHY TYRFCSQFSK GWAIFGFYE RRTVNMLTSF CGALHVCFIT PSFPVDTSNQ FVLQLRPELQ DALISIIDHY TYRFCSQFSK GWAIFGFYE RRTVNMLTSF CGALHVCFIT PSFPVDTSNQ FVLQLRPELQ EALISIIDHY
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hWQTFVYIYD ADHGLSVLQR VLDTAAEKNW QVTAVNILTT TEEGYRMLFQ DLEKKKERLV WDCESERLN KWQKFWIYD ADHGLSVLQK VLDTAAEKNW QVTAVNILTT TEEGYRMLFQ DLEKKKERLV WDCESERLN hWQTFLYIYD ADRGLSVLQR VLDTAAEKNW QVTAVNILTT TEEGYRMLFQ DLEKKKERLV WDCESERLN
*
*
AILGQIVKLE KNGIGYHYIL ANLGFMDIDL NKFKESGRNV TGFQLVNYTD TIPARIMQQW RTSDSRDHTR AILGQIIKLE KNGIGYHYIL ANLGFMDIDL NKFKESGANV TGFQLVNYTD TIPAKIMQQW KNSDARDHTR AILGQIVKLE KNGIGYHYIL ANLGFMDIDL NKFKESGANV TGFQLVNYTD TIPARIMQQW RTSDARDHTP ‘l’DWKRPKYTS ALTYDGVKVM AEAFQSLRRQ RIDISRRGNA GDCLANPAVP WGQGIDIQRA LQQVRFEGLT LQWKRPKYTS ALTYDGVKVM AEAFQSLRRQ RIDISRRGNA GDCLANPAVP WGQGIDIQRA LQQVRFEGLT VDWKRPKYTS ALTYDGVKVM AEAFQSLRRQ RIDISRRGNA GDCLANPAVP WGQGIDIQRP. LQQVRFEGLT GNVQFNEKGR ZNVQFNEKGR 2NVQFNEKGR
*
*
*
RTNYTLHVIE MKHDGIRKIG YWNEDDKFVP AATDAQAGGD NSSVCNRTYI VTTILEDPYV RTNYTLHVIE MKHDGIRKIG YWNEDDKFVP AATDAQAGGD NSSVQNRTYI VTTILEDPYV RTNYTLHVIE MKHDGIRKIG YWNEDDKFVP AATDAQAGGD NSSVQNRTYI VTTILEDPY\‘
MLKKNANQFE GNDRYECYCV ELAAEIAKHV GYSYRLEIVS DGKYGARDPD TKAWNGMVGE MLKKNANQFE GNDRYEGYCV ELAAEIAKHV GYSYRLEIVS DGKYGARDPD TKAWNGMVGE MLKKNANQFE GNDRYEGYCV ELAAEIAKHV GYSYRLEIVS DGKYGARDPD TKAWNGMVGE
SVYGRADVAV LVYGRADVAL’ LVYGRADVAi’
APLTITLVRE EVI DFSKPFM SLGISIMIKK PQKSKPGVFS FLDPLAYEIW MCIV a Y IGV SWJ,FLVSRF APLTITLVRE EVIDFSKPFM SLGISIMIKK PQKSKPGVFS FLDPLAYETW MCIV U Y I GV S W U S R F APLTITLVRE EVIDFSKPFM SLGISIMIKK PQKSKPGVFS FLDPLA-BYIG V V SWLFLVSRF SPYEWHSEEF EECRDQTTSD QSNEL W F S L W O G WSPRSL S G R I V G C W FFSPYEWHSEEF EECRDQTTSD Q S N E m N S LWFST,GaE%lc, OGCDLSPRSL S G R I V G ~ WFFTT.= SP‘r‘EWHSEEF EECRDQTTSD Q S N E m N S LW F S T , W OGCDLsPnsL SGRIVGrn m m W
L L L
T T T
0
4-
V ERMVSPIESA EDLAKQTEIA YGTLEAGSTK EFFRRSKIAV FEKMWTYMKS AEPSVFVRTT V ERMVSPIESA EDLAKQTEIA YGTLEAGSTK EFFRRSKIAV FEKMWTYMKS AEPSVF‘JRTT V ERMVSPIESA EDLAKQTEIA YGTLEAGSTK EFFRRSKIAV FEKMWTYMKS AEPSVFVRTT
0
0
v
FEGMIRVRKS KGFYAYLLES TMNEYIEQRK PCDTMKVGGN LDSKGYGIAT PKGSALRNPV NLAVLKLNEQ EEGMIRVRKS KGKYAYLLES TMNEYIEQRK PCDTMKVGGN LDSKGYGIAT PKGSALRNPV NLAVLKLNEQ EEGMIRVRKS KGKYAYLLES TMNEYIEQRK PCDTMKVGGN LDSKGYGIAT PKGSALRNPV NLAVLKLNEQ GLLDKLKNKW WYDKGECGTG GGDSKDKTSA LSLSNVAGVF YILIGGLGLA MLV U E F C Y KSRSESKRMK GLLDKLKNKW WYDKGECGSG GGDSKDKTSJ LSLSNVAGVF YILIGCLGLA MT,VATLEFCY KSRSESKRMK GLLDKLKNKW WYDKGECGSG GGDSKDKTSA LSLSNVAGVF YILIGGLGLA MLVALIEFCY KSRSESKRMK GFCLIPQQSI NEAIRTSTLP RNSGAGASOG GGSGENGRW SQDFPKSMQS IPCMSHSSGM PLGATG‘ CFCLIPQQSI NEAIRTSTLP RNSGAGASSG 0-SGENGRW SHDFPKSMQS IPCMSHSSGM PLGATGL YFCLIPQQSI NEAIRTSTLF RPTSGAGASOG SOSGENGRW SQDFPKSMQS IPCMSHSSSK PLG.\TGL
Figure 2 4lignment of deduced amino acid sequences for the rat, human and mouse glutamate receptor subunit CIuRT Amino acid ditterences between human and rat or human and mouse are in bold Cleavage of the signal peptide i s predicted at position indicated with the arrom Four proposed transmembrane domalns are underlined Potentlal extracellular N-glycosylation sites are indicated & ith a5terisks Putative phosphorylation sltes are indicated by a cross (Ca*/calmoduline-dependent proteinkinase Ill and dot5 (proteiri Linase C I Putative spice S i t < ’ 1 5 indicated with a triangle
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HUMAN GLUTAMATE RECEPTOR cDNA
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Figure 3 Cellular localization of human glutamate receptor GIuR1 and mRNA in the hippocampal formation. (a, b) Dark-field photomicrographs of hippocampal formation after hybridization with a probe in the anti-mRNA sense orientation (a) or mRNA sense orientation (b). (c, d) Light-field photomicrographs of pyramidal cells i n the CA3 region of hippocampal formation (c) and granule cells of the dentate gyrus (d) after hybridization with an anti-mRNA sense orientation probe. (e, f) Dark-field photomicrographs of pyramidal cells in the CA2 region of the hippocampal formation (e) and of the enthorinal cortex if) after hybridization with an anti-mRNA sense orientation probe. CA=cornu ammonis, DG=dentate gyrus. Scale bars: in a for a and b, 560 pm: in d for c and d, 40 prn: in e, 140 p m , in f, 280 prn.
was cloned and sequenced. It codes for a 906 amino acid protein that shares 98.2% amino acid identity (1 6 aa changes) with the rat GluRl and 98.4% with mouse GluRl . The identity between the human and rat nucleotide sequences is 89.4%.
Similar levels of amino acid identity have been found between human and bovine a, and PI subunits of the GABA-A receptor (99 and 98% respectively) (Schofield et a/., 1991) and between the human and rat p2 subunit of the neuronal
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21 6
M.-C. POTIER
Figure 4 Autoradiogram of human chromosomes flow sorted onto nitrocellulose tilters and hybridized with the EcoRl/Sacl 1:agmcnl ot KR4
acetylcholine receptor (97%) (EMBL X53179 and EMBL 104636 respectively). In addition, a putative consensus splicing sequence has been detected (Fig. 2)which could be used in alternative splicing tor creating subunit heterogeneity as in the rat iSommer et a/., 1990). Results from Northern blot analysis indicate that human GluRl i s expressed in human hippocampus, cerebellum and cortex. The size of the human and rat transcripts were found to be the same 15.2 kb). A difference in intensity of the hybridization signal between human and rat brain was observed. Harrison et a / . (1990), using oligonucleotide probes from the rat sequence on human brain, reported similar findings. Our results confirm that this difference is due to lower levels ot mRNA in human brain. By in situ hybridisation on human hippocampal formation, GluRl mRNA were found t o be concentrated i n granule and pvramidal cells, supporting results obtained with rat probes (Harrison et a/., 1990). Binding studies of ('H)kainate to membranes from normal or Alzheimer brains show no difference in cortex and hippocampus (Cowburn et a/., 19891. However, quantitative ligand binding autoradiography revealed an increase of (-'H)kainate binding sites in hippocampus of Alzheimer patients (Geedes et a/., 1985). Also, a 70% increase ot binding was observed i n the frontal of cortex 1-2 i z11ti m e r pat i e n t s, n/hic h cor re1ated with the number of senile plaques (Chalmers et a/., 1990). Although in a recent report a slight increase of
Er AL mRNA was tound i n hippocampus of Alzheimer patients compared to control (Harrison et a/., 19901, in this study, no apparent difference of hybridization could be detected either in the hippocampus by in situ hybridization or in cortex by Northern blot analysis. Dot blot analysis of flow sorted human chromosomes has proved to be a useful method for chromosomal localization of DNA sequences (Lebo et a/., 1985). Using this technique, the human GIuR1 gene has been assigned to chromosome 5. In conclusion, we have shown that the CluRl receptor exists in the human. Its amino acid sequence is highly homologous to the rat sequence. The gene is expressed in human brain and localized on chromosome 5. It should be useful in studying neurotoxicity and other neuropathological processes.
MATERIALS A N D METHODS c D N A library construction and screening A previously described non-amplified hippocampal cDNA library was used (Coedert et a/., 1989). Sets ot two replica nitrocellulose filters ( I 00,000 plaques) were screened under high stringency conditions (at 42 "C overnight in 50% formamide, 4xSSC (2OxSSC=0.3 M trisodium citrate, 3 M NaCIj, 50 m M sodium phosphate buffer pH=7, 120 ,ugh1 heparin, lOO,ug/ml acid/base cleaved salmon sperm DNA, 10x Denhardt's solution) with two rat CluRl cDNA probes prepared as follows: polymerase chain reaction (PCR) arnplification was carried out on first strand cDNA synthesised irom rat cortical poly(A')RNA with reverse transcriptase (murrne BRL) and oligodeoxynucleotides corresponding to nucleotides -71 to -48 and 1323 to 1347 for the sequence encoding the N terminal half of the rat CluRl receptor and nucleotides 1353 to 1376 and 2644 to 2667 for the sequence encoding the C-terminal half of the rat sequence (Hollmann et a/., 1989) PCR products were purified from agarose gels and labelled by the random priming method (Feinberg and Vogelstein, 19831. Filters were washed twice in 2xSSC, O.1'X1 SDS at room temperature and twice in O.lxSSC, 0.1% SDS a t 6 O T . Filters were exposed to Kodak XAR-5 X-ray film at -80 "C overnight. Twelve clones were plaque purified. The full-length cDNA clone I K K ~ J was used for all further experiments
Sequence determination The < D N A clone KR4 w a s subcloned into t h e EcoRI site ot h l l i mp18 and i t s sequence determined on both strand5 by the dideoxy chain termination reaction using Sequenase Vercion 2 0 (USB, Cleveland, Ohio) and synthetic oligonucleotides ( 1 9 tor -strand and 14 tor +strandl Sequence analviis wa5 pertocmed according to Staden (1990)
In situ hybridisation in human brain The c D N A clone KR4 was used lor ,n s,!u h\hrtdrsairon on
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H U M A N GLUTAMATE RECEPTOR cDNA
brain tissues (hippocampal formation) from two patients: one that died from Alzheimer's disease (82 years old) and a control patient who had died without any neurological or psychiatric disorders (76 years old). Hybridizations were carried out overnight at 42 "C in 50% formamide, 4xSSC, 50 m M sodium phosphate buffer pH=7, 120 p d m l heparin and 100 pghl acid/base cleaved salmon sperm D N A . Clone KR4 was used for making a single-stranded D N A probe as previously described (Coedert, 1987). KR4 was subcloned into the EcoRl site of M 1 3 rnp19. Sense and antisense orientations were determined by sequencing. The sections were washed for 1 hr in 2xSSC at room temperature and for 1 hr in 1 xSSC at 55 "C. Slides were dehydrated in 70% ethanol, dried and dipped in llford K5 emulsion and kept at -20 "C for four weeks. They were developed in Kodak D19 developer, stained with cresyl violet, dehydrated, cleaned and mounted in DPX. Northern blot analysis Poly(A+)RNA from a whole rat brain, cerebellum and cortex from a 76-year-old control patient, cortex and hippocampus from a 66-year-old control patient, cortex from an 80-year-old control patient and cortex from Alzheimer patients (87 and 82 years old) were prepared by a modification of the guanidinium isothiocyanate hot phenol technique, electrophoresed on a 2.2 M formaldehyde 0.9% agarose gel and transferred directly onto a nylon membrane (Hybond-N) as described before (Coedert, 1987). Membranes were prehybridized 'and hybridized at high stringency (see screening of c D N A library) to a single stranded D N A "P probe (anti mRNA sense orientation of KR4). Results were normalized using the mouse /%actin cDNA clone as in Coedert (1987).
Flow sorted chromosome dot blots Chromosomes from human lymphoblastoid cell lines of a normal male were prepared according to the method previously described (Carter et a/., 1990). Once in suspension, they were stained with chromomycin A, (40pg/ml) and Hoechst 33258 (2 p g r n l ) for t w o hours at 4 "C. Sodium citrate and sodium sulphite were added prior to flow analysis at 10 m M and 25 rnM respectively. Flow cytometry analysis was performed on a FACStar plus flow sorter (Becton Dickinson). Ten thousand of each chromosome type were sorted onto nitrocellulose filter disks (25 mm, 0 . 2 p m pore, millipore); two chromosome types as separate dots per disk. Filters were denatured, neutralized, baked, prehybridized and then hybridized to a single stranded DNA probe. An EcoRI/Sacl fragment corresponding to nucleotide sequence -1 04 to 1020 was used instead of the EcoRl fragment because it gave a higher signal/noise ratio when hybridized to human genomic DNA.
ACKNOWLEDGEMENTS We are grateful to D r M. Goedert for providing his human hippocampus cDNA library, for molecular biology advice and useful comments on the manuscript, and to Dr N. Unwin for discussions. N.P.C. is supported by a grant from the Medical Research Council of Great Eritian.
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(Received 74th M a y 1997 and in revised form 2nd September 1997)
REFERENCES Bettler, B., Boutler, I., Hermans-Borgmeyer, I., O'SheaGreenfield, A., Deneris, E.S., Moll, C., Borgmeyer, U., Hollmann, M. and Heineman, S. (1990). Cloning of a novel glutamate receptor subunit, GIuR5: expression in the nervous system during development. Neuron 5 , 583-595. Boutler, I., Hollrnann, M., O'Shea-Greenfield A., Hartley, M., Deneris, E., Maron, C. and Heineman, S. (1990). Molecular cloning and functional expression of glutamate receptor subunit genes. Science 249,1033-1037. Carter, N., Ferguson-Smith, M.E., Affara, N.A., Briggs, H. and Ferguson-Smith, M.A. (1990). Study of X chromosome abnormality in XX males using bivariate flow karyotype analysis and flow sorted dot blots. Cytometry 11, 202-207. Chalrners, D.T., Dewar, D., Graham, D.I., Brooks, D.N. and McCulloch, J. (1 990). Differential alterations of cortical glutamatergic binding sites in senile dementia of the Alzheimer type. Proc. Natl. Acad. Sci. USA 87, 1352-1 356. Choi, D.W. (1988). Glutamate neurotoxicity and diseases of tke nervous system. Neuron 1, 623-634. Cowburn, R.F., Hardy, J.A., Briggs, R.S. and Roberts, P.J. (1989). Characterisation, density, and distribution of kainate receptors i n normal and Alzheimer's disease human brain. /. Neurochem. 5 2 , 140-1 47. I. and Egebjerg, J., Bettler, B., Herrnans-Borgmeyer, Heinemann, S. (1991). Cloning of a c D N A for a glutamate receptor subunit activated by kainate but not AMPA. Nature 351, 745-748. Feinberg, A.P. and Vogelstein, €3. (1 983). A technique for radiolabeling D N A restriction fragments to high specific activity. A n d . Biochem. 132, 6-13. Call, C., Sumikara, K. and Lynch C . (1 990). Levels of mRNA for a putative kainate receptor are affected by seizures. Proc. Natl. Acad. Sci. USA 87, 7643-7647. Geddes, J.W., Monaghan, D.T., Cotman, C.W., Lott, I.T., Kim, R.C. and Chang Chui, H. (1985). Plasticity of hippocampal circuitry i n Alzheimer's disease. Science 230, 1179-1 181. Goedert, M. (1987). Neuronal localisation of amyloid beta protein precursor mRNA in normal brain and in Alzheimer's disease. EMBO). 6 , 3627-3632. Goedert, M., Spillantini, M.C., lakes, R., Rutherford, D. and Crowther, R.A. (1989). Multiple isoforms of human microtubule-associated protein Tau: sequences and localization in neurofibrillary tangles of Alzheimer's disease. Neuron 3, 51 9-526. Harrison, P.J., McLaughlin, D. and Kerwin, R.W. (1991). Decreased hippocampal expression of a glutamate receptor gene in schizophrenia. Lancet 337, 450-452. Harrison, P.J., Barton, A.J.L., Najlerahim, A. and Pearson, R.C.A. (1 990). Distribution of a kainate/AMPA receptor mRNA i n normal and Alzheimer brain. NeuroReport 1, 149-1 52. Hollmann, M., O'Shea-Creeniield, A., Rogers, S.W. and Heinemann, 5 . (1989). Cloning by functional expression of a member of the glutamate receptor family. Nature 342, 643-648. Keinanen, K., Wisden, W., Sommer, B., Werner, P., Herb, A., Verdoorn, A,, Sakman, 6. and Seeburg, P. (1990). A family of AMPA-selective glutamate receptors. Science 249, 556-560. Kerwin, R.W., Patel, S., Meldrum, B.S., Czudek, C. and Reynolds, G.P. (1 988). Asymmetrical loss of glutamate receptor subtype in left hippocampus in schizophrenia. Lancet, i, 583-584. Lebo, R.V., Tolan D.R., Bruce, B.D., Cheung, M.-C. and Kan,
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Y . W . (1 985). Spot-blot analysis of sorted chromosomes designs a fructose intolerance disease locus to chromosome 9. Cytornetry 6, 478-483. Maragos, W.F., Creenamyre, J.T., Pennev, 1.6. and Young, A.B. ( 1 987). Glutarnate dysfunction i n Alzheimer’s disease: an hypothesis. Trends Neurosci. 10, 65-68. Masu, M., Tanabe, Y., Tsuchida, K., Shiigemoto, R. and Nakanishi, S. (1991). Sequence and expression of a metabotropic glutamate receptor. Nature 349, 760-765. Nakanishi, N., Shneider, N.A. and Axel, R. (1990).A family of glutamate receptor genes: evidence for the formation of heteromultimeric receptors with distinct channel properties. Neuron 5 , 569-581. Sakirnura, K., Bujo, H., Kushiya, E., Araki, K., Yamazaki, M., Yamazaki, M., Meguro, H., Warashina, A., Numa, S. and Mishina, M. (1990). Functional expression from cloned cDNAs of glutamate receptor species responsive to kainate and quisquaiate. FEBS Left. 272, 73-80.
Schofield, P.R., Pritchett, D.B., Sontheimer, H., Kettenmann, H. and Seeburg, P.H. (1991). Sequence and expression of a human CABA-A receptor a1 and pl subunits. FEBS Lett. 244,
361-364. Sommer, B., Keinanen, K., Verdoorn, T.A., Wisden, W., Burnashev, N., Herb, A., Kohler, M., Takagi, T., Sakman, 6. and Seeburg, P.H. (1990). Flip and flop: a cell-specific functional switch in glutamate-operated channels of the CNS. Science 249, 1580-1 585. Staden, R . (1990). An improved sequence handling package that runs on the Apple Macintosh. Cabios 6, 387-393. Werner, P., Voigt, M., Keinanen, K., Wisden, W. and Seeburg, P.H. (1991).Cloning of a putative high-affinity kainate receptor expressed predominantly i n hippocampal CA3 cells. Nature 351, 742-744.
DNA Sequence-]. DNA Sequencing and Mapping, Vol. 2, pp. 21 1-2 18 Reprints available directly from the publisher Photocopying permitted by license only
0 1992 Harwood Academic Publishers CmbH Printed in the United Kingdom
The human glutamate receptor cDNA GluR1: cloning, sequencing, expression and localization to chromosome 5 M.-C. POTIER*, M.G. SPILLANTINI and N.P. CARTERt Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England and t Department ofPathology, University of Cambridge, Cambridge CB2 1 QP, England EMBL Data Library Accession No. X58633
The rat glutamate receptor i s a 907 amino acid transmembrane protein. Using the rat GluRl cDNA as a probe, we have isolated cDNA clones from a human hippocampal cDNA library. Sequence of a full length cDNA clone revealed 98,2% and 89.4% identity to the rat sequence at the amino acid and nucleotide levels respectively. The human cDNA clone detected an RNA transcript in human cerebral cortex, hippocampus and cerebellum, similar to that seen in rat. In situ hybridization experiments showed that human CluRl mRNA i s present in granule and pyramidal cells in the hippocampal formation and that there i s no apparent difference of distribution between control patient and patient with Alzheimer's disease. Dot blot analysis of flow-sorted human chromosomes showed that the CluRl gene maps to chromosome 5. KEY WORDS: glutamate receptor, human chromosome 5, in 5 i f U hybridization, human brain
INTRODUCTION L-glutamate i s the major excitatory neurotransmitter in the central nervous system of mammals. It interacts with two types of membrane receptors: the ionotropic receptors which form transmembrane channels permeant to cations and the metabotropic receptors which are coupled to G protein and stimulate inositol phosphate/Ca2+ intracellular pathways. lonotropic receptors can be classified according
*Corresponding author, present address: Centre National de la Recherche Scientifique, Laboratoire de Physiologie Nerveuse, 91 198 Gif-sur-Yvette Cedex, France.
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to their pharmacological properties. NMDA (Nmethyl-D-aspartate) receptors are coupled to large conductance channels permeant to monovalent cations and Ca" whereas AMPA/kainate (AMPA: a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors are permeant to monovalent cations only, mainly Na' under physiological conditions. The amino acid sequences of some non-NMDA glutamate receptors from rodent brain have been obtained through expression cloning. A rat metabotropic receptor consists of 1199 amino acids containing 7 putative transmembrane domains with no sequence similarities to conventional G protein-coupled receptors (Masu et a/., 19911. AMPA/kainate receptors are heterooligomers of at least three classes of related subunits of about 900 amino acids each with 4 transmembrane domains (Hollmann et a/.,l989; Keinanen et a/., 1990; Boulter et a/.,1990; Sommer et a/., 1990; Nakanishi et a/., 1990; Bettler et a/., 1990; Sakimura et a/., 1990; Egebjerg et a/., 1991; Werner e t a / . , 1991). The four known members of the first class (GIuRI to GkuR4) share 56 to 73% amino acid identity. The three known members of the second class (GIuR5-1, GluR5-2 and GluR6) share 80% amino acid identity with each other. These two classes of subunits are 40% identical. Finally, the only member of the third class i s a high affinity kainate receptor (KA-1) sharing 30% amino acid identity with other classes. Giutamate receptors are involved in experimental neurotoxicity (Choi, 1988) and have been
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M.-C. POTIER ET AL.
implicated in pathological processes. After electrolytic seizure in rat, the GIuR1 mRNA i s downregulated in specific brain regions (Gall et a/., 1990). A 7'0% decrease of GluRl mRNA has recently been reported in hippocampus of schizophrenic patients, although an increase in specific kainate binding sites had been demonstrated previously (Harrison el a / . , 1991; Kerwin et a/., 1988). Finally, glutamate receptors have been shown to be modified in Alzheimer's disease (Maragos et a / . , 1987'; Harrison et a/., 1990). In the present report, we describe the cloning, sequencing, expression and chromosomal localization of the human GluRl receptor cDNA.
RESULTS Isolation of human kainate receptor cDNA clones Screening of 100,000 plaques from a human hippocampal cDNA library with a radiolabelled PCR product derived from the nucleotide sequence encoding the C terminal half of the rat glutamate receptor GluRl gave 18 positive clones (KR1 to KR18). Twelve clones were plaque purified after three rounds of screening with a radiolabelled PCR product derived from the nucleotide sequence encoding the N terminal half of the rat GluRl. Six clones had an insert of more than 2 kb and two 'KR4 and KR9) had an insert of about 3 kb. Sequencing of human GluRl cDNA clone K R 4 The 3 kb EcoRl fragment of KR4 was subcloned into M13 mp18 and sequenced on both strands. It consisted of an open reading frame of 2722 nucleotides following with 197 nucleotides of 5 ' untranslated sequence and ending with 74 nucleotides of 3 ' untranslated sequence after the inframe stop codon (Fig. 1 ). The open reading frame codes for a protein of 906 amino acids which shows 98.2% identity with the rat GluRl receptor and 98.4% identity with the mouse GluRl receptor (Fig. 2). Comparison of the human and rat nucleotide sequences shows 89.4% conservation . The deduced protein sequence contains a putat i v e signal peptide o i 18 amino acids at its N terminus. There are 6 putative glycosylation sites, and 4 potential phosphorylation sites: one for Ca'+/calmodul ine-dependent protein kinase I 1 and three for protein kinase C. 4nalvsis ot the deduced amino acid sequence
for hydrophobicity suggests the presence ot four putative transmembrane segments (M, to M4). These sequence data will appear in the EMB L/G e n Ba n k/D DBJ N uc Ieot id e Sequence Da t abases under the accession number X58633 H U M A N GluRl cDNA. Expression of GluRl in normal and Alzheimer brains The distribution of GluRl transcripts was studied by in situ hybridization in the human hippocampal formation and by Northern blot analysis using single stranded D N A probes in the anti-mRNA sense orientation. Northern blot analysis of poly(A+)RNAfrom cortex, hippocampus and cerebellum of two control patients, cortex of two patients who died of Alzheimer's disease and rat brain showed the same major band of 5.2 kb as determined by Hollman et a / . (19891, stronger in rat than i n human (data not shown). Distribution of hybridization positive cells was high in the dentate gyrus and lower in the cornu ammonis with no apparent difference between the fields (Fig. 3a). Hybridization was specific, since only background labelling was detected when a probe in the mRNA sense orientation was used (Fig. 3b). The same qualitative distribution of hybridization positive cells was observed in both control and Alzheimer brains. There was a slrong labelling of granule celli-in the dentate gyrus (Fig. 3d) and pyramidal cells throughout the cornu ammonis (Fig. 3c and e) and enthorinal cortex (Fig. 3f) although not all granule or pyramidal cells were positive. Chromosomal localization The chromosonal localization of GluRl was studied by hybridization to flow sorted human chromosomes. All human chromosomes could be sorted separately, except for chromosomes 9 to 12. Dot blot analysis of flow sorted chromosomes showed that the single stranded radiolabelled Sac1 fragment of KR4 hybridized only to the left dot of the filter containing chromosomes 5 and 6. indicating localisation to chromosome 5 (Fig. 4).
DISCUSSION A cDNA clone encoding the human equivalent ot one subunit of the rat glutamate receptor (CIuR1)
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HUMAN GLUTAMATE RECEPTOR cDNA
CGAAAACAACACCCAGAACAGCGACAAGAATAAAGGGAAAGGGGGGGAAACACCAAATCTATGAITGGACCTGGGCTTCTTTTTCGCCAATCCAAAAAGGAAT M Q E I F A F F C T G F L C A V V G A N F P N N I Q I G G L F P N Q Q S Q E E A ATGCAGCACATTTITCCCTTCTTCIGCACCGGIITCCTAGGCGCGGTAGTAGGTGCCAATTTCCCCAACAAIAICCAGATCGGGGGATTATTTCCAAACCAGCAGTCACAGGAACAIGCT 10
20
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SO
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A F R F A L S Q L T E P P K L L P Q I D I V N I S D S F E ~ I Y R F C S Q F S X GCTTTTACATIICCITTGTCGCAACTCACAGAGCCCCCGAAGCTGCTCCCCCAGATIGATATIGTGAACATCAGCCACAGCTTTGAGAIGACCIATAGATTCTGTTCCCAGTTCTCCAAA 130
150
lU0
160
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G V W A I F G F Y E R R T V N H L T S F C G A L E V C F I T P S F P V D T S N Q CCAGTCTATGCCAICTITGCGTTTIATGAACGIAGGACTGTCAACATGCTGACCTCCTTTTGTGGCGCCCTCCACGTCTGCTICATTACGCCGAGCTTICCGGTTGAIACATCAAATCAG 250
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280
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F V L Q L R P E L Q D A L I S I I D E Y K U Q X F V Y I Y D A D R G L S V L Q X TTTCTCCTTCAGCTGCGCCCTGAACTGCAGGATGCCCTCATCAGCATCATTGACCATTACAAGTGGCAGAAATTTGTCTACATTTATGATGCCGACCGGGGCTTAICCGTCCIGCAGAAA 370
V
L
D
390
380
T
A
A
E
K
N
U
410
400
Q
V
T
A
V
N
(20
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L
T
T
E
E
450
UP0
130
T
G
Y
R
M
L
F
Q
4 60
D
L
E
410
K
K
K
480
E
R
L
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CTCCTCCATACAGCTGCTGAGAAGAACTGGCAGGTGACAGCAGTCAACATTTTGACAACCACAGAGCAACGATACCGGATGCTCTTTCAGGACCTGGAGAAGAAAAAGGAGCGGCTGGTG
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V V O C E S E R L N A I L G Q I I X L E K N G I G Y ~ Y I L A N L G F M D I D L GTGGTCGACIGTGAATCAGAACGCCTCAATGCTATCTTGGGCCAGATTATAAAGCTACAGAAGAATGGCATCGGCIACCACTACATTCTTGCAAATCTGGGCTTCA~GGACATIGACTTA 620
610
650
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67 0
680
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N K F X E S G A N V T C F Q L V N Y T D T I P A K I H Q Q U K N S D A R D E T R AACAAATTCAACGAGACTGCCGCCAAIGTGACAGGTTTCCAGCTGGTGAACTACACAGACACTATTCCGGCCAAGATCATCCACCAGTCGAAGAATAGTGATGCTCCAGACCACACACGG 730
740
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V D U K R P K Y T S A L T Y D C V X V H A E A F Q S L R R Q R I D I S R R G N A CTCCACICGAACACACCCAAGTACACCTCTGCGCTCACCTACGAIGGGCIGAAGGTGATGGCTGAGCCTTTCCACAGCCTGCCGAGCCACACAA~TGATATATCTCGCCGCGGGAATGCT 850
860
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890
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C D C L A N P A V P U G Q G I D I Q R A L Q Q V R F E G L T C N V Q F N E K G R CCCCATTCTCTCCCTAACCCAGCTGTTCCCTGGGGCCAAGGGATCGACATCCAGAGAGCTCTGCAGCAGGTCGCGTTTGAAGCTTTAACACGAAACCTGCAGTT~AATGAGAAAGGACGC 97 0
980
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1000
1010
1020
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R T N Y T L B V I E H K E D C I R K I G Y W N E D D K ~ V P A A T D A Q A G G D CCCACCAACTACACCCTCCACGTCATTCAAATGAAACATCACGGCATCCCAAACATTGCTTACTCCAATCAACArGATAACTTTCTCCCTGCACCCACCCATGCCCAACCTCCCGCCCAT 1090
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1110
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N S S V Q N R T Y I V T T I L E D P Y V U L K X N A N Q ~ C C N D R X E C Y C AATTCAACTCTTCACAACACAACATACATCGTCACAACAATCCTAGAAGATCCTTATCTGATGCTCAACAACAACCCCAATCACTTTGACCCCAATCACCCTTACCACGGCTACTCTC~A 1210
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E L A A E I A K E V G Y S Y R L E I V S D G X Y G A R D P D T K A U N C M V G E GACCTGCCGCCAGAGATTGCCAAGCACGTGGGCTAC~CCTACCGTCTGGAGATIGTCAGTGATGGAAAATACGGAGCCCGAGACCCTCACACGAAGGCCTGGAATGCCATGGTGGCACAG 1330
1340
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L V Y G R A D V A V A P L T I T L V R E C V I D F S K P F U $ L G I S I H I K K CTCGTCTATCCAAGAGCACATGTGGCTGTGGCTCCCCTTACTATCACTTTGGICCGGGAAGAAGTTATAGATTTCTCCAAACCATTTATCAGTTTGGGCATCTCCATCATGATTAAAAAA 1460
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P Q K S K P C V F S F L D P L A Y E I U H C I V F A Y I G V S V V L F L V S R F CCACACAAATCCAAGCCCGGIGTCITCTCCTTCCTTGATCCTTTGGCTTAIGAGATTTGGAIGTGCATTGTTTTTGCCTACATTGGAGTGAGTGTTGTCCTCTTCCTGGTCAGCCGCTTC 1510
1580
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S P Y E W B S E E F E E C R D Q T T S D Q S N E F G I F N S L U F S L G A F M Q AGTCCCIATGAATCGCACAGTGAAGAGTTTGAGGAAGGACGGGACCAGACAACCAGTGACCAGTCCAATGAGTTTGGGATATTCAACAGTTIGTGGTTCTCCCTGCGACCCTTCATGCAG 1690
1700
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1120
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Q G C D I S P R S L S G R I V C C V W U F F T L I I I S S Y T A N L A A F L T V CAACGATCTGACATITCTCCCAGGTCCCTGTCTGGTCGCATCGICGGTGGCGTCTCGTGGT~CTTCACCTTAATCATCATCTCCTCATATACACCCAATCTCGCCGCCTTCCTGACCGTG 1810
1820
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lP00
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E R H V S P I E S A E D L A K Q T E I A Y G T ~ E A G S T K E F F R R S K I A GAGAGGATGGTGTCICCCATTGAGAGTGCAGAGCACCTAGCGAAGCAGACAGAAATTCCCTACGGGACGCTGGAAGCAGGATCTACTAAGGAGTTCTTCACGAGGTCTAAAATTGCAGTG 1930
1940
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F E K H W T Y M K S A E P S V F V R T T E E G U I R V R K S K G X Y A Y L L E S TITCAGAAGATCTCCACATACATGAAGICAGCAGAGCCATCAGTTITTGTGCGGACAACAGAGGAGGGGATCATTCGAGIGAGGAAATCCAAAGGCAAATATGCCTACCTCCTGGAGTCC 2050
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? H N E Y I E Q R K P C D T M K V C C N L D S K G Y G I ~ T P K G S A L R N P V ACCAIGAATGACTACATICAGCAGCGGAAACCCTGTGACACCATGAAGGTGGGAGGTAACTTGGATTCCAAAGGCTATGGCATTGCAACACCCAAGCGGTCTGCCCTGAGAAATCCAGTA 2170
2180
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2280
N L A V L K L N E Q C L L D K L K N X U U Y D X G E C G S G G G D S K D K I S A AACCTCGCAGTGITAAAACTGAACGAGCAGGGGCTTTTGGACAAATTGAAAAACAAATGGTGGTACGACAAGGGCGAGTGCGGCAGCGGGGGAGGTGATTCCAAGGACAAGACAAGCGCI 2290
L
S
L
2300
S
N
V
A
2310
G
V
F
2320
Y
I
2330
L
I
G
G
L
2340
G
L
A
2350
H
L
V
2360
A
L
I
2370
E
F
C
Y
2380
K
S
R
2390
S
Z
S
2400
K
R
H
K
CICAGCCTCAGCAATGICCCACGCGTGTTCTACATCCTGATCGGAGGACTTGGACTAGCCATGCTGGTTGCCTTAATCGAGTTCTGCTACAAATCCCGTAGTGAATCCAAGCGGATGAAG 2420
2410
G
F
C
L
I
P
Q
2U30
Q
S
I
2440
N
E
A
2450
I
R
T
S
2460
T
L
P
2470
R
N
S
2480
G
A
G
A
2490
S
S
G
2500
G
S
G
2510
E
N
G
R
2520
V
V
S
CCITITICTTIGATCCCACAGCAATCCATCAACGAAGCCATACGGACATCGACCCTCCCCCGCAACAGCGGGGCAGGAGCCAGCAGCGGCGGCAGTGGAGAGAATCGTCGCGIGGTCAGC 2530
2540
2550
2560
2570
2580
2590
2600
2610
2620
2630
2640
~ D F P K S M Q S I P C H S E S S G H P L G A T ~ ~ ~ C A T C A C T T C C C C A A C I C C A T ~ ~ A A I ~ ~ A I I ~ ~ T T ~ ~ A T ~ A ~ ~ ~ A ~ A ~ T T C A G ~ ~ A T ~ C C C T T G G G A ~ C C A C 2~1~3 0~ A T T G T A 2 1A4C0 T G G A G C2 A 7 5G0A T G C A G2A7 6C0C C C I T G G G G A G C A 2680 2690 2700 2110 2120 2650 2660 2670 TCCCCAGCCCCAICCCAAACCCIICAGTGCCAAAAACA~CAACAAAATGAAACGCAACCGCAAI~C 2770
2780
2790
2800
2810
2820
Figure 1 Nucleotide and deduced amino acid sequence of cDNA clone KR4 encoding the human glutamate receptor subunit GluRl. The amino acid sequence i s shown above the nucleotide sequence. Nucleotides are numbered in the 5 ' to 3 ' direction beginning with the first residue of the initiation codon. The in-frame stop codon i s indicated with an asterisk.
214
M.-C. POTIER ET AL.
Rat Human
Mouse
b
MPYIFAFFCT GFLGAW AN FPNNIQIGGL FPNQQSQEHA AFRFALSQLT EPPKLLPQID I V ~ S C T F E M MQHIFAFFCT GFLGAWGAN FPNNIQIGGL FPNQQSQEHA AFRFALSQLT EPPKLLPQID IVNISDSFEK MPYIFAFFCT GFLGAWGAN FPNNIQIGGL FPNQQSQEHA AFRFALSQLT EPPKLLPQID IVNISDTFEK TYRFCSQFSK GWAIFGFYE RRTVNMLTSF CGALHVCFIT PSFPVDTSNQ FVLQLRPELQ EALISIIDHY TYRFCSQFSK GWAIFGFYE RRTVNMLTSF CGALHVCFIT PSFPVDTSNQ FVLQLRPELQ DALISIIDHY TYRFCSQFSK GWAIFGFYE RRTVNMLTSF CGALHVCFIT PSFPVDTSNQ FVLQLRPELQ EALISIIDHY
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hWQTFVYIYD ADHGLSVLQR VLDTAAEKNW QVTAVNILTT TEEGYRMLFQ DLEKKKERLV WDCESERLN KWQKFWIYD ADHGLSVLQK VLDTAAEKNW QVTAVNILTT TEEGYRMLFQ DLEKKKERLV WDCESERLN hWQTFLYIYD ADRGLSVLQR VLDTAAEKNW QVTAVNILTT TEEGYRMLFQ DLEKKKERLV WDCESERLN
*
*
AILGQIVKLE KNGIGYHYIL ANLGFMDIDL NKFKESGRNV TGFQLVNYTD TIPARIMQQW RTSDSRDHTR AILGQIIKLE KNGIGYHYIL ANLGFMDIDL NKFKESGANV TGFQLVNYTD TIPAKIMQQW KNSDARDHTR AILGQIVKLE KNGIGYHYIL ANLGFMDIDL NKFKESGANV TGFQLVNYTD TIPARIMQQW RTSDARDHTP ‘l’DWKRPKYTS ALTYDGVKVM AEAFQSLRRQ RIDISRRGNA GDCLANPAVP WGQGIDIQRA LQQVRFEGLT LQWKRPKYTS ALTYDGVKVM AEAFQSLRRQ RIDISRRGNA GDCLANPAVP WGQGIDIQRA LQQVRFEGLT VDWKRPKYTS ALTYDGVKVM AEAFQSLRRQ RIDISRRGNA GDCLANPAVP WGQGIDIQRP. LQQVRFEGLT GNVQFNEKGR ZNVQFNEKGR 2NVQFNEKGR
*
*
*
RTNYTLHVIE MKHDGIRKIG YWNEDDKFVP AATDAQAGGD NSSVCNRTYI VTTILEDPYV RTNYTLHVIE MKHDGIRKIG YWNEDDKFVP AATDAQAGGD NSSVQNRTYI VTTILEDPYV RTNYTLHVIE MKHDGIRKIG YWNEDDKFVP AATDAQAGGD NSSVQNRTYI VTTILEDPY\‘
MLKKNANQFE GNDRYECYCV ELAAEIAKHV GYSYRLEIVS DGKYGARDPD TKAWNGMVGE MLKKNANQFE GNDRYEGYCV ELAAEIAKHV GYSYRLEIVS DGKYGARDPD TKAWNGMVGE MLKKNANQFE GNDRYEGYCV ELAAEIAKHV GYSYRLEIVS DGKYGARDPD TKAWNGMVGE
SVYGRADVAV LVYGRADVAL’ LVYGRADVAi’
APLTITLVRE EVI DFSKPFM SLGISIMIKK PQKSKPGVFS FLDPLAYEIW MCIV a Y IGV SWJ,FLVSRF APLTITLVRE EVIDFSKPFM SLGISIMIKK PQKSKPGVFS FLDPLAYETW MCIV U Y I GV S W U S R F APLTITLVRE EVIDFSKPFM SLGISIMIKK PQKSKPGVFS FLDPLA-BYIG V V SWLFLVSRF SPYEWHSEEF EECRDQTTSD QSNEL W F S L W O G WSPRSL S G R I V G C W FFSPYEWHSEEF EECRDQTTSD Q S N E m N S LWFST,GaE%lc, OGCDLSPRSL S G R I V G ~ WFFTT.= SP‘r‘EWHSEEF EECRDQTTSD Q S N E m N S LW F S T , W OGCDLsPnsL SGRIVGrn m m W
L L L
T T T
0
4-
V ERMVSPIESA EDLAKQTEIA YGTLEAGSTK EFFRRSKIAV FEKMWTYMKS AEPSVFVRTT V ERMVSPIESA EDLAKQTEIA YGTLEAGSTK EFFRRSKIAV FEKMWTYMKS AEPSVF‘JRTT V ERMVSPIESA EDLAKQTEIA YGTLEAGSTK EFFRRSKIAV FEKMWTYMKS AEPSVFVRTT
0
0
v
FEGMIRVRKS KGFYAYLLES TMNEYIEQRK PCDTMKVGGN LDSKGYGIAT PKGSALRNPV NLAVLKLNEQ EEGMIRVRKS KGKYAYLLES TMNEYIEQRK PCDTMKVGGN LDSKGYGIAT PKGSALRNPV NLAVLKLNEQ EEGMIRVRKS KGKYAYLLES TMNEYIEQRK PCDTMKVGGN LDSKGYGIAT PKGSALRNPV NLAVLKLNEQ GLLDKLKNKW WYDKGECGTG GGDSKDKTSA LSLSNVAGVF YILIGGLGLA MLV U E F C Y KSRSESKRMK GLLDKLKNKW WYDKGECGSG GGDSKDKTSJ LSLSNVAGVF YILIGCLGLA MT,VATLEFCY KSRSESKRMK GLLDKLKNKW WYDKGECGSG GGDSKDKTSA LSLSNVAGVF YILIGGLGLA MLVALIEFCY KSRSESKRMK GFCLIPQQSI NEAIRTSTLP RNSGAGASOG GGSGENGRW SQDFPKSMQS IPCMSHSSGM PLGATG‘ CFCLIPQQSI NEAIRTSTLP RNSGAGASSG 0-SGENGRW SHDFPKSMQS IPCMSHSSGM PLGATGL YFCLIPQQSI NEAIRTSTLF RPTSGAGASOG SOSGENGRW SQDFPKSMQS IPCMSHSSSK PLG.\TGL
Figure 2 4lignment of deduced amino acid sequences for the rat, human and mouse glutamate receptor subunit CIuRT Amino acid ditterences between human and rat or human and mouse are in bold Cleavage of the signal peptide i s predicted at position indicated with the arrom Four proposed transmembrane domalns are underlined Potentlal extracellular N-glycosylation sites are indicated & ith a5terisks Putative phosphorylation sltes are indicated by a cross (Ca*/calmoduline-dependent proteinkinase Ill and dot5 (proteiri Linase C I Putative spice S i t < ’ 1 5 indicated with a triangle
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HUMAN GLUTAMATE RECEPTOR cDNA
215
Figure 3 Cellular localization of human glutamate receptor GIuR1 and mRNA in the hippocampal formation. (a, b) Dark-field photomicrographs of hippocampal formation after hybridization with a probe in the anti-mRNA sense orientation (a) or mRNA sense orientation (b). (c, d) Light-field photomicrographs of pyramidal cells i n the CA3 region of hippocampal formation (c) and granule cells of the dentate gyrus (d) after hybridization with an anti-mRNA sense orientation probe. (e, f) Dark-field photomicrographs of pyramidal cells in the CA2 region of the hippocampal formation (e) and of the enthorinal cortex if) after hybridization with an anti-mRNA sense orientation probe. CA=cornu ammonis, DG=dentate gyrus. Scale bars: in a for a and b, 560 pm: in d for c and d, 40 prn: in e, 140 p m , in f, 280 prn.
was cloned and sequenced. It codes for a 906 amino acid protein that shares 98.2% amino acid identity (1 6 aa changes) with the rat GluRl and 98.4% with mouse GluRl . The identity between the human and rat nucleotide sequences is 89.4%.
Similar levels of amino acid identity have been found between human and bovine a, and PI subunits of the GABA-A receptor (99 and 98% respectively) (Schofield et a/., 1991) and between the human and rat p2 subunit of the neuronal
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21 6
M.-C. POTIER
Figure 4 Autoradiogram of human chromosomes flow sorted onto nitrocellulose tilters and hybridized with the EcoRl/Sacl 1:agmcnl ot KR4
acetylcholine receptor (97%) (EMBL X53179 and EMBL 104636 respectively). In addition, a putative consensus splicing sequence has been detected (Fig. 2)which could be used in alternative splicing tor creating subunit heterogeneity as in the rat iSommer et a/., 1990). Results from Northern blot analysis indicate that human GluRl i s expressed in human hippocampus, cerebellum and cortex. The size of the human and rat transcripts were found to be the same 15.2 kb). A difference in intensity of the hybridization signal between human and rat brain was observed. Harrison et a / . (1990), using oligonucleotide probes from the rat sequence on human brain, reported similar findings. Our results confirm that this difference is due to lower levels ot mRNA in human brain. By in situ hybridisation on human hippocampal formation, GluRl mRNA were found t o be concentrated i n granule and pvramidal cells, supporting results obtained with rat probes (Harrison et a/., 1990). Binding studies of ('H)kainate to membranes from normal or Alzheimer brains show no difference in cortex and hippocampus (Cowburn et a/., 19891. However, quantitative ligand binding autoradiography revealed an increase of (-'H)kainate binding sites in hippocampus of Alzheimer patients (Geedes et a/., 1985). Also, a 70% increase ot binding was observed i n the frontal of cortex 1-2 i z11ti m e r pat i e n t s, n/hic h cor re1ated with the number of senile plaques (Chalmers et a/., 1990). Although in a recent report a slight increase of
Er AL mRNA was tound i n hippocampus of Alzheimer patients compared to control (Harrison et a/., 19901, in this study, no apparent difference of hybridization could be detected either in the hippocampus by in situ hybridization or in cortex by Northern blot analysis. Dot blot analysis of flow sorted human chromosomes has proved to be a useful method for chromosomal localization of DNA sequences (Lebo et a/., 1985). Using this technique, the human GIuR1 gene has been assigned to chromosome 5. In conclusion, we have shown that the CluRl receptor exists in the human. Its amino acid sequence is highly homologous to the rat sequence. The gene is expressed in human brain and localized on chromosome 5. It should be useful in studying neurotoxicity and other neuropathological processes.
MATERIALS A N D METHODS c D N A library construction and screening A previously described non-amplified hippocampal cDNA library was used (Coedert et a/., 1989). Sets ot two replica nitrocellulose filters ( I 00,000 plaques) were screened under high stringency conditions (at 42 "C overnight in 50% formamide, 4xSSC (2OxSSC=0.3 M trisodium citrate, 3 M NaCIj, 50 m M sodium phosphate buffer pH=7, 120 ,ugh1 heparin, lOO,ug/ml acid/base cleaved salmon sperm DNA, 10x Denhardt's solution) with two rat CluRl cDNA probes prepared as follows: polymerase chain reaction (PCR) arnplification was carried out on first strand cDNA synthesised irom rat cortical poly(A')RNA with reverse transcriptase (murrne BRL) and oligodeoxynucleotides corresponding to nucleotides -71 to -48 and 1323 to 1347 for the sequence encoding the N terminal half of the rat CluRl receptor and nucleotides 1353 to 1376 and 2644 to 2667 for the sequence encoding the C-terminal half of the rat sequence (Hollmann et a/., 1989) PCR products were purified from agarose gels and labelled by the random priming method (Feinberg and Vogelstein, 19831. Filters were washed twice in 2xSSC, O.1'X1 SDS at room temperature and twice in O.lxSSC, 0.1% SDS a t 6 O T . Filters were exposed to Kodak XAR-5 X-ray film at -80 "C overnight. Twelve clones were plaque purified. The full-length cDNA clone I K K ~ J was used for all further experiments
Sequence determination The < D N A clone KR4 w a s subcloned into t h e EcoRI site ot h l l i mp18 and i t s sequence determined on both strand5 by the dideoxy chain termination reaction using Sequenase Vercion 2 0 (USB, Cleveland, Ohio) and synthetic oligonucleotides ( 1 9 tor -strand and 14 tor +strandl Sequence analviis wa5 pertocmed according to Staden (1990)
In situ hybridisation in human brain The c D N A clone KR4 was used lor ,n s,!u h\hrtdrsairon on
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H U M A N GLUTAMATE RECEPTOR cDNA
brain tissues (hippocampal formation) from two patients: one that died from Alzheimer's disease (82 years old) and a control patient who had died without any neurological or psychiatric disorders (76 years old). Hybridizations were carried out overnight at 42 "C in 50% formamide, 4xSSC, 50 m M sodium phosphate buffer pH=7, 120 p d m l heparin and 100 pghl acid/base cleaved salmon sperm D N A . Clone KR4 was used for making a single-stranded D N A probe as previously described (Coedert, 1987). KR4 was subcloned into the EcoRl site of M 1 3 rnp19. Sense and antisense orientations were determined by sequencing. The sections were washed for 1 hr in 2xSSC at room temperature and for 1 hr in 1 xSSC at 55 "C. Slides were dehydrated in 70% ethanol, dried and dipped in llford K5 emulsion and kept at -20 "C for four weeks. They were developed in Kodak D19 developer, stained with cresyl violet, dehydrated, cleaned and mounted in DPX. Northern blot analysis Poly(A+)RNA from a whole rat brain, cerebellum and cortex from a 76-year-old control patient, cortex and hippocampus from a 66-year-old control patient, cortex from an 80-year-old control patient and cortex from Alzheimer patients (87 and 82 years old) were prepared by a modification of the guanidinium isothiocyanate hot phenol technique, electrophoresed on a 2.2 M formaldehyde 0.9% agarose gel and transferred directly onto a nylon membrane (Hybond-N) as described before (Coedert, 1987). Membranes were prehybridized 'and hybridized at high stringency (see screening of c D N A library) to a single stranded D N A "P probe (anti mRNA sense orientation of KR4). Results were normalized using the mouse /%actin cDNA clone as in Coedert (1987).
Flow sorted chromosome dot blots Chromosomes from human lymphoblastoid cell lines of a normal male were prepared according to the method previously described (Carter et a/., 1990). Once in suspension, they were stained with chromomycin A, (40pg/ml) and Hoechst 33258 (2 p g r n l ) for t w o hours at 4 "C. Sodium citrate and sodium sulphite were added prior to flow analysis at 10 m M and 25 rnM respectively. Flow cytometry analysis was performed on a FACStar plus flow sorter (Becton Dickinson). Ten thousand of each chromosome type were sorted onto nitrocellulose filter disks (25 mm, 0 . 2 p m pore, millipore); two chromosome types as separate dots per disk. Filters were denatured, neutralized, baked, prehybridized and then hybridized to a single stranded DNA probe. An EcoRI/Sacl fragment corresponding to nucleotide sequence -1 04 to 1020 was used instead of the EcoRl fragment because it gave a higher signal/noise ratio when hybridized to human genomic DNA.
ACKNOWLEDGEMENTS We are grateful to D r M. Goedert for providing his human hippocampus cDNA library, for molecular biology advice and useful comments on the manuscript, and to Dr N. Unwin for discussions. N.P.C. is supported by a grant from the Medical Research Council of Great Eritian.
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(Received 74th M a y 1997 and in revised form 2nd September 1997)
REFERENCES Bettler, B., Boutler, I., Hermans-Borgmeyer, I., O'SheaGreenfield, A., Deneris, E.S., Moll, C., Borgmeyer, U., Hollmann, M. and Heineman, S. (1990). Cloning of a novel glutamate receptor subunit, GIuR5: expression in the nervous system during development. Neuron 5 , 583-595. Boutler, I., Hollrnann, M., O'Shea-Greenfield A., Hartley, M., Deneris, E., Maron, C. and Heineman, S. (1990). Molecular cloning and functional expression of glutamate receptor subunit genes. Science 249,1033-1037. Carter, N., Ferguson-Smith, M.E., Affara, N.A., Briggs, H. and Ferguson-Smith, M.A. (1990). Study of X chromosome abnormality in XX males using bivariate flow karyotype analysis and flow sorted dot blots. Cytometry 11, 202-207. Chalrners, D.T., Dewar, D., Graham, D.I., Brooks, D.N. and McCulloch, J. (1 990). Differential alterations of cortical glutamatergic binding sites in senile dementia of the Alzheimer type. Proc. Natl. Acad. Sci. USA 87, 1352-1 356. Choi, D.W. (1988). Glutamate neurotoxicity and diseases of tke nervous system. Neuron 1, 623-634. Cowburn, R.F., Hardy, J.A., Briggs, R.S. and Roberts, P.J. (1989). Characterisation, density, and distribution of kainate receptors i n normal and Alzheimer's disease human brain. /. Neurochem. 5 2 , 140-1 47. I. and Egebjerg, J., Bettler, B., Herrnans-Borgmeyer, Heinemann, S. (1991). Cloning of a c D N A for a glutamate receptor subunit activated by kainate but not AMPA. Nature 351, 745-748. Feinberg, A.P. and Vogelstein, €3. (1 983). A technique for radiolabeling D N A restriction fragments to high specific activity. A n d . Biochem. 132, 6-13. Call, C., Sumikara, K. and Lynch C . (1 990). Levels of mRNA for a putative kainate receptor are affected by seizures. Proc. Natl. Acad. Sci. USA 87, 7643-7647. Geddes, J.W., Monaghan, D.T., Cotman, C.W., Lott, I.T., Kim, R.C. and Chang Chui, H. (1985). Plasticity of hippocampal circuitry i n Alzheimer's disease. Science 230, 1179-1 181. Goedert, M. (1987). Neuronal localisation of amyloid beta protein precursor mRNA in normal brain and in Alzheimer's disease. EMBO). 6 , 3627-3632. Goedert, M., Spillantini, M.C., lakes, R., Rutherford, D. and Crowther, R.A. (1989). Multiple isoforms of human microtubule-associated protein Tau: sequences and localization in neurofibrillary tangles of Alzheimer's disease. Neuron 3, 51 9-526. Harrison, P.J., McLaughlin, D. and Kerwin, R.W. (1991). Decreased hippocampal expression of a glutamate receptor gene in schizophrenia. Lancet 337, 450-452. Harrison, P.J., Barton, A.J.L., Najlerahim, A. and Pearson, R.C.A. (1 990). Distribution of a kainate/AMPA receptor mRNA i n normal and Alzheimer brain. NeuroReport 1, 149-1 52. Hollmann, M., O'Shea-Creeniield, A., Rogers, S.W. and Heinemann, 5 . (1989). Cloning by functional expression of a member of the glutamate receptor family. Nature 342, 643-648. Keinanen, K., Wisden, W., Sommer, B., Werner, P., Herb, A., Verdoorn, A,, Sakman, 6. and Seeburg, P. (1990). A family of AMPA-selective glutamate receptors. Science 249, 556-560. Kerwin, R.W., Patel, S., Meldrum, B.S., Czudek, C. and Reynolds, G.P. (1 988). Asymmetrical loss of glutamate receptor subtype in left hippocampus in schizophrenia. Lancet, i, 583-584. Lebo, R.V., Tolan D.R., Bruce, B.D., Cheung, M.-C. and Kan,
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Y . W . (1 985). Spot-blot analysis of sorted chromosomes designs a fructose intolerance disease locus to chromosome 9. Cytornetry 6, 478-483. Maragos, W.F., Creenamyre, J.T., Pennev, 1.6. and Young, A.B. ( 1 987). Glutarnate dysfunction i n Alzheimer’s disease: an hypothesis. Trends Neurosci. 10, 65-68. Masu, M., Tanabe, Y., Tsuchida, K., Shiigemoto, R. and Nakanishi, S. (1991). Sequence and expression of a metabotropic glutamate receptor. Nature 349, 760-765. Nakanishi, N., Shneider, N.A. and Axel, R. (1990).A family of glutamate receptor genes: evidence for the formation of heteromultimeric receptors with distinct channel properties. Neuron 5 , 569-581. Sakirnura, K., Bujo, H., Kushiya, E., Araki, K., Yamazaki, M., Yamazaki, M., Meguro, H., Warashina, A., Numa, S. and Mishina, M. (1990). Functional expression from cloned cDNAs of glutamate receptor species responsive to kainate and quisquaiate. FEBS Left. 272, 73-80.
Schofield, P.R., Pritchett, D.B., Sontheimer, H., Kettenmann, H. and Seeburg, P.H. (1991). Sequence and expression of a human CABA-A receptor a1 and pl subunits. FEBS Lett. 244,
361-364. Sommer, B., Keinanen, K., Verdoorn, T.A., Wisden, W., Burnashev, N., Herb, A., Kohler, M., Takagi, T., Sakman, 6. and Seeburg, P.H. (1990). Flip and flop: a cell-specific functional switch in glutamate-operated channels of the CNS. Science 249, 1580-1 585. Staden, R . (1990). An improved sequence handling package that runs on the Apple Macintosh. Cabios 6, 387-393. Werner, P., Voigt, M., Keinanen, K., Wisden, W. and Seeburg, P.H. (1991).Cloning of a putative high-affinity kainate receptor expressed predominantly i n hippocampal CA3 cells. Nature 351, 742-744.
