Neurochem. Int. Vol. 21, No. 1, pp. 91-98, 1992
0197-0186/9255.00+0.00 Copyright ~) 1992 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
S T R U C T U R E A N D E X P R E S S I O N OF H U M A N A N D R A T D2 D O P A M I N E R E C E P T O R G E N E S KAZUAKI ARAKI, l RYOZO KUWANO, ~* KEN MORII, ~* SHIGENOBU HAYASHI, 3 SHINSEI MINOSHIMA, 4 NOBUYOSHI SHIMIZU, 4 TAKASHI KATAGIRI, 2 HIROSHI USUI, 2 TOSHIRO KUMANISHI 2 a n d YASUO TAKAHASHIt~" Departments of 'Neuropharmacology and 2Neuropathology, Brain Research Institute, Niigata University, Niigata 951 3Saigata National Hospital, Saigata, Ohgata-machi, Nakakubiki-gun, Niigata 949-31 4Department of Molecular Biology, Keio University School of Medicine, Shinanomachi, Shinjuku-ku, Tokyo 160, Japan (Received 26 August 1991 ," accepted 12 November 1991)
Abstract--D2 dopamine receptor may be related with the pathogenesis of Parkinson's disease and schizophrenia. Furthermore, the antipsychotic drugs have high affinity for D2 dopamine receptor. We carried out the cloning of the genomic DNA for human D2 dopamine receptor and clarified the structure of this gene. Our isolated gene spans about l 5 kbp and consists of seven exons interrupted by six introns. However, putative first exon was not yet identified. Spot blot hybridization analysis of cell sorter fractionated human chromosomal DNA with D2 receptor genomic DNA revealed the localization of this gene in the chromosome 11 fraction. We analyzed human genomic DNA by Southern blot hybridization with D2 dopamine receptor genomic DNA as a probe, but so far we could not find RFLP. Northern blot analyses of brain RNA of several animals and rat brain RNA after various treatments were carried out. Developmental changes of D2 dopamine receptor mRNA were observed in the rat brains.
D o p a m i n e and dopamine receptors may be involved in the m o v e m e n t and emotion balances. D2 dopamine receptor has high affinity for the antipsychotic drugs. This receptor may also be related to the pathogenesis of Parkinson's disease and schizophrenia (Seeman, 1980; Seeman and Niznik, 1990). We have attempted to analyze the structure of the dopamine receptor gene and changes of this structure in the schizophrenia. Recently we reported the purification of bovine brain D2 dopamine receptor protein (Nishizawa et al., 1988 ; Usui et al., 1990). Further we anlayzed the D2 dopamine receptor expressed in Xenopus oocyte after injection of poly(A) R N A extracted from the bovine striata by using electrophysiological procedure (Sakai et al., 1990). Bunzow et al. (1988) published a paper on c D N A cloning of rat D2 dopamine receptor. Then we preliminarily reported the cloning and the structure of human D2 dopamine gene (Kuwano et al., 1990; Araki et al., 1990, 1991). In this paper we
describe all our data including the cloning, structure, Southern blot analysis and chromosomal localization of human D2 dopamine receptor gene and Northern blot analysis of D2 dopamine receptor m R N A . We also describe the developmental changes of rat brain m R N A for D2 dopamine receptor. EXPERIMENTAL PROCEDURES
Probes
Four oligodeoxynucleotides (I, I1, III and IV) corresponding to the four regions of the rat D2 dopamine receptor cDNA isolated by Bunzow et aL (1988) were synthesized on an Applied Biosystems DNA synthesizer. Oligodeoxynucleotides were labeled with ['~-32p]ATP by T4 polynucleotide kinase and used for screening of human genomic DNA library and for Northern and Southern blot analyses. Genomic screening anti sequencing
EMBL 3 human genomic DNA libraries, which were constructed from human leucocyte genomic DNA or were purchased from Clontech Laboratories (Palo Alto, Calif., U.S.A.), were screened by plaque hybridization with the above four oligodeoxynucleotide probes. The DNA fragments from the positive phage clones were digested with several restriction endonucleases and the restriction mapping was carried out. These DNA fragments were subcloned into
*Present address: Research Laboratory for Molecular Biology, Niigata University, Niigata 951, Japan. t Author to whom all correspondence should be addressed. 91
92
KAZUAKI ARAKI el al.
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Fig. 1. Southern blot hybridization of h u m a n and rat genomic DNA. Probe is synthetic oligodeoxynucleotide IV. Endonucleases used for digestion of genomic D N A were H (HindlII), E (EcoRI) and B (BamHI). A, h u m a n ; B, rat.
Blot analyses R N A was isolated from the rat brain by the guanidinium thiocyanate/cesium chloride procedure (Chirgwin et al., [979; Yamakuni el al., 1988). Northern blot hybridization was done as described previously (Thomas, 1980). Highmolecular weight total genomic D N A was prepared from the h u m a n placenta by the procedure of Blin and Stafford (1976). Southern blot hybridization was carried out with the genomic D N A fragments labeled with [:~-~2p]dCTP by a random-primed D N A labeling kit (Southern, 1975).
p U C 8 vector and used for D N A nucleotide sequencing with M a x a m Gilbert method (Maxam and Gilbert, 1980) or dideoxy method (Sanger et al., 1977). Exon intron organization was determined on the basis of the restriction map and D N A nucleotide sequence.
Chromosomal mappin9 H u m a n metaphase chromosomes were prepared from h u m a n B lymphoblast cell line, GM00130B, as described previously (Taira et al., 1989). C h r o m o s o m e s stained with propidium iodide were excited under 488 n m light with an LP520 optical filters and sorted by a FACS 440 cell sorter (Beckton Dickinson). C h r o m o s o m e s stained with Hoechst 33258 were also sorted by a FACS cell sorter. Spot blot hybridization was performed as described previously. Assignment of chromosomes in each fraction was carried out with chromosome-specific D N A probe. Filter disks were hybridized with the ~2P-labeled h u m a n D2 dopamine receptor genomic DNA.
RESULTS ANt) DISCUSSION
Southern blot hybridization After high-molecular weight DNA was prepared a n d d i g e s t e d w i t h several r e s t r i c t i o n e n d o n u c l e a s e s , S o u t h e r n blot a n a l y s i s o f this d i g e s t e d D N A w a s per-
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Fig. 2. Restriction map of the gene for the h u m a n D2 dopamine receptor. The solid boxes indicate the exons and the straight lines between each exon show the introns. Relevant endonuclease sites were shown as follows : E, EcoRl ; B, BamHI ; K, Kpnl ; Bg, BgllI ; S, Stul ; H, Hindlll. Some D N A fragments u3ed for sequencing are shown under the restriction map.
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D2 dopamine receptors
93
10 20 30 40 50 60 TT&AGGAGGT GTTTOTTGAA TGAAATAAAT AAATOATGGA CCAGCTCTGT CTAAGCCCAC
2110 2120 2130 2140 3180 AGTGCTCAGG CcAGGGACCC AGAGCCAGOT eeee*~e*e* eeew * * *
70 60 60 100 110 120 CTTTATATGC ATTTTTAAAA AATTGTGTTT CCCCATTTGT CCTACCAOCT GTAAAAACTT
2170 2180 2190 2200 2210 2220 TGACCCTGTG GTGTTTGCAG GAGTCTTCGA GGGGGAAAGG GAGGGGCCAG TGAGATGGGT
130
140
160
160
170
100
2230
2240
2250
2260
2160 t***
2270
2260
GCTAGCAAAA OAGTGGCAGA AGGCCCAACA TTTTT&GAAA ATTCTTAGCC GTAAACCTTA
GGCTGATGCC TAGGA&CTTO TCCGGCTTTA CCCAGGCCCT CTGCCTCTGG TCOAGGAGGC__
190 200 210 220 220 240 AATCCCOAOT TGGAAAATTC TCATTATTAA ATGCCAAGSG TTGGTCTTCC TAGAGSCCTC
2290 2300 2310 2320 2330 2340 TGCCCGGCGA GCCCAGGAGC TGGAGATGGA GATGCTCTCC AGCACCAGCC CACGCGAGAG
260 260 270 260 290 300 TGCAAGAGGC CCTCTCACCG CCACCTTGTG CCCATTTTTC CTC-CCAGAGC CTGCCACCCA
2350 2360 2370 3380 2390 2400 GACCCGGTAC AGCCCCATCC CTCCCAGCCA CCACCAGCTG ACTCTCCCCG ACCCGTCCCA
310 320 330 340 360 360 GTOGCTCCAC CCCCCTGATG GATCCACTOA ATCTGTCCTG GTSTGATGAT GATCTGGAGA
2410 2420 2430 3440 2450 2460 CCATGGTCTC CACAGCACTC CCGACAGCCC CGCCAAACCA GAGAAGAATG GGCAT~..CAA
370 380 300 400 410 420 GGCAGAACTG GAGCCGGCCC TTCAACGGGT CAGACGGGAA GGCGGACAOA CCCCACTACA
2470 2480 2490 2500 2510 2520 AOACCACCCC AAGATTGCCA AGATCTTTGA GATCCAGACC ATGCCCAATG GCAAAACCCG
430 440 450 460 470 480 ACTACTATGC CACACGGCTC ACCCTGCTCA TCGCTGTCAT CGTCT7CGGC AACGTGCTGG
2530 2540 3660 2560 2570 2590 GACCTCCCTC AAGACCATOA GCCGTAGGAA GCTCTCCCAG CAGASGGAGA AGAAAGCCAC
490 500 810 520 630 540 TGTGCATGGC TGTGTCCCGC GAGAAGGCGC TGCAGACCAC CACCAACTAC CTOATCGTCA
2590 2600 2610 2620 2630 2640 TCAGATGCTC GCCATT~;TTC TCGGTGAGTC= GGCCCTGGCG CTGGCCACAG CCGGTCTGTG
650 560 670 560 590 600 GCCTTGCAOT GGCCGACCTC CTCGTCGCCA CACTGGTCAT GCCCTGGGTT GTCTACCTOG
2650 3660 2670 2680 2690 2700 AAAGGTCCCA TTCCCTGCC8 TGTCCTTCCT AACCATGGCT Oeee*~eeee eeeeee * *
610 620 630 640 650 860 AGGTAGGTGG GCCCCTTGCC AGCACTTTCT (;CAGCA(]GGC CCTGCACTGG ACACeeee*~,
2710 2720 2730 2740 2750 2750 AAGACCGGGA CTCCTACAGT CCTTCTGTGG CTACAGCCCA CCGTCTTGGC ATACGAGCCA
670
680
000
700
710
730
2770 2790 2790 2800 2810 2820 GGGCGCACTO OGTGTGGGTG TTCCCAGCCG TGCCTCCCCG GCTCTGGGGA CCAGCCTGAC
730
740
750
760
770
780
2830 2840 2650 2850 2870 2800 CATGCCCTCT CCCCCAGGCG TGTTCATCAT CTGCTGGCTG CCCTTCTTCA TCACACACAT
AC&AGACTTO CAGCTGCCTC CTGAGTCTGT OGCCTCATCG GCTCCAGGAG TACCAGGCT& 790
800
610
820
630
640
CAGGACCT&A GCTAATCTCC CACTCCTOCT OTCCATCCAT TATGTTGCTT TGTCCCC~
2890 2900 3910 2930 2030 2940 CCTOAACATA CACTGTGACT GCAACATCCC GCCTGTCCTG TACAGCGCCT TCACGTGGCT
850 660 670 680 590 900 TGGTAGGTGA GTGGAAATTC AGCAGGATTC ACTGTGACAT CTTCGTCACT CTGGACGTCA
2950 3960 3970 2980 3990 3000 GGGCTATGTC AACAGCGCCG TGAACCCCAT CATCTACACC ACCTTCAACA TTGAGTTCCG
910 920 930 940 860 960 TG~TG~GC~C GGCGAGCATC CTGAACTTGT GTGCCATCAG CATCGACAGG TGAGCCCAGC
3010 3020 3030 3040 3050 3060 C/~AGGCCTTC CTGAAGATCC TCCACTGCTG ACTCTGCTGC CTGCCCGCAC AGCAOCCTGC
970
960
090
1000
1010
1020
CAGGGTGGAA GAGGTTGCTC TGGCCACTCA CCATTCTGCC TGCCCCAGCT TTTCTCAAAG
3070
3080
3090
3100
3110
3120
TTCCCACCTC CCTGCCCAGG CCkGCCAGCC TCACCCTTGC GAACCGTGAG CAGGAAGGCC
1030 1040 1060 1060 1070 1080 CCAGGCTGTG TCTCGTGTCT GTOATGCTGT OCAGCTGGCT GTGTCCATGG GGGTCCATGT
3130 3140 3150 3100 3170 3180 TGGGTGOATC GGCCTCCTCT TCACCCCG3C AGGCCCTGCA GTOTTCGCTT GGCTCCATGC
1090 1100 1110 1120 1130 1140 GTTTGTGTGT ATGGGTGTGC ATOTGTGTAT OTTTGGCTAG GAGAGCACAC ACTCCCTAAA
3190 3200 3310 3220 3230 3240 TCCTCACTGC CCGCACACCC TCACTCTGCC AGGGCAGTGC TSGTGAGCTG GGCATGGTAC
1160 1160 1170 1180 1190 1200 GAGAAGCCAT CTATGGACAG TGATGCTGCC 8AGCATACTC AGACAGTGAC TOGTkCAAAA 1210
1220
1230
1240
1250
1260
2260 3260 3370 3280 3290 3300 CAGCCCTGGG GCTGGGCCCC CCAGCTCAGG GGCAGCTCAT AGAGTCCCCC CTCCCACCTC 3310 3320 3330 3340 3350 3360 CAGTCCCCCT ATCCTTGGCA CCAAAOATGC AGCCGCCTTC CTTGACCTTC CTCTGGGGCT
GGGGAAAGAT TOTGATTCTG ACGGAACCCC TAGCTCTAGC ACATCTTACT CATGTACCTT 1270
1280
1290
1300
1310
1320
]330 1340 1350 1360 1370 1300 GTGAGGGTGT CT¢TOGTGTG TGCATATTGT GGAGTGAGGG OTCCCTGGGC CTCCACCCCA 1390 1400 1410 1420 1430 1440 GATTCAGGGT CCCCCGCCCG TTGCAGC;[AC ACAGCTGTGG CCATGCCCAT GCTGTACAAT 1450 1460 1470 1480 1490 1600 ACGCGCTACA GCTCCAAGCG CCGGGTCACC GTCATGATCT CCATCGTCTG GGTCCTGTCC
1510 1520 1530 1540 1650 IS60 TTCACCATCT CCTGCCCACT CCTCTTCGGA CTCAATAACG CAGGTACATT CT3CTTTGCT w 1670 1580 1500 1600 ]6]0 1520 TGCCTGAGGC CkCCTOGCCC TGGGCCTGCT CCCTCGAAGG GCCCCTGAGG GAkCAGkGTC 1630 1640 1650 1660 1670 CTGGCACAGA CATGGGTOGA AACCAATGGS # * * * * * * * * * * * * * * * * * * * * e * 1690
1700
1710
1720
1730
3370 3380 3390 3400 3410 3420 CTAGGGTTGC TGGAGCCTGA GTCAGGGCCC AGAGGCTGAG TTTTCTGTTT GTGGGGCTTG 3430 3440 3460 3460 3470 3460 GCGTGGAGCA GGCGGTGGGG AGAGATGGAC AGTTCACACC CTGCAAGGCC CACAGGAGCC 3490 3500 3610 3520 3530 3540 AAGCAAGCTC TCTTGCCGAG GAGCCAGCCA ACTTCAGTCC TGGGAGACCC ATGTAAATAC 3650 3560 3570 3580 3590 3600 CACTGCAGGT TGGACCCCAG AGATTCCCAA GCCAAAAACC 7TAGCTCCCT CCCGACCCCG 3610 3620 3630 3640 3550 3660 ATGTGGACCT CTACTTTCCA GGCTAGTCCG GACCCACCTC AC¢CCGTTAC AC~TCCCCAA 3870 3690 3090 3700 3710 3720 GTGGTTTCCA CATGCTCTGA GAAGAGGAGC CCTCATCTTG AAGGGCCCAG GAGGGTCTAT
1680
3730 3740 3750 3760 3770 3780 GGGGAGAGGA ACTCCTTGGC CTAGCCCACC CTTCTGCCTT CTGACGGCCC TGCAATGTAT
1740
3790 3800 3610 3820 3630 3640 CCCTTCTCAC AGCACATGCT GGCCAGCCTG OGGCCTGGCA ~GGAGGTCAG GCCCTGGAAC
*
CCOGTOGGCC CGCTGACTCC CTGCCTGCCC CGGGCTCCCT CCCCCAGSCC AGAACGAGTG 1750
1760
1770
1760
1790
1800
CATCATTGCC AACCCGGCCT TCGTGGTCTA CTCCTCCATC GTCTCCTTCT ACGTOCCCTT 1610 1610 1830 1840 1650 1680 CATTGTCACC CTGCTGGTCT ACATCAAGA7 CTACATTGTC CTCCGCAGAC GCCGCAAGCG 1870 1880 1890 1900 1910 1920 AGTCAACACC AAACGCAGCA GCCGAGCTTT CAGGGCCCAC CTGAGGGCTC CACTAAAGG_.T
38s0 3860 3870 3860 3890 3900 TCTATCTGGG CTAGGGGACA TCAGAGGTTC TTTGAGGGAC TGCCTCTGCC ACACTCTGAC 3010
1990
2000
2010
2020
2030
2040
TTGTGTACCA TGTOCTGGCT CACTCCAC/~ GGCAACTGTA CTCACCCCGA GGACATGAAA 2050 3060 2070 3080 3090 2100 CTCTGCACCG TTATCATGAA GTCTAATGGG AGTTTCCCAG TGSACAOGCG GAGAGTSG=~A
3930
3940
3950
3960
3970 3080 3090 4000 4010 4020 TCCACTGCCT CTGCCTTAGA GGACCCACGG CTAAGAGGCT GCTGAAAACC ATCTGGCCTG 4030
1930 1940 1950 1960 1970 1980 CTCAAGACAC CCCCCAACCe e * * * * * * * * * * * * * e * * * * * * * e e * e e * * * * * s e e e e * * e
3920
GCAAAACCAC TTTCCTTTTC TATTCCTTCT CCCCTTTCCT CTCTCCTGTT TCCCAACCCT
4040
4060
4060
4070
4080
GCCTGGCCCT GCCCTGAGGA AGGAGGGGAA GCTCGAGCTT GGGAGAGCCC CTGGGGCCTA 4090 4100 4110 4120 4130 4140 GACTCTGTAA CATCACTATC CATGCACGAA ACTkATAAAk CTTTGACGAG TGACCTTCCA
4160 4100 4170 41ao 4190 4200 GGACCCCTGG GTAGAAGGCA GCAGTGCCAC TTCTGTGCI'T GGCATTCAAG TATAGGAAGA
Fig. 3. Nucleotide sequence of h u m a n D2 dopamine receptor gene. Exon sequences were underlined. The inside portions of each intron were omitted. The junctional sequences were double underlined.
M
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2110
2120
2130
2140
2150
2160
2330 2240 2250 2260 2270 2280 GTGGACCTCTACTTTCCAGGCTAGTCCGGACCCACCTCACCCCGTTACAGCTCCCCAAGT
2170 2180 2190 2200 2210 2220 CTGCAGGTTGGACCCCAGAGATTCCCAAGCCAAAAACCTTAGCTCCCTCCCGACCCCGAT
GCAAGCTCTCTTGCCGAGGAGCCAGGCAACTTCAGTCCTGGGAGACCCATGTAAATACCA
T
1510
M
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TGAACAT ACACTGTGACTGCAACATCCCGCCTGTCCTGTACAGCGCCTTCACGTGGCTGG N I H C D C N I P P V L Y $ A F T W L G
K
2420
243(~
2440
2450
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CTTCTCACAGC ACATGCTGGCCAGCCTGGGGCCTGGCAGGGAGGTCAGGCCCTGG A ACTC
2410
2350 2350 2370 2380 2390 2400 GGAGAGGAACTCCTTGGCCTAGCCCACCCTTCTGCCTTCTGACGGCCCTGCAATGTATCC
K
2080
145O 1460 147n 14110 149O 1500 TCGCC ATTGTTCTCGGCGTGTTCATCATCTGCTGGCTGCCCTTCTTCATCACACACATCC A I V L G V F 1 I C W I. P F F I T H I L
A
2070
2290 2300 2310 2320 2330 234{* GGTTTCCACATGCTCTGAGAAGAGGAGCCCTCATCTTGAAGGGCCCAGGAGGGTCTATGG
l
2060
GTGGAGCAGGCGGTGGGGAGAGATGGACAGTTCACACCCTGCAAGGCCCACAGGAGGCA^
2050
1990 2000 2010 2020 2030 2040 AGGGTTGCTGGAGCCTOAGTCAGGGCCCAGAGGCTGAGTTTTCTGTTTGTGGGGCTTGGC
1930 1940 1950 1960 1970 1980 GTCCCCCTATCCTTGGCACCAAAGATGCAGCCGCCTTCCTTGACCTTCCTCTGGGGCTCT
1870 1880 1890 19OO 1910 1920 GCCCTGGGGCTGGGCCCCCCAGCTCAGGGGCAGCTCATAGAGTCCCCCCTCCCACCTCCA
1810 1820 1830 1840 1850 ]86,, CTCACTGCCCGCACACCCTCACTCTGCCAGGGCAGTGCT AGTG AGCTGGGCATGGT ACC A
1750 1760 177o 178o 179O I 8n(, GGTGGATCGGCCTCCTCTTC ACCCCGGCAGGCCCTGCAGTGTTCGCTTGGCTCC ATGCTC
12~t0 1400 1410 1420 1430 ) 44h TCAAGACCATGAGCCGTAGGAAGCTCTCCCAGCAGAAGGAGAAGAAAGCCACTCAGATGC
K
CCAAGATTGCCAAGATCTTTGAGATCCAGACCATGCCCAATGGCAAAACCCGGACCTCCC
1330
TCC ACAGCACTCCCGACAGCCCCGCCAAACCAGAGAAGAATGGGCATGCCAA AGACCACC H S T P D S P A K P E K N G H A K D H P
1270
1210 1220 1230 1240 1250 1200 ACAGCCCCATCCCTCCCAGCCACCACCAGCTGACTCTCCCCGACCCGTCCCACCATGGTC S P I P P S H H q L T g P D P S H H G L
GAGCCCAGGAGCTGGAGATGGAGATGCTCTCCAGCACC A(]CCCACCCGAGAGGACCCGGT A Q g L E M E N L 8 8 T S P P E R T R Y
I
1670 16~o AC AGC AGCCTGCT T
1690 1700 1710 1720 173o 174l) CCC ACCTCCCTGCCC AGGCCAGCCAGCCTCACCCTTGCGAACCGTGAGC AGGA,~GGCCT G
f
1630 164U 1650 1660 AGGCCTTCCTGAAGATCCTCCACTGCTGACTCTGCTGCCTGCCCGC A F L K 1 L H C
1570 1580 1590 1600 1610 1620 GCTATGTCAACAGCGCCGTGAACCCCATCATCT ACACCACC TTCAACATTG AGTTCCGC A y V N $ A V N P I I Y T T F N I E F R K
Fig. 4. Nucleotide sequence and deduced amino acid sequence of the human D2 dopamine receptor mRNA. The amino acids were expressed as a single letter. Seven transmembrane regions were underlined.
610 620 630 1540 6~O 5K41 AGGTGGTAGGTGAGTGGAAATTCAGCAGGATTCACTGTGACATCTTCGTCACTCTGGACG V V G E W R F $ R I H C D I F V T L 0 V
550 560 570 880 590 600 GCCTTGCAGTGGCCGACCTCCTCGTCGCCACACTGGTCATGCCCTGGGTTGTCT ACCTGG L A ¥ A D L L V A T L V N P V V V Y L E
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490 500 510 620 530 540 TGTGC ATGGCTGTGTCCCGCGAGA AGGCGCTGCAGACCACCACCAACTACCTGATCGTCA
D
1090 1100 1110 |120 1130 I140 TTATCATGAAGTCTAATGGGAGTTTCCCAGTGAACAGGCGGAGAGTAGAGGCTGCCCGGC
y
430 440 45O 450 470 480 ACTACTATGCCACACGGCTC ACCCTGCTCATCGCTGTCATCGTCTTCGGCAACGTGCTGG Y Y A T H L T L L I A V I V F G N V L V
I~
1030 1040 I050 1080 InTO 108{, ACCTGAGGGCTCCACTAAAGGGCAACTGT ACTCACCCCGAGGACATGAAACTCTGCACCG L R A P L K G N C T H P E D M K I. C T V
S
370 380 39O 400 410 42O GGCAGAACTGGAGCCGGCCCTTCAACGGGTCAGACGGGAAGGCGGACAGACCCC ACT ACA Q N W S R P F N G S D G K A D R P H Y N
L
970 980 990 IOOO 1{110 1020 TCCTCCGCAGACGCCGCAAGCGAGTCAACACCAAACGCAGCAGCCGAGCTTTCAGGGCCC L R R R R K R V N T K R S S R A F R A H
N
R
L
310 320 330 340 350 360 GTGGCTCCACCGCCCTGATGGATCCAC~'GAATCTGTCCTGGT ATGATGATGATCTGGAGA
P
910 920 930 940 950 960 TCGTCTCCTTCTACGTGCCCTTCATTGTCACCCTGCTGGTCT ACATCAAGATCTACATTG V S F Y V P F I V T g L V Y I K I Y I V
250 250 270 280 290 300 TGCAAGAGGCCCTCTCACCGCCACCTTGTGCCCATTTTTCCTGCCAGAGCCTGCCACCCA
D
B50 860 §70 880 89(1 90O ATAACGCAGACCAGAACGAGTGC ATCATTGCCAACCCGGCCTTCGTGGTCTACTCCTCCA N A D Q N E C I I A N p A F V V Y $ S I
210 220 230 240 ATTAAATGCC AAGAGTTGG7 CTTCCT AGAGACCTC
190 2 O0 AATCCCGAGTTGGAAkATTCTCATT
H
790 800 810 820 830 84O TGATCTCCATCGTCTGGGTCCTGTCCTTCACCATCTCCTGCCCACTCCTCTTCGGACTCA I S I V W V L S F T I S C P L L F G L N
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720 74O 750 760 770 78O CTGTGGCCATGCCCATGCTGTACAATACGCGCTACAGCTCCAAGCGCCGGGTCACCGTCA V A N P N L Y N T R Y $ S K R R V T V H
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~70 6B0 690 7OO 710 TCATGATGTGCACGGCGAGCATCCTGAACTTGTGTGCCATCAGCATCGACAGGT
130 140 150 160 170 I~O GCTAGCAAAAGAGTGGCAGAAGGCCCAACATTTTTAGAAAATTCTTAGCCGTAAACCTTA
O 2 I CTTTATAT~ATTTTTAA~AATTGTGT~GCCCATT~CCTACCA~TGTAAAAA~T~
TTAAGGAGG~GTTTGTTG~TGAAATAA~AAATGATG~CCAGCTCT~CTAAGCCC~
D2 dopamine receptors
95
414 I
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Fig. 5. Hydrophilicity profile of human D2 dopamine receptor (414). I-VI! under the profile indicate the transmembrane regions.
formed with a probe IV. As shown in Fig. 1, the blot analysis of human genomic digested DNA showed the presence of a single hybridizing band, suggesting the presence of a single copy of human D2 dopamine receptor gene per haploid genome. Figure 1 also shows the blot analysis of rat genomic digested DNA, indicating the presence of a single or two bands. Exon intron organization receptor gene
of human D2 dopamine
As described in the section of Experimental Procedures, a human genomic DNA library was screened by plaque hybridization with four oligodeoxy-
nucleotides as a probe and three positive clones were isolated. These cloned DNAs were used for further analyses. Figure 2 shows the restriction map, the subcloned fragments and exon-intron organization. Nucleotide sequencing of the DNA fragments from the clones revealed the structure of the D2 dopamine receptor gene. This gene isolated spans about 15 kbp in length and appeared to be composed of six exons separated by five introns. Recently several groups found an additional amino acid sequence of rat D2 receptor produced by alternative splicing of the new exon (Giros et al., 1989; Chio et al., 1990). Since the nucleotide sequences of
2 A
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Flow karyotype of GM130B with Prl
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Fig. 6. Localization of the human D2 dopamine receptor gene on the flow-sorted chromosomes from the human B lymphoblast line GM00130B. 1 : (A) Propidium iodide-stained flow-karyotype.(B) Hybridization of human D2 dopamine receptor genomic DNA to spots containing sorted chromosomes. 2 : (A) Hoechst 33258-stained flow-karyotype. (B) Hybridization of human D2 dopamine receptor genomic DNA to spots containing sorted chromosomes.
96
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Fig. 8. Northern blot hybridization of rat brain RNA intoxicated with methylmercury. Total RNA (20 #g) from control and intoxicated rat brains were used. Conditions were as described in Fig. 7.
e
)
on the rat gene of O'Malley et al. (1990). Exon intron organization was not observed in most other Gprotein-coupled receptor gene family (for example, ~and/Y-adrenergic receptors, muscarinic A C h receptor, etc.) except opsin genes (Nathan, 1987). Therefore, it is important that D2 dopamine receptor gene has the exon intron organization.
Fig. 7. Northern blot analysis of brain RNA from several animals. Poly(A) ~ RNAs (5 pg) of each animal brain were used• Probe is the BamHI-AccI fragment of rat D2 dopamine receptor cDNA. B, bovine ; R, rat ; M, mouse : H, human.
Homoloy.v between receptors
some introns in our D N A fragments were already determined, these sequences were again surveyed, focusing A G - G T junctional sequence. The results indicated the presence of an additional exon between 4th exon and 5th exon. This additional exon contains 87 bp sequence coding 29 amino acid residues• In conclusion, it was found that two human D2 dopamine receptor m R N A s are produced by alternative splicing of this exon. Two human D2 dopamine receptor proteins were translated from these two m R N A s , producing D2 (443) and D2 (414) (Figs 3 and 4). These results are in agreement with the data of Grandy et al. (1989b). At the 3' end of this gene, poly(A) signal was found at the 3' side o f e x o n 7. Probably, one more additional exon remains to be identified at the 5' end of this human D2 receptor gene, considering the data
rat and human
D2 dopamine
The connection of the exons of human D2 dopamine receptor gene produced the nucleotide sequence corresponding to that of this receptor m R N A . The amino acid sequence of this receptor protein was deduced from the nucleotide sequence of m R N A (Fig. 4). The receptor proteins were composed of414 amino acid and 443 amino acid residues. The high homology of amino acid sequence was found between rat and human D2 dopamine receptor (about 95%), showing the decrease of one amino acid and changes of 19 amino acids in the human receptor. Hydrophilicity profile of human D2 dopamine receptor was examined, indicating the presence of seven hydrophobic regions (Fig. 5). These hydrophobic regions
t 1
5
10
15
20
50
6 0 days
Fig. 9. Developmental changes of rat D2 dopamine receptor mRNA. Total RNA (20 ,ug) isolated from rat brains of different developmental stages was spotted onto nitrocellulose filters and hybridized with ~2P-labeled D2 dopamine receptor cDNA as shown in Fig. 7.
D2 dopamine receptors suggest the presence of seven transmembrane regions. This profile showed that the structure of D2 dopamine receptor was similar to that of other G-protein coupled receptors with seven transmembrane regions. C h r o m o s o m a l localization
Since the chromosomal localization of this receptor gene was not yet identified at that time, where we isolated this gene, we attempted to assign this chromosomal localizatioaa by the spot blot hybridization procedure with D2 dopamine receptor genomic D N A on the flow-sorted chromosomes. The results are shown in Fig. 6. The fraction containing chromosome 11 shows the presence of positive signal. This data is in agreement with that of Grandy et al. (1989a) using a completely different method. SearchJbr RFLP
As the next step, we carried out Southern blot analysis of human genomic D N A to find R F L P with D2 dopamine receptor gene. Several restriction endonucleases were used to digest the human genomic DNA. Until now, RFLP with Mspl, TaqI, BglII, HindIII, EcoR1 and BamH1 was not observed. According to the report of Grandy et al. (1989a), RFLP was found in the human genomic D N A digested with TaqI. However, we could not yet confirm this data. For the examination of the pathogenesis of schizophrenia and Parkinson's disease, we should find RFLP in the human genomic DNA. Northern blot analys&
Figure 7 shows the results of Northern blot hybridization analysis of poly(A) + R N A from several animal brains. These m R N A s for D2 dopamine receptor from rat, bovine and human brains were found to be a single m R N A species of approximatley 2.5 kb in length. However, the mouse m R N A s were composed of two m R N A species, one of which was considerably larger than 2.5 kb. The significance of this larger m R N A in the mouse brain was not yet clarified. We examined the changes of D2 dopamine receptor m R N A in the rat and hamster brains after various treatments. The m R N A from the brains of the hamsters after Creutzfeldt-Jakob disease infection, did not show significant change. However, the D2 dopamine receptor m R N A of the brains of the rats with methylmercury intoxication showed slight decrease. Methylmercury (10 mg/kg wt/day) was injected subcutaneously for 7 days into the rat and then allowed the rat for a further 7 days until the intoxication symptom appeared (Fig. 8).
97
Developmental changes of this m R N A were also examined in the rats. Although fetal brains do not contain significant amounts of this mRNA, the newborn rats showed the significant signal. This signal gradually increased until 60 days after birth (Fig. 9). These results may correspond to the developmental changes of the movement and emotion in the rats. Recently cDNAs or genes for D1, D3, D4 and D5 dopamine receptors were isolated (Dearry et al., 1990 ; Sokoloffet al., 1990 ; Van Tol et al., 1991 ; Sunahara et al., 1991). The regional distributions of five dopamine receptors were examined by in situ hybridization histochemistry and it was found that each receptor showed different distribution patterns in the brain. We should examine the expressions of five dopamine receptor genes and their changes in various conditions using their specific probes. The human and rat genes for these receptors should be cloned and analyzed, although genes for human D1, D4 and D5 dopamine receptors were recently isolated (Sunahara et al., 1990, 1991 ; Van Tol et al., 1991).
REFERENCES
Araki K., Kuwano R. and Takahashi Y. (1990) Gene structure of human D2 dopamine receptor. A. Rep. Pharmacopsychiat. Res. Fdn. 21,233--237. Araki K., Kuwano R., Hayashi S. and Takahashi Y. (1991) Structure and expression of human and rat dopamine receptor genes. J. Neurochem. 57 (Suppl), S90D. Blin N. and Stafford D. W. (1976) Isolation of high molecular-weight DNA. Nucleic Acids Res. 3, 2303 2308. Bunzow J. R., Von Tol H. H. M., Grandy D. K., Albert P., Salon J., Christie M., Machida C. A., Neve K. A. and Civelli O. (1988) Cloning and expression of a rat D2 dopamine receptor cDNA. Nature 336, 783-787. Chio C. L., Hess G. F., Graham R. S. and Huff R. M. (1990) A second molecular form of D2 dopamine receptor in rat and bovine caudate nucleus. Nature 343, 266-269. Chirgwin J. M., Przybyla A. E., MacDonald R. J. and Rutter W. J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294~5299. Dearry A., Gingrich J. A., Falardeau P., Fremeau R. T. Jr, Bates M. D. and Caron M. G. (1990) Molecular cloning and expression of the gene for a human DI dopamine receptor. Nature 347, 72-76. Giros B., Sokoloff P., Mortres M.-P., Riou J.-F., Emorine L. and Schwartz J.-C. (1989) Alternative splicing directs the expression of two D2 dopamine receptor isoforms. Nature 342, 923-926. Grandy D. K., Litt M., Allen L., Bunzow J. R., Marchionni M., Makam H., Reed L., Magenis R. E. and Civelli O. (1989a) The human dopamine D2 receptor gene is located on chromosome 11 at q22-q23 and identifies Taql RFLP. Am. J. Hum. Genet. 45, 778-785. Grandy D. K., Marchionni M. A., Makam H., Stofko R. E., Alfano ZM., Frothingham L., Fischer J. B., Burke-Howie K. J., Bunzow J. R., Server A. C. and Civelli O. (1989b)
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Cloning of the cDNA and gene tor a human D2 dopamine receptor. Proc. natn Acad. Sci., U.S.A. 86, 9762--9766. Kuwano R., Araki K. and Takahashi Y. (1990) Structural analysis of human D2 dopamine receptor gene. Neurosci. I~es. 11 Suppl.~ S120. Maxam A. M. and Gilbert W. (1980) Sequencing-endlabeled DNA with base-specific chemical cleavages. Methods Enzym. 65, 499 560. Nathan J. 0987) Molecular biology of visual pigments. A. Rev. Neurosci. 10, 163--194. Nishizawa Y., Sato M., Hayashi S. and Takahashi Y. (1988) Purification of receptors binding antipsychotics: comparison with D2 dopamine receptor. In: Pharmacology and Functional Regulation o f Dopaminergic Neuron (Beart P. M., Woodruff G. N. and Jackson D. M., eds), pp. 92 95. Macmillan, London. O'Malley K. L., Mack K. J., Gandelman K.-Y. and Todd R. D. (1990) Organization and expression of the rat D 2A receptor gene : identification of alternative transcripts and a variant donor splice site. Biochemistry 29, 1367 1371. Sakai Y., Sekiguchi M., Okamoto K., Kuwano R. and Takahashi Y. (1990) Dopamine receptors expressed in the Xenopus oocytes injected with bovine striatal mRNA. Molec. Brain Res. 7, 183 187. Sanger F., Nicklen S. and Coulson A. R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. natn Acad. Sci., U.S.A. 74, 5463 5467. Seeman P. (1980) Dopamine receptor. Pharmac. Rez~. 32, 229 313. Seeman P. and Niznik H. B. (1990) Dopamine receptors and transporters in Parkinson's disease and schizophrenia. F A S E B J . 4, 2737 2744. Sokoloff P., Giros B., Martres M.-P., Bouthenet M.-L. and Schwartz J.-C. (1990) Molecular cloning and characterization of a novel dopamine receptor (D3) as a target for neuroleptics. Nature 347, 146 151.
Southern E. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. molec. Biol. 98, 503 517. Sunahara R. K., Niznik H. B., Weiner D. M., Stormann T. M., Brann M. R., Kennedy J. L., Gelernter J. E., Rozmabel R., Yang Y.~ Israel Y., Seeman P. and O'Dowd B. F. (1990) Human dopamine D2 receptor encoded by an intronless gene on chromosome 5. Nature 347, 8(~83. Sunahara R. K., Guan H.-C., O'Dowd B. F., Seeman P., Laurier L. G., Ng G., George S. R., Torchia J., Van Tol H. H. M. and Niznik H. B. (1991) Cloning of the gene for a human dopamine D5 receptor with higher affinity for dopamine than D1. Nature 350, 614 619. Taira M., Kudoh J., Minoshima S., Iizasa T., Shimada H., Shimizu Y., Tatibana M. and Shimizu N. (1989) Localization of human phosphoribosylpyrophosphate synthetase subunit I and II genes to different regions of the X chromosome and assignment of two PRPSI-related genes to autosomes. Somat. Cellmolec. Genet. 15, 29 37. Thomas P. S. (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. natn Acad. Sei., U.S.A. 77, 5201 5205. Usui H., Takahashi Y., Maeda N., Mitsui H., Isobe T., Okuyama T., Nishizawa Y. and Hayashi S. (1990) Purification of D2 dopamine receptor using photoaffinity labeling, HPLC and preparatives SDS-PAGE. J. Chromat. 515~ 375 384. Van Tol H. H. M., Bunzow J. R., Guan H.-C., Sunahara R. K., Seeman P., Niznik H. B. and Civelli O. (1991) Cloning of the gene for a human dopamine D4 receptor with high affinity for the antipsychotic clozapine. Nature 350, 610 614. Yamakuni T., Kuwano R., Araki K., Usui, H., lnoue Y. and Takahashi Y. (I 988) Developmental and regional changes ofmRNA for a cerebellar protein (spot 35) in the rat brain. J. Neurochem. 50, 282-284.
0197-0186/9255.00+0.00 Copyright ~) 1992 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
S T R U C T U R E A N D E X P R E S S I O N OF H U M A N A N D R A T D2 D O P A M I N E R E C E P T O R G E N E S KAZUAKI ARAKI, l RYOZO KUWANO, ~* KEN MORII, ~* SHIGENOBU HAYASHI, 3 SHINSEI MINOSHIMA, 4 NOBUYOSHI SHIMIZU, 4 TAKASHI KATAGIRI, 2 HIROSHI USUI, 2 TOSHIRO KUMANISHI 2 a n d YASUO TAKAHASHIt~" Departments of 'Neuropharmacology and 2Neuropathology, Brain Research Institute, Niigata University, Niigata 951 3Saigata National Hospital, Saigata, Ohgata-machi, Nakakubiki-gun, Niigata 949-31 4Department of Molecular Biology, Keio University School of Medicine, Shinanomachi, Shinjuku-ku, Tokyo 160, Japan (Received 26 August 1991 ," accepted 12 November 1991)
Abstract--D2 dopamine receptor may be related with the pathogenesis of Parkinson's disease and schizophrenia. Furthermore, the antipsychotic drugs have high affinity for D2 dopamine receptor. We carried out the cloning of the genomic DNA for human D2 dopamine receptor and clarified the structure of this gene. Our isolated gene spans about l 5 kbp and consists of seven exons interrupted by six introns. However, putative first exon was not yet identified. Spot blot hybridization analysis of cell sorter fractionated human chromosomal DNA with D2 receptor genomic DNA revealed the localization of this gene in the chromosome 11 fraction. We analyzed human genomic DNA by Southern blot hybridization with D2 dopamine receptor genomic DNA as a probe, but so far we could not find RFLP. Northern blot analyses of brain RNA of several animals and rat brain RNA after various treatments were carried out. Developmental changes of D2 dopamine receptor mRNA were observed in the rat brains.
D o p a m i n e and dopamine receptors may be involved in the m o v e m e n t and emotion balances. D2 dopamine receptor has high affinity for the antipsychotic drugs. This receptor may also be related to the pathogenesis of Parkinson's disease and schizophrenia (Seeman, 1980; Seeman and Niznik, 1990). We have attempted to analyze the structure of the dopamine receptor gene and changes of this structure in the schizophrenia. Recently we reported the purification of bovine brain D2 dopamine receptor protein (Nishizawa et al., 1988 ; Usui et al., 1990). Further we anlayzed the D2 dopamine receptor expressed in Xenopus oocyte after injection of poly(A) R N A extracted from the bovine striata by using electrophysiological procedure (Sakai et al., 1990). Bunzow et al. (1988) published a paper on c D N A cloning of rat D2 dopamine receptor. Then we preliminarily reported the cloning and the structure of human D2 dopamine gene (Kuwano et al., 1990; Araki et al., 1990, 1991). In this paper we
describe all our data including the cloning, structure, Southern blot analysis and chromosomal localization of human D2 dopamine receptor gene and Northern blot analysis of D2 dopamine receptor m R N A . We also describe the developmental changes of rat brain m R N A for D2 dopamine receptor. EXPERIMENTAL PROCEDURES
Probes
Four oligodeoxynucleotides (I, I1, III and IV) corresponding to the four regions of the rat D2 dopamine receptor cDNA isolated by Bunzow et aL (1988) were synthesized on an Applied Biosystems DNA synthesizer. Oligodeoxynucleotides were labeled with ['~-32p]ATP by T4 polynucleotide kinase and used for screening of human genomic DNA library and for Northern and Southern blot analyses. Genomic screening anti sequencing
EMBL 3 human genomic DNA libraries, which were constructed from human leucocyte genomic DNA or were purchased from Clontech Laboratories (Palo Alto, Calif., U.S.A.), were screened by plaque hybridization with the above four oligodeoxynucleotide probes. The DNA fragments from the positive phage clones were digested with several restriction endonucleases and the restriction mapping was carried out. These DNA fragments were subcloned into
*Present address: Research Laboratory for Molecular Biology, Niigata University, Niigata 951, Japan. t Author to whom all correspondence should be addressed. 91
92
KAZUAKI ARAKI el al.
(B)
E
(A)
E
H
B
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H
B /H
/B
Kb
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-
-
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~
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0.5--
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~
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Fig. 1. Southern blot hybridization of h u m a n and rat genomic DNA. Probe is synthetic oligodeoxynucleotide IV. Endonucleases used for digestion of genomic D N A were H (HindlII), E (EcoRI) and B (BamHI). A, h u m a n ; B, rat.
Blot analyses R N A was isolated from the rat brain by the guanidinium thiocyanate/cesium chloride procedure (Chirgwin et al., [979; Yamakuni el al., 1988). Northern blot hybridization was done as described previously (Thomas, 1980). Highmolecular weight total genomic D N A was prepared from the h u m a n placenta by the procedure of Blin and Stafford (1976). Southern blot hybridization was carried out with the genomic D N A fragments labeled with [:~-~2p]dCTP by a random-primed D N A labeling kit (Southern, 1975).
p U C 8 vector and used for D N A nucleotide sequencing with M a x a m Gilbert method (Maxam and Gilbert, 1980) or dideoxy method (Sanger et al., 1977). Exon intron organization was determined on the basis of the restriction map and D N A nucleotide sequence.
Chromosomal mappin9 H u m a n metaphase chromosomes were prepared from h u m a n B lymphoblast cell line, GM00130B, as described previously (Taira et al., 1989). C h r o m o s o m e s stained with propidium iodide were excited under 488 n m light with an LP520 optical filters and sorted by a FACS 440 cell sorter (Beckton Dickinson). C h r o m o s o m e s stained with Hoechst 33258 were also sorted by a FACS cell sorter. Spot blot hybridization was performed as described previously. Assignment of chromosomes in each fraction was carried out with chromosome-specific D N A probe. Filter disks were hybridized with the ~2P-labeled h u m a n D2 dopamine receptor genomic DNA.
RESULTS ANt) DISCUSSION
Southern blot hybridization After high-molecular weight DNA was prepared a n d d i g e s t e d w i t h several r e s t r i c t i o n e n d o n u c l e a s e s , S o u t h e r n blot a n a l y s i s o f this d i g e s t e d D N A w a s per-
Ikb
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.
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Fig. 2. Restriction map of the gene for the h u m a n D2 dopamine receptor. The solid boxes indicate the exons and the straight lines between each exon show the introns. Relevant endonuclease sites were shown as follows : E, EcoRl ; B, BamHI ; K, Kpnl ; Bg, BgllI ; S, Stul ; H, Hindlll. Some D N A fragments u3ed for sequencing are shown under the restriction map.
B
D2 dopamine receptors
93
10 20 30 40 50 60 TT&AGGAGGT GTTTOTTGAA TGAAATAAAT AAATOATGGA CCAGCTCTGT CTAAGCCCAC
2110 2120 2130 2140 3180 AGTGCTCAGG CcAGGGACCC AGAGCCAGOT eeee*~e*e* eeew * * *
70 60 60 100 110 120 CTTTATATGC ATTTTTAAAA AATTGTGTTT CCCCATTTGT CCTACCAOCT GTAAAAACTT
2170 2180 2190 2200 2210 2220 TGACCCTGTG GTGTTTGCAG GAGTCTTCGA GGGGGAAAGG GAGGGGCCAG TGAGATGGGT
130
140
160
160
170
100
2230
2240
2250
2260
2160 t***
2270
2260
GCTAGCAAAA OAGTGGCAGA AGGCCCAACA TTTTT&GAAA ATTCTTAGCC GTAAACCTTA
GGCTGATGCC TAGGA&CTTO TCCGGCTTTA CCCAGGCCCT CTGCCTCTGG TCOAGGAGGC__
190 200 210 220 220 240 AATCCCOAOT TGGAAAATTC TCATTATTAA ATGCCAAGSG TTGGTCTTCC TAGAGSCCTC
2290 2300 2310 2320 2330 2340 TGCCCGGCGA GCCCAGGAGC TGGAGATGGA GATGCTCTCC AGCACCAGCC CACGCGAGAG
260 260 270 260 290 300 TGCAAGAGGC CCTCTCACCG CCACCTTGTG CCCATTTTTC CTC-CCAGAGC CTGCCACCCA
2350 2360 2370 3380 2390 2400 GACCCGGTAC AGCCCCATCC CTCCCAGCCA CCACCAGCTG ACTCTCCCCG ACCCGTCCCA
310 320 330 340 360 360 GTOGCTCCAC CCCCCTGATG GATCCACTOA ATCTGTCCTG GTSTGATGAT GATCTGGAGA
2410 2420 2430 3440 2450 2460 CCATGGTCTC CACAGCACTC CCGACAGCCC CGCCAAACCA GAGAAGAATG GGCAT~..CAA
370 380 300 400 410 420 GGCAGAACTG GAGCCGGCCC TTCAACGGGT CAGACGGGAA GGCGGACAOA CCCCACTACA
2470 2480 2490 2500 2510 2520 AOACCACCCC AAGATTGCCA AGATCTTTGA GATCCAGACC ATGCCCAATG GCAAAACCCG
430 440 450 460 470 480 ACTACTATGC CACACGGCTC ACCCTGCTCA TCGCTGTCAT CGTCT7CGGC AACGTGCTGG
2530 2540 3660 2560 2570 2590 GACCTCCCTC AAGACCATOA GCCGTAGGAA GCTCTCCCAG CAGASGGAGA AGAAAGCCAC
490 500 810 520 630 540 TGTGCATGGC TGTGTCCCGC GAGAAGGCGC TGCAGACCAC CACCAACTAC CTOATCGTCA
2590 2600 2610 2620 2630 2640 TCAGATGCTC GCCATT~;TTC TCGGTGAGTC= GGCCCTGGCG CTGGCCACAG CCGGTCTGTG
650 560 670 560 590 600 GCCTTGCAOT GGCCGACCTC CTCGTCGCCA CACTGGTCAT GCCCTGGGTT GTCTACCTOG
2650 3660 2670 2680 2690 2700 AAAGGTCCCA TTCCCTGCC8 TGTCCTTCCT AACCATGGCT Oeee*~eeee eeeeee * *
610 620 630 640 650 860 AGGTAGGTGG GCCCCTTGCC AGCACTTTCT (;CAGCA(]GGC CCTGCACTGG ACACeeee*~,
2710 2720 2730 2740 2750 2750 AAGACCGGGA CTCCTACAGT CCTTCTGTGG CTACAGCCCA CCGTCTTGGC ATACGAGCCA
670
680
000
700
710
730
2770 2790 2790 2800 2810 2820 GGGCGCACTO OGTGTGGGTG TTCCCAGCCG TGCCTCCCCG GCTCTGGGGA CCAGCCTGAC
730
740
750
760
770
780
2830 2840 2650 2850 2870 2800 CATGCCCTCT CCCCCAGGCG TGTTCATCAT CTGCTGGCTG CCCTTCTTCA TCACACACAT
AC&AGACTTO CAGCTGCCTC CTGAGTCTGT OGCCTCATCG GCTCCAGGAG TACCAGGCT& 790
800
610
820
630
640
CAGGACCT&A GCTAATCTCC CACTCCTOCT OTCCATCCAT TATGTTGCTT TGTCCCC~
2890 2900 3910 2930 2030 2940 CCTOAACATA CACTGTGACT GCAACATCCC GCCTGTCCTG TACAGCGCCT TCACGTGGCT
850 660 670 680 590 900 TGGTAGGTGA GTGGAAATTC AGCAGGATTC ACTGTGACAT CTTCGTCACT CTGGACGTCA
2950 3960 3970 2980 3990 3000 GGGCTATGTC AACAGCGCCG TGAACCCCAT CATCTACACC ACCTTCAACA TTGAGTTCCG
910 920 930 940 860 960 TG~TG~GC~C GGCGAGCATC CTGAACTTGT GTGCCATCAG CATCGACAGG TGAGCCCAGC
3010 3020 3030 3040 3050 3060 C/~AGGCCTTC CTGAAGATCC TCCACTGCTG ACTCTGCTGC CTGCCCGCAC AGCAOCCTGC
970
960
090
1000
1010
1020
CAGGGTGGAA GAGGTTGCTC TGGCCACTCA CCATTCTGCC TGCCCCAGCT TTTCTCAAAG
3070
3080
3090
3100
3110
3120
TTCCCACCTC CCTGCCCAGG CCkGCCAGCC TCACCCTTGC GAACCGTGAG CAGGAAGGCC
1030 1040 1060 1060 1070 1080 CCAGGCTGTG TCTCGTGTCT GTOATGCTGT OCAGCTGGCT GTGTCCATGG GGGTCCATGT
3130 3140 3150 3100 3170 3180 TGGGTGOATC GGCCTCCTCT TCACCCCG3C AGGCCCTGCA GTOTTCGCTT GGCTCCATGC
1090 1100 1110 1120 1130 1140 GTTTGTGTGT ATGGGTGTGC ATOTGTGTAT OTTTGGCTAG GAGAGCACAC ACTCCCTAAA
3190 3200 3310 3220 3230 3240 TCCTCACTGC CCGCACACCC TCACTCTGCC AGGGCAGTGC TSGTGAGCTG GGCATGGTAC
1160 1160 1170 1180 1190 1200 GAGAAGCCAT CTATGGACAG TGATGCTGCC 8AGCATACTC AGACAGTGAC TOGTkCAAAA 1210
1220
1230
1240
1250
1260
2260 3260 3370 3280 3290 3300 CAGCCCTGGG GCTGGGCCCC CCAGCTCAGG GGCAGCTCAT AGAGTCCCCC CTCCCACCTC 3310 3320 3330 3340 3350 3360 CAGTCCCCCT ATCCTTGGCA CCAAAOATGC AGCCGCCTTC CTTGACCTTC CTCTGGGGCT
GGGGAAAGAT TOTGATTCTG ACGGAACCCC TAGCTCTAGC ACATCTTACT CATGTACCTT 1270
1280
1290
1300
1310
1320
]330 1340 1350 1360 1370 1300 GTGAGGGTGT CT¢TOGTGTG TGCATATTGT GGAGTGAGGG OTCCCTGGGC CTCCACCCCA 1390 1400 1410 1420 1430 1440 GATTCAGGGT CCCCCGCCCG TTGCAGC;[AC ACAGCTGTGG CCATGCCCAT GCTGTACAAT 1450 1460 1470 1480 1490 1600 ACGCGCTACA GCTCCAAGCG CCGGGTCACC GTCATGATCT CCATCGTCTG GGTCCTGTCC
1510 1520 1530 1540 1650 IS60 TTCACCATCT CCTGCCCACT CCTCTTCGGA CTCAATAACG CAGGTACATT CT3CTTTGCT w 1670 1580 1500 1600 ]6]0 1520 TGCCTGAGGC CkCCTOGCCC TGGGCCTGCT CCCTCGAAGG GCCCCTGAGG GAkCAGkGTC 1630 1640 1650 1660 1670 CTGGCACAGA CATGGGTOGA AACCAATGGS # * * * * * * * * * * * * * * * * * * * * e * 1690
1700
1710
1720
1730
3370 3380 3390 3400 3410 3420 CTAGGGTTGC TGGAGCCTGA GTCAGGGCCC AGAGGCTGAG TTTTCTGTTT GTGGGGCTTG 3430 3440 3460 3460 3470 3460 GCGTGGAGCA GGCGGTGGGG AGAGATGGAC AGTTCACACC CTGCAAGGCC CACAGGAGCC 3490 3500 3610 3520 3530 3540 AAGCAAGCTC TCTTGCCGAG GAGCCAGCCA ACTTCAGTCC TGGGAGACCC ATGTAAATAC 3650 3560 3570 3580 3590 3600 CACTGCAGGT TGGACCCCAG AGATTCCCAA GCCAAAAACC 7TAGCTCCCT CCCGACCCCG 3610 3620 3630 3640 3550 3660 ATGTGGACCT CTACTTTCCA GGCTAGTCCG GACCCACCTC AC¢CCGTTAC AC~TCCCCAA 3870 3690 3090 3700 3710 3720 GTGGTTTCCA CATGCTCTGA GAAGAGGAGC CCTCATCTTG AAGGGCCCAG GAGGGTCTAT
1680
3730 3740 3750 3760 3770 3780 GGGGAGAGGA ACTCCTTGGC CTAGCCCACC CTTCTGCCTT CTGACGGCCC TGCAATGTAT
1740
3790 3800 3610 3820 3630 3640 CCCTTCTCAC AGCACATGCT GGCCAGCCTG OGGCCTGGCA ~GGAGGTCAG GCCCTGGAAC
*
CCOGTOGGCC CGCTGACTCC CTGCCTGCCC CGGGCTCCCT CCCCCAGSCC AGAACGAGTG 1750
1760
1770
1760
1790
1800
CATCATTGCC AACCCGGCCT TCGTGGTCTA CTCCTCCATC GTCTCCTTCT ACGTOCCCTT 1610 1610 1830 1840 1650 1680 CATTGTCACC CTGCTGGTCT ACATCAAGA7 CTACATTGTC CTCCGCAGAC GCCGCAAGCG 1870 1880 1890 1900 1910 1920 AGTCAACACC AAACGCAGCA GCCGAGCTTT CAGGGCCCAC CTGAGGGCTC CACTAAAGG_.T
38s0 3860 3870 3860 3890 3900 TCTATCTGGG CTAGGGGACA TCAGAGGTTC TTTGAGGGAC TGCCTCTGCC ACACTCTGAC 3010
1990
2000
2010
2020
2030
2040
TTGTGTACCA TGTOCTGGCT CACTCCAC/~ GGCAACTGTA CTCACCCCGA GGACATGAAA 2050 3060 2070 3080 3090 2100 CTCTGCACCG TTATCATGAA GTCTAATGGG AGTTTCCCAG TGSACAOGCG GAGAGTSG=~A
3930
3940
3950
3960
3970 3080 3090 4000 4010 4020 TCCACTGCCT CTGCCTTAGA GGACCCACGG CTAAGAGGCT GCTGAAAACC ATCTGGCCTG 4030
1930 1940 1950 1960 1970 1980 CTCAAGACAC CCCCCAACCe e * * * * * * * * * * * * * e * * * * * * * e e * e e * * * * * s e e e e * * e
3920
GCAAAACCAC TTTCCTTTTC TATTCCTTCT CCCCTTTCCT CTCTCCTGTT TCCCAACCCT
4040
4060
4060
4070
4080
GCCTGGCCCT GCCCTGAGGA AGGAGGGGAA GCTCGAGCTT GGGAGAGCCC CTGGGGCCTA 4090 4100 4110 4120 4130 4140 GACTCTGTAA CATCACTATC CATGCACGAA ACTkATAAAk CTTTGACGAG TGACCTTCCA
4160 4100 4170 41ao 4190 4200 GGACCCCTGG GTAGAAGGCA GCAGTGCCAC TTCTGTGCI'T GGCATTCAAG TATAGGAAGA
Fig. 3. Nucleotide sequence of h u m a n D2 dopamine receptor gene. Exon sequences were underlined. The inside portions of each intron were omitted. The junctional sequences were double underlined.
M
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1930 1940 1950 1960 1970 1980 GTCCCCCTATCCTTGGCACCAAAGATGCAGCCGCCTTCCTTGACCTTCCTCTGGGGCTCT
1870 1880 1890 19OO 1910 1920 GCCCTGGGGCTGGGCCCCCCAGCTCAGGGGCAGCTCATAGAGTCCCCCCTCCCACCTCCA
1810 1820 1830 1840 1850 ]86,, CTCACTGCCCGCACACCCTCACTCTGCCAGGGCAGTGCT AGTG AGCTGGGCATGGT ACC A
1750 1760 177o 178o 179O I 8n(, GGTGGATCGGCCTCCTCTTC ACCCCGGCAGGCCCTGCAGTGTTCGCTTGGCTCC ATGCTC
12~t0 1400 1410 1420 1430 ) 44h TCAAGACCATGAGCCGTAGGAAGCTCTCCCAGCAGAAGGAGAAGAAAGCCACTCAGATGC
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1570 1580 1590 1600 1610 1620 GCTATGTCAACAGCGCCGTGAACCCCATCATCT ACACCACC TTCAACATTG AGTTCCGC A y V N $ A V N P I I Y T T F N I E F R K
Fig. 4. Nucleotide sequence and deduced amino acid sequence of the human D2 dopamine receptor mRNA. The amino acids were expressed as a single letter. Seven transmembrane regions were underlined.
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250 250 270 280 290 300 TGCAAGAGGCCCTCTCACCGCCACCTTGTGCCCATTTTTCCTGCCAGAGCCTGCCACCCA
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D2 dopamine receptors
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Fig. 5. Hydrophilicity profile of human D2 dopamine receptor (414). I-VI! under the profile indicate the transmembrane regions.
formed with a probe IV. As shown in Fig. 1, the blot analysis of human genomic digested DNA showed the presence of a single hybridizing band, suggesting the presence of a single copy of human D2 dopamine receptor gene per haploid genome. Figure 1 also shows the blot analysis of rat genomic digested DNA, indicating the presence of a single or two bands. Exon intron organization receptor gene
of human D2 dopamine
As described in the section of Experimental Procedures, a human genomic DNA library was screened by plaque hybridization with four oligodeoxy-
nucleotides as a probe and three positive clones were isolated. These cloned DNAs were used for further analyses. Figure 2 shows the restriction map, the subcloned fragments and exon-intron organization. Nucleotide sequencing of the DNA fragments from the clones revealed the structure of the D2 dopamine receptor gene. This gene isolated spans about 15 kbp in length and appeared to be composed of six exons separated by five introns. Recently several groups found an additional amino acid sequence of rat D2 receptor produced by alternative splicing of the new exon (Giros et al., 1989; Chio et al., 1990). Since the nucleotide sequences of
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Fig. 6. Localization of the human D2 dopamine receptor gene on the flow-sorted chromosomes from the human B lymphoblast line GM00130B. 1 : (A) Propidium iodide-stained flow-karyotype.(B) Hybridization of human D2 dopamine receptor genomic DNA to spots containing sorted chromosomes. 2 : (A) Hoechst 33258-stained flow-karyotype. (B) Hybridization of human D2 dopamine receptor genomic DNA to spots containing sorted chromosomes.
96
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on the rat gene of O'Malley et al. (1990). Exon intron organization was not observed in most other Gprotein-coupled receptor gene family (for example, ~and/Y-adrenergic receptors, muscarinic A C h receptor, etc.) except opsin genes (Nathan, 1987). Therefore, it is important that D2 dopamine receptor gene has the exon intron organization.
Fig. 7. Northern blot analysis of brain RNA from several animals. Poly(A) ~ RNAs (5 pg) of each animal brain were used• Probe is the BamHI-AccI fragment of rat D2 dopamine receptor cDNA. B, bovine ; R, rat ; M, mouse : H, human.
Homoloy.v between receptors
some introns in our D N A fragments were already determined, these sequences were again surveyed, focusing A G - G T junctional sequence. The results indicated the presence of an additional exon between 4th exon and 5th exon. This additional exon contains 87 bp sequence coding 29 amino acid residues• In conclusion, it was found that two human D2 dopamine receptor m R N A s are produced by alternative splicing of this exon. Two human D2 dopamine receptor proteins were translated from these two m R N A s , producing D2 (443) and D2 (414) (Figs 3 and 4). These results are in agreement with the data of Grandy et al. (1989b). At the 3' end of this gene, poly(A) signal was found at the 3' side o f e x o n 7. Probably, one more additional exon remains to be identified at the 5' end of this human D2 receptor gene, considering the data
rat and human
D2 dopamine
The connection of the exons of human D2 dopamine receptor gene produced the nucleotide sequence corresponding to that of this receptor m R N A . The amino acid sequence of this receptor protein was deduced from the nucleotide sequence of m R N A (Fig. 4). The receptor proteins were composed of414 amino acid and 443 amino acid residues. The high homology of amino acid sequence was found between rat and human D2 dopamine receptor (about 95%), showing the decrease of one amino acid and changes of 19 amino acids in the human receptor. Hydrophilicity profile of human D2 dopamine receptor was examined, indicating the presence of seven hydrophobic regions (Fig. 5). These hydrophobic regions
t 1
5
10
15
20
50
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Fig. 9. Developmental changes of rat D2 dopamine receptor mRNA. Total RNA (20 ,ug) isolated from rat brains of different developmental stages was spotted onto nitrocellulose filters and hybridized with ~2P-labeled D2 dopamine receptor cDNA as shown in Fig. 7.
D2 dopamine receptors suggest the presence of seven transmembrane regions. This profile showed that the structure of D2 dopamine receptor was similar to that of other G-protein coupled receptors with seven transmembrane regions. C h r o m o s o m a l localization
Since the chromosomal localization of this receptor gene was not yet identified at that time, where we isolated this gene, we attempted to assign this chromosomal localizatioaa by the spot blot hybridization procedure with D2 dopamine receptor genomic D N A on the flow-sorted chromosomes. The results are shown in Fig. 6. The fraction containing chromosome 11 shows the presence of positive signal. This data is in agreement with that of Grandy et al. (1989a) using a completely different method. SearchJbr RFLP
As the next step, we carried out Southern blot analysis of human genomic D N A to find R F L P with D2 dopamine receptor gene. Several restriction endonucleases were used to digest the human genomic DNA. Until now, RFLP with Mspl, TaqI, BglII, HindIII, EcoR1 and BamH1 was not observed. According to the report of Grandy et al. (1989a), RFLP was found in the human genomic D N A digested with TaqI. However, we could not yet confirm this data. For the examination of the pathogenesis of schizophrenia and Parkinson's disease, we should find RFLP in the human genomic DNA. Northern blot analys&
Figure 7 shows the results of Northern blot hybridization analysis of poly(A) + R N A from several animal brains. These m R N A s for D2 dopamine receptor from rat, bovine and human brains were found to be a single m R N A species of approximatley 2.5 kb in length. However, the mouse m R N A s were composed of two m R N A species, one of which was considerably larger than 2.5 kb. The significance of this larger m R N A in the mouse brain was not yet clarified. We examined the changes of D2 dopamine receptor m R N A in the rat and hamster brains after various treatments. The m R N A from the brains of the hamsters after Creutzfeldt-Jakob disease infection, did not show significant change. However, the D2 dopamine receptor m R N A of the brains of the rats with methylmercury intoxication showed slight decrease. Methylmercury (10 mg/kg wt/day) was injected subcutaneously for 7 days into the rat and then allowed the rat for a further 7 days until the intoxication symptom appeared (Fig. 8).
97
Developmental changes of this m R N A were also examined in the rats. Although fetal brains do not contain significant amounts of this mRNA, the newborn rats showed the significant signal. This signal gradually increased until 60 days after birth (Fig. 9). These results may correspond to the developmental changes of the movement and emotion in the rats. Recently cDNAs or genes for D1, D3, D4 and D5 dopamine receptors were isolated (Dearry et al., 1990 ; Sokoloffet al., 1990 ; Van Tol et al., 1991 ; Sunahara et al., 1991). The regional distributions of five dopamine receptors were examined by in situ hybridization histochemistry and it was found that each receptor showed different distribution patterns in the brain. We should examine the expressions of five dopamine receptor genes and their changes in various conditions using their specific probes. The human and rat genes for these receptors should be cloned and analyzed, although genes for human D1, D4 and D5 dopamine receptors were recently isolated (Sunahara et al., 1990, 1991 ; Van Tol et al., 1991).
REFERENCES
Araki K., Kuwano R. and Takahashi Y. (1990) Gene structure of human D2 dopamine receptor. A. Rep. Pharmacopsychiat. Res. Fdn. 21,233--237. Araki K., Kuwano R., Hayashi S. and Takahashi Y. (1991) Structure and expression of human and rat dopamine receptor genes. J. Neurochem. 57 (Suppl), S90D. Blin N. and Stafford D. W. (1976) Isolation of high molecular-weight DNA. Nucleic Acids Res. 3, 2303 2308. Bunzow J. R., Von Tol H. H. M., Grandy D. K., Albert P., Salon J., Christie M., Machida C. A., Neve K. A. and Civelli O. (1988) Cloning and expression of a rat D2 dopamine receptor cDNA. Nature 336, 783-787. Chio C. L., Hess G. F., Graham R. S. and Huff R. M. (1990) A second molecular form of D2 dopamine receptor in rat and bovine caudate nucleus. Nature 343, 266-269. Chirgwin J. M., Przybyla A. E., MacDonald R. J. and Rutter W. J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294~5299. Dearry A., Gingrich J. A., Falardeau P., Fremeau R. T. Jr, Bates M. D. and Caron M. G. (1990) Molecular cloning and expression of the gene for a human DI dopamine receptor. Nature 347, 72-76. Giros B., Sokoloff P., Mortres M.-P., Riou J.-F., Emorine L. and Schwartz J.-C. (1989) Alternative splicing directs the expression of two D2 dopamine receptor isoforms. Nature 342, 923-926. Grandy D. K., Litt M., Allen L., Bunzow J. R., Marchionni M., Makam H., Reed L., Magenis R. E. and Civelli O. (1989a) The human dopamine D2 receptor gene is located on chromosome 11 at q22-q23 and identifies Taql RFLP. Am. J. Hum. Genet. 45, 778-785. Grandy D. K., Marchionni M. A., Makam H., Stofko R. E., Alfano ZM., Frothingham L., Fischer J. B., Burke-Howie K. J., Bunzow J. R., Server A. C. and Civelli O. (1989b)
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Cloning of the cDNA and gene tor a human D2 dopamine receptor. Proc. natn Acad. Sci., U.S.A. 86, 9762--9766. Kuwano R., Araki K. and Takahashi Y. (1990) Structural analysis of human D2 dopamine receptor gene. Neurosci. I~es. 11 Suppl.~ S120. Maxam A. M. and Gilbert W. (1980) Sequencing-endlabeled DNA with base-specific chemical cleavages. Methods Enzym. 65, 499 560. Nathan J. 0987) Molecular biology of visual pigments. A. Rev. Neurosci. 10, 163--194. Nishizawa Y., Sato M., Hayashi S. and Takahashi Y. (1988) Purification of receptors binding antipsychotics: comparison with D2 dopamine receptor. In: Pharmacology and Functional Regulation o f Dopaminergic Neuron (Beart P. M., Woodruff G. N. and Jackson D. M., eds), pp. 92 95. Macmillan, London. O'Malley K. L., Mack K. J., Gandelman K.-Y. and Todd R. D. (1990) Organization and expression of the rat D 2A receptor gene : identification of alternative transcripts and a variant donor splice site. Biochemistry 29, 1367 1371. Sakai Y., Sekiguchi M., Okamoto K., Kuwano R. and Takahashi Y. (1990) Dopamine receptors expressed in the Xenopus oocytes injected with bovine striatal mRNA. Molec. Brain Res. 7, 183 187. Sanger F., Nicklen S. and Coulson A. R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. natn Acad. Sci., U.S.A. 74, 5463 5467. Seeman P. (1980) Dopamine receptor. Pharmac. Rez~. 32, 229 313. Seeman P. and Niznik H. B. (1990) Dopamine receptors and transporters in Parkinson's disease and schizophrenia. F A S E B J . 4, 2737 2744. Sokoloff P., Giros B., Martres M.-P., Bouthenet M.-L. and Schwartz J.-C. (1990) Molecular cloning and characterization of a novel dopamine receptor (D3) as a target for neuroleptics. Nature 347, 146 151.
Southern E. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. molec. Biol. 98, 503 517. Sunahara R. K., Niznik H. B., Weiner D. M., Stormann T. M., Brann M. R., Kennedy J. L., Gelernter J. E., Rozmabel R., Yang Y.~ Israel Y., Seeman P. and O'Dowd B. F. (1990) Human dopamine D2 receptor encoded by an intronless gene on chromosome 5. Nature 347, 8(~83. Sunahara R. K., Guan H.-C., O'Dowd B. F., Seeman P., Laurier L. G., Ng G., George S. R., Torchia J., Van Tol H. H. M. and Niznik H. B. (1991) Cloning of the gene for a human dopamine D5 receptor with higher affinity for dopamine than D1. Nature 350, 614 619. Taira M., Kudoh J., Minoshima S., Iizasa T., Shimada H., Shimizu Y., Tatibana M. and Shimizu N. (1989) Localization of human phosphoribosylpyrophosphate synthetase subunit I and II genes to different regions of the X chromosome and assignment of two PRPSI-related genes to autosomes. Somat. Cellmolec. Genet. 15, 29 37. Thomas P. S. (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. natn Acad. Sei., U.S.A. 77, 5201 5205. Usui H., Takahashi Y., Maeda N., Mitsui H., Isobe T., Okuyama T., Nishizawa Y. and Hayashi S. (1990) Purification of D2 dopamine receptor using photoaffinity labeling, HPLC and preparatives SDS-PAGE. J. Chromat. 515~ 375 384. Van Tol H. H. M., Bunzow J. R., Guan H.-C., Sunahara R. K., Seeman P., Niznik H. B. and Civelli O. (1991) Cloning of the gene for a human dopamine D4 receptor with high affinity for the antipsychotic clozapine. Nature 350, 610 614. Yamakuni T., Kuwano R., Araki K., Usui, H., lnoue Y. and Takahashi Y. (I 988) Developmental and regional changes ofmRNA for a cerebellar protein (spot 35) in the rat brain. J. Neurochem. 50, 282-284.
