Journal qfNeurochrmistry Raven Press, Ltd., New York 8 1991 International Society for Neurochemistry

The Mouse Dopamine D2* Receptor Gene: Sequence Homology with the Rat and Human Genes and Expression of Alternative Transcripts Kenneth J. Mack, *Richard D. Todd, and ./.Karen L. O’Malley Department of Neurology und Waisman Center,forMental Retardation, University of Wisconsin, Madison, Wisconsin; and Departments of *Psychiatry, *Genetics, ?Anatomy, and TNeurobiology, Washington University, St. Louis, Missouri, U.S.A.

~

Abstract: To understand the possible involvement of dopamine receptors in the pathogenesis of various neurological disorders, we have cloned and sequenced a dopamine D ~ A receptor gene from the mouse. A mouse genomic library was screened with probes derived from the published sequence of a rat D2Areceptor cDNA. Using restriction endonuclease mapping, Southern blotting, and DNA sequencing, we have determined the cDNA sequence and genomic organization of the mouse D ~ receptor A gene. Unlike other guanine nucleotide-binding protein-coupled receptors, but similar to its rat and human counterparts, the mouse D ~ receptor A gene has seven introns and spans at least 30 kb of genomic DNA. The mouse D2Asequence shows 99% amino acid homology with the rat and 95% amino acid homology with the human sequence. As would be predicted, sequence differences are significantly more frequent outside of the hypothesized

transmembrane spanning domain regions of the protein. Using the polymerase chain reaction with primers made from neighboring exons, we have identified two alternatively spliced D2Atranscripts in the mouse. However, in contrast to the other species studied, the mouse expresses primarily the mRNA representing the larger, 444-amino-acid form of the receptor. Mouse pituitary expresses only the mRNA of the 444-amino-acid form of the D ~ receptor. A Hence, the mouse may offer the best model to study the in vivo physiology of the long form of the D2, receptor. Key Words: Dopamine D2 receptor-Mouse-Gene homology-Alternative splicing. Mack K. J. et al. The mouse dopamine D2Areceptor gene: Sequence homology with the rat and human genes and expression of alternative transcripts. J. Neurochem. 57, 795801 (1991).

Dopamine receptors have been widely studied owing to their proposed roles in the treatment and etiology of many neurological and psychiatric disorders. Traditional pharmacological and physiological studies have defined two principal types of dopamine receptors, D 1 and D2, each with distinct pharmacological binding profiles, signal transduction systems, and sites of localization (reviewed by Creese et al., 1983; Niznik, 1987). Recently, at least four molecular forms of D2-like receptor coding sequences have been described. Originally, the molecular sequence of a rat D2 receptor cDNA (called here D2A415) was described (Bunzow et al., 1988). Later, the product of a second D2 receptor gene (D2,) (Todd et al., 1989) was reported. Subsequently, the gene encoding the D2A415 cDNA was found

to be alternatively spliced to form a second mRNA, D2A444 (Dal Toso et al., 1989;Giros et al., 1989;Grandy et al., 1989; Monsma et al., 1989; Selbie et al., 1989; Chio et al., 1990; Eidne et al., 1990; O’Malley et al., 1990). In contrast, some authors (see, e.g., Dal Toso et al., 1989) have referred to the alternatively spliced forms of this originally described D2 gene as D2A (called D2A444here) and D2B (called D2A4I here). More recently, a third type of D2 receptor gene was described, called D3 (Sokoloff et al., 1990). The D3 receptor has only 52% overall identity with the D ~ A receptor yet exhibits a clear D2-like pharmacological profile (Sokoloff et al., 1990). To understand the gene structure and expression of the D2 receptors, the D2A gene was cloned and sequenced from rat (O’Malley et al., 1990), human

Received October 24, 1990; revised manuscript received January 10, 1991; accepted February 4, 1991. Address correspondence and reprint requests to Dr. K. L. OMalley at Departments of Anatomy and Neurobiology, Washington University, St. Louis, MO 63 1 10, U.S.A.

Abbreviations used: G protein, guanine nucleotide-binding protein; PCR, polymerase chain reaction.

795

K. J. MACK ET AL.

796

(Gandelman et al., 1991), and now mouse genomic libraries. The mouse gene was specifically chosen because of the relative ease of making inbred strains, the available techniques for producing transgenic animals, the wealth of preexisting data on transcription factors in this species, and the ability to use cross-species comparisons to identify important, highly conserved sequences. In this study, we now report on the organization of the mouse DzAreceptor, its sequence homology with rat and human receptor sequences, and the expression of alternative forms in the mouse nervous system.

EXPERIMENTAL PROCEDURES Materials Most enzymes were purchased from Promega. Sequenase was purchased from U.S. Biochemical. Nylon membranes were from Schleicher & Schuell, and radionucleotides were from Amersham. NIH 3T3 mouse genomic libraries were either purchased from Stratagene or made as previously described (Todd et al., 1989).

Isolation of a mouse D2Agene Oligonucleotide primers from the rat cDNA sequence were used to initially screen the mouse genomic library. Subsequent clones were obtained by walking using radiolabeled end fragments of the initial clone. To clone exon 1, a polymerase chain reaction (PCR) product derived from the first 96 bp of the rat cDNA clone (Bunzow et al., 1988) was used as a probe. Labeling of probes, hybridization, and washing conditions were performed in accordance with standard methods (Sambrook et al., 1989). Oligonucleotides were as described by OMalley et al. ( 1990).

DNA sequence analysis Oligonucleotides derived from rat cDNA and genomic sequence were used to identify relevant genomic restriction fragments. After subcloning, exon and intron boundries were sequenced on both strands with denatured double-stranded DNA as a template for primer-extended synthesis (Chen and Seeburg, 1985). Sequence information was analyzed using programs from Intelligenetics.

PCR To obtain exon 1, 1 pg of rat genomic DNA was used as a template in amplification reactions (Sambrook et al., 1989). Oligonucleotides oD2,-225 (S’dGGCTGCCGGAGGGGCGGC) and oD2,-2 1 5 (S’dCTTCAAGCCATATGGCGC) were used to prime synthesis. Reaction products were separated on a 12% polyacrylamide gel, and a fragment of the appropriate size was excised, electroeluted, and ethanol-precipitated. For amplification of RNA, 1.25 pg of total RNA (Chomczynski and Sacchi, 1987) was reverse-transcribed (Krug and Berger, 1987) with an oligonucleotide (oD2,-298; SdGGGCAGCCAGCAGATGATGAACAC) complementary to exon 8. The resultant cDNA was used for PCR with a primer identical to exon 5 (oD2,-232; 5’dTACATCGTCCTCCGGAAGCGCCGGAA) to show the alternatively spliced forms. Temperatures for the PCR protocols were 93°C for 30 s, 61°C for 120 s, and 72°C for 180 s. The mRNA levels were standardized by using complementary oligonucleotide primers to the 18s fragment of rRNA (Chan et al., 1984). Oligonucleotides were end-labeled and then added to the PCR mixtures. PCR products were separated on a 12% polyacrylamide gel, the gel was dried down and exposed to x-ray film, and quantification was done using a densitometer.

RESULTS AND DISCUSSION Organization of the D2Agene A partial genomic clone encoding exons 7 and 8 was isolated using oligonucleotide probes derived from the rat D2A sequence (Bunzow et al., 1988). Using conventional walking techniques, a 12-kbclone containing exons 2-8 was isolated. Two additional walking clones encoding 18 kb 5’ of exon 2 did not contain exon I . A PCR product corresponding to the rat exon 1 sequence was subsequently used to rescreen the mouse genomic library. A nonoverlapping clone, containing exon 1, was isolated in this fashion. As shown in Fig. 1 , the mouse D2A gene spans at least 30 kb in length, with exons 2-8 clustered in 1 1 kb of genome. Intron 1 is at least 18 kb in length. This is similar in organization

-

A

FIG. 1. Mouse dopamine D2A receptor gene structure. A: Restriction enzyme map of the mouse D2Agene. B, BarnHI; R, EcoRI; X, Xba. B: Exon map of the mouse gene. Coding regions are in black; noncoding regions are hatched. Exon 1 and exon 2 are separated by at least 18 kb. Exon 6 (open box) is alternatively spliced. C Rat (O’Malley et al., 1990) and human (Gandelman et al., 1991) DZA genes are shown for comparison with that of the mouse.

B

-1

X B R

BXB

X

X B

RBB

II I

I II

I

I I

11 I

2

Mouse/# -,

C

2

1

Human Rat

3 4

5 6

7

I I

~n

I

3

4

5

A// 2

3

4

5

I D

I

I

I O

6

6

7

7

0

8

0

I H1 Kb

J . Neurochem.. Vol. 57, No. 3, 1991

797

MOUSE D2A RECEPTOR HOMOLOGY AND EXPRESSION TABLE 1. Exonlintron boundary sequences for the mouse D2A gene Exon/intron junctions Exon

Size (bp)

1 2 3 4 5 6 7 8

>310 316 110 137 191 87 33 1 1263

Donor TTGAAG-" CTGGAG284 CGACAG395 ACACAGS3' CTCAAG722 AGAATGSo9 TTCTTGII~O

CACTGC1331

Intron size (-kb)

Acceptor gtaagaactggt gtaggtcttggg gtgaggacatcc gEaagtctggcc gtctccaacctc gtaagtgttcag gtgagtaagctc

tctcccggccag ttttatctccag ttgcctttgcag ctattcccctag ctcactccacag tctctgcttcag ccctcttcccag

. . .aataaaaccttgacaacactcatccacgg

-30AGCCGT 285GTGGTG 396GTACAC 532ACCAGA 723GGCAAC 8

1

0

'I4'GTGTGT

~

~

>18 4.1 0.8 1.3 0.7 L ~.8 1.7

cacttggtgctgacactgtcacactacctt

-

-

Exon sequences are shown in uppercase letters; 3' untranslated and intron sequences are in lowercase. Superscript numbers represent nucleotide positions of exon/intron boundaries in the D2* cDNA sequence. Consensus polyadenylation signals are underlined. An asterisk marks the variant donor splice site present in the rat and mouse but not the human sequences.

to the rat and human D2Agenes (O'Malley et al., 1990; Gandelman et al., 1991) (Fig. 1). In contrast to most guanine nucleotide-binding protein (G protein)-coupled receptor genes, which are intronless (or have an intron only in their 5' untranslated regions), the D2.4 gene is relatively large, with multiple introns and alternatively spliced forms. Each intron/exon boundary was sequenced several times (Table 1). The position of all boundaries was conserved in all three species. Of note is that intron 4 contains a variant donor splice site sequence, similar to the rat but not the human D2A gene (O'Malley et al., 1990). Sequence of mouse cDNA: homology with rat and human clones Expressed mouse D2A sequences were compiled from the genomic clones (Fig. 2). The 5' untranslated sequence represents the mouse analog of the longest human cDNA reported (Selbie et al., 1989). In the coding regions, the mouse gene shares 95% amino acid and 90% nucleic acid sequence homology with the human coding regions. The rat sequence is almost identical, with 99% amino acid and 97% nucleic acid homology (Fig. 2). Because the G protein-coupled receptor family is characterized by the presence of seven conserved transmembrane spanning domains, it would be expected that sequence differences between species would primarily fall outside these domains. Of the 2 1 amino acid differences between the human and mouse sequences, 23.8% (five) were in transmembrane domains, and 72.6% (16) were outside these regions. After correction for the number of amino acid residues in the transmembrane and nontransmembrane spanning domains, this represents a significant deficiency of amino acid differences in transmembrane regions (x2= 4.37; p < 0.05, df= 1). Most of the amino acid changes between the three species are conservative with respect to charge. Several differences, however, are of potential structural significance. Most notable is the use of an

arginine at amino acid 89 in transmembrane I1 of the mouse instead of a proline for the rat and human. The differencesin coding regions for transmembrane spanning and nonspanning are not as pronounced at the nucleic acid sequence level. There are no significant differences in the number of nucleotide changes among mouse, rat, and human transmembrane and nontransmembrane coding regions. For the two most divergent species, mouse and human, there are 86 differences of 834 nontransmembrane region nucleotides ( 10.3%) and 45 differences of 498 transmembrane region nucleotides (9.0%). As would be expected, the majority of differences were in the third codon position. There were significantly fewer differences in codon positions 1 and 2, but these also did not differ between nontransmembrane and transmembrane regions. The alternatively spliced exon 6 is somewhat more divergent: Of 87 nucleotides, there are 13 differences between the mouse and human ( 14.9%).Three of these are in codon position 1, whereas 10 are in codon position 3. Overall, there is a trend for more DNA sequence differences in the nontransmembrane domain coding regions of the receptor. This species homology compares favorably with other similar genes. For example, another gene involved in dopaminergic transmission, the tyrosine hydroxylase gene, shows 9 1% amino acid homology between the human and rodent forms (Coker et al., 1988). The muscarinic MI receptor gene, another member of the G protein-coupled receptor family, shows 98% amino acid homology between the mouse and rat and 97%homology between the mouse and human (Bonner et al., 1988; Shapiro et al., 1988). Similar to the results presented here, there were some amino acids conserved between rat and human sequences but not between rat and mouse sequences. Expression of multiple transcripts To determine if exon 6 was alternatively transcribed in the mouse, mouse mRNA was reverse-transcribed with D2A-specificprimers, and the cDNA was used in

J . Neurochem.. Vol. 57, No. 3, 1991

~

~

~

798

K. J. MACK ET A L . -340

CTGGTCGCCTCTCGCGCAGCGCCGACTGCCCGCCTCCCCG CCTGGTCCCCGGCCTGCGCTCCCGCCCTCCCGCCCCGCCTCGCCCTGCCCCGCCGCGGCC CGTCCACTGCTCCCCGCGGGCCCGAGCCGGCCGAGCGGCTGCCGCCGGGGATCTGAACGG CGCGGCGGGGCCGGGAGCCGAGGGACCCGCAGAGGGGACCGGCGGCCCCGGACGGCTGCC GGAGGGGCGGCCGTGCGTGGATGCGGCGGGAGCTGGAAGCCTCGAGCAGCCGGCGCCTTC

- 6 0 TCTGGCCCCGGGCGCCCTATGGCTTGAAGAGCCGTGCCGTGCCACCCAGTGGCCCCACTGCCCCA 0 ATGGATCCACTGAACCTGTCCTGGTACGATGATGATCTGGAGAGGCAGAACTGGAGCCGG

M

MetAspProLeuAsnLeuSerTrpTyrAspAspAspLeuGluArgGluAsnTrpSerArg

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

R

H

60 CCCTTCAATGGGTGCGAAGGGAACTACGCCAGACAGGC~CCACTACAACTACTATGCCATGCTG

M

ProPheAsnGlyCysGluGlyLysProAspArgProHisTyrAsnTyrTyrAlaMetLeu Ser. Ala. . . . . SerAsp Ala . . . . . . . . . Thr .

. . . .

R

H

. . . . . . . . . .

. . . .

120 CTCACCCTCCTCATCTTTATCATCGTCTTTGGCAATGTGCTGGTGTGCATGGCTGTATCC

M

LeuThrLeuLeuIlePheIleIleValPheGlvAsnValLeuValC~sMetAlaValSer

. . . . . . . . . . . . . . . . . . . . . . . . . AlaVal . . . . . . . . . . . . .

R

H

180 AGAGAGAAGGCTTTGCAGACCACCACCAACTACCTACCTGATAGTCAGCCTCGCTGTGGCCGAT

M

ArgGluLysAlaLeuGlnThrThrThrAsnTyrLeuIleValSerLeuAlaValAlaAs~

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

R

H

2 4 0 CTTCTGGTGGCCACACTGGTTATGCGCTGGGTCGTCTATCTGGAGGTGGTGGGTGAGTGG

M R

~LeuValAlaThrLeuValMetArqTrpValValTvrLeuGluValValGlyGluTrp

. . . . . . . . Pro. . . . . . . . . . . . . . . . . . .

H 300

M R H 360

. . . . . . . . . .

AAATTCAGCAGCATTCACTGTGACATCTTTGTCACTCTGGATGTCATGATGTGCACAGCA LysPheSerSerIleHisCysAspIlePheValThrLeuAs~ValMetMetCvsThrAla Arg Arg

. . . . . .

M

Pro.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

AGCATCTTGAACCTGTGTGCCATCAGCATCGACAGGTACA~AGCTGTGGC~ATGCCTATG SerIleLeuAsnLeuCysAlaIleSerIleAspArgTyrThrAlaValAlaMetProMe~

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

R H

4 2 0 TTGTATAACACACGCTACAGCTCCAAGCGCCGAGTTACTGTCATGATCGCCATTGTCTGG

LeuTyrAsnThrArgTyrSerSerLysArgArgValThrValMetIleAlaIleValTr~

M R

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ser. . .

H 480

M R

H

GTCCTGTCCTTCACCATCTCTTGCCCACTGCTCTTTGGACTC~CAACACAGACCAGAAT ValLeuSerPheThrIleSerCysProLeuLeuLeuPheGlyLeuAsnAsnThrAspGlnAsn

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ala. . .

5 4 0 GAGTGTATCATTGCCAACCCTGCCTTCGTGGTCTACTCCTCCATCGTCTCGTTCTACGTG

M

GluCysIleIleAlaAsnProAlaPheValValTvrSerSerIleValSerPheTyrVal

. . . . . . . . . . . . . . . . . . . .

R H

. . . . . . . . . . . . . . . . . . . .

600 C C C T T C A T C G T C A C C C T G C T G G T C T A T A T C A A A A T C T A C A G C G T C G G

M

ProPheIleValThrLeuLeuValTyrIleLysIleLysIleTyrIleValLeuArgLysArgArg

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Arg.

R

H

.

660 AAGCGGGTCAACACCAAGCGTAGCAGCCGAGCTTTCAGAGCCAACCTG~GACACCACTC

LysArgValAsnThrLysArgSerSerArgAlaPheArgAlaAsnLeuLysThrproLeu

M

R H

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H i s . ArgAla . .

7 2 0 AAGGGCAACTGTACCCACCCTGAGGACATGAAACTCTCTGCACCGTTATCATGAAGTCTAAT

M R

H

LysGlyAsnCysThrHisProGluASp~etLysLeuCysThrValIleMetLysSerA~n

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

799

MOUSE D2,4 RECEPTOR HOMOLOGY AND EXPRESSION 7 8 0 GGGAGTTTCCCAGTGAACAGGCGGAGAATGGATCCTGCCCGCCGAGCTCAGGAGCTGGAA

GlySerPheProValAsnArgArgArgMetAspProAlaArqArqAlaGlnGluLeuGlu

M R H

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . ValGlu . . . . . . . . .

8 4 0 ATGGAGATGCTGTCAAGCACCAGCCCCCCAGAGAGGACCCGGTATAGCCCCATCCCTCCC

MetGluMetLeuSerSerThrSerProProGluArgThrArqTyrSerProIleProPro

M R H

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

900

M R H

AGTCACCACCAGCTCACTCTCCCCGATCCATCCCACCACGGTCTACATAGCAACCCTGAC SerHisHisGlnLeuThrLeuProAspProSerHisHisGlyLeuHisSerAsnProAsp

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

960 AGTCCTGCCAAACCAGAAAAGAATGGGCATTGTCAATCCCAGGATTGCCAAG

M R H

SerProAlaLysProGluLysAsnGlyHisAlaLysIleValAsnProArgIleAlaLys

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AspHis . Lys . . .

1020 TTCTTTGAGATCCAGACCATGCCCAATGGCAAAACCCGGACCTCCCTTAAGACGATGAGC

M

PhePheGluIleGlnThrMetProAsnGlyLysThrArqThrSerLeuLysThrMetSer

R H

Ile

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 0 8 0 CGCAGGAAACTCTCCCAGCAGAAGGAGAAG~GCCACTCAGATGCTTGCCATTGTTC~T

M R H

ArqArqLysLeuSerGlnGlnLysGluLysLysAlaThrGlnMetLeuAlaIleValLeu

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . .

1 1 4 0 GGTGTGTTCATCATCTGCTGGCTGCCCTTCTTCATCACGCACATCCTGAATATACACTGT

M R

H 1200

M R H

GlYValPheIlelleCysTr~LeuProPhePheIleThrH~sIleLeuAsnIleH~sCys

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

GACTGCAACATCCCACCAGTCCTCTACAGCGCCTTCACATGGCTGGGCTATGTCAACAGT AspCysAsnIleProProValLeuTyrSerAlaPheThrTr~LeuGlyTyrValAsnSer

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1260 GCCGTGAACCCCATCATCTATACCACCTTCAACATTGAGTTCCGCAATCCACGGCCTTCATGAAG

M R H

AlaValAsnProIleIleTyrThrThrPheAsnIleGluPheArqLysAlaPheMetLys

. . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

Leu.

1 3 2 0 ATCCTGCACTGCTGAGTCTGCCCCTTGCCTGCACAGCAGCTGCTTGCCGCCTCCCTGCCT

M R H

IleLeuHisCys

. . .

. . .

1380 AGGCAGGCCAGACCTCATCCCTGCAAGCTGTGGGCAGAAAGGCCCAGATGGACTCGGCCT TCTCTTGACCCTGCAGGCTCTGCAGTGTTAGCTTGGCTCGGTGCCCCTCTCTGCCCACAC

ACCCTTATCCTGCCAGGGTAGGGCAAGGGAGACTGGTATCTTACCAGCTCTTGGGGTTGG ATCCATGGCTCAGAGCAGCTCACAGAGTGCCCCTTTCACATGCAGATCCTGTCTCCTTGG CACCAAAGAAGCAGCAGCCTTCCTTGACCTTCCTCTCAGGCACGG~GCTAGCTCAGTAG 1680

CGGAGCACACCTTGATTGTTGGCTTGGCCTGGCCCTTGCTTGCCTATGTTGGATCAGGTG

GTAGAAGAGAAGGACAGTTCTTACTTTACAGGGACCACATAGG~GCAGGGAACATGCC AAGGCCTCCAGGTGACGTTAGTGTCGGGAGACACACATAAACACCAGGTAGCTCCACGGA CCCCAGAGAAACTGAGGCTGAAAATCTGTTTTCTGTTTTCCACCCCAACTCTAGTGTGAATCCCTAC TTTCCATAGCAGTGGGTATTGCTATGTTCTCCACTGTTATAGAATCCCATGGTGTTTCTG 1980 TACCCTTCGGGGAAAACAACTCTAATCCTCAAGGGCCCCAAGAGAGACTGTAAAGAGAAA AATAGCCGATTTCCCTCTACCCTCCAATCCACTCCACTCCGCCACTTCTTGACATACATTGGACA TAGCCATTCCCCACAGCAGATGCTGGACAGCCTGGGAAGTTGAGCCTTGGACCAGTGTTG GAGCTGAAGTTGGAGGTGGTAACTTGGGGCTCTTGGGCGGGGGGTGTTGATATCTTCTCT CTCCCAAGTCTCTTCTCTGCCAGTGCCTCTGCCTTAGAGGAGGCTGTGGATGGGGCTGCT 2 2 8 0 GGGGCTGCTGATACCATTGGGTCTGGCCCTGAGTGAGGGTGGGGAAGCTGCAGCTTGGAG

GGGTCTGGGCTCCAACCTCTGTAACATCACCATACATCGCACCAAACCAA~~~CCTTG 2 4 0 0 ACAACACTCA FIG. 2. Opposite page and above: Mouse sequence and comparison of the deduced amino acid sequence of the D2A444 receptor in the mouse, rat, and human. Beneath the mouse D2* nucleotide sequence is the amino acid sequence of the mouse (M),rat (R), and human ( H ) dopamine D ~ receptors. A The transmembrane domains are underlined. The alternatively spliced exon is in boldface. Only amino acid

differences between the rat or the human and the mouse are shown. J.

Neiinichiw ,

L ‘ d . 57, “ I .

3. 1991

800

K. J. MACK ET AL. TABLE 2. Ratio of D2A4441D2A415 in mouse, rat, and human tissues

STANDARD CURVE 100

80 v)

3

/

C 70z

Tissue Species

Whole brain

Basal ganglia

Midbrain

Pituitary

Mouse Rat Human

4.1 k 0.4 1.5 rt 0.1 ND

6.7 -t 1.4 1.5 k 0.2 1.5 k 0.3

3.9 k 0.5 0.5 kO.2 1.2 & 0.2

>204 7.6 k 1.7 1.0 k 0.1

60U

2

50-

k U

40-

a 3020

-

0

1

2

3

4

MICROGRAMS RNA

FIG. 3. DNA amplification standard curve for mouse basal ganglion RNA. indicated amounts of total RNA were reverse-transcribed and amplified for 30 cycles as described in the text. The correlation coefficient was 0.97 for the primer set oD2-298/oD2-232.

a PCR analysis. To determine if the amount of PCR product was linearly related to the amount of starting material, from 0.06 to 2 pg of basal ganglia mRNA was reverse-transcribed in separate reactions. The relationship between the amount of starting material and PCR product was linear (Fig. 3), and it was determined that up to 35 cycles was still within the exponential component of the amplification curve. To verify that the PCR products represented D2, sequence, the PCR products were blotted onto nitrocellulose and probed with an internal D2,-specific primer. These Southern blots confirmed the D2, nature of the PCR products. A PCR primer set spanning exons 5 and 7 generated two products, corresponding to the alternatively spliced forms of the mRNA, i.e., containing or excluding exon 6 (Fig. 4). However, in comparison to the rat and human, the exon 6-containing form of the D2Areceptor

The ratio between the D2A444and the D2A415forms (the amount of D2,, divided by the amount of D2A415) of the dopamine receptor is shown. A value of > 1 indicates that the 444-amino-acid form is predominant, whereas a value of < I indicates that the 415-aminoacid form is predominant. For human tissues, putamen and substantia nigra were used to represent the basal ganglia and midbrain, respectively. Rat data are from OMalley et al. (1990). Human data are from Gandelman et al. (1991). ND, not determined. 'No D2A41S transcripts were detected in mouse pituitary. The sensitivity of the assay would have allowed detection of levels of I-5% of the D2A444levels.

(444 amino acids) is more predominant in mouse whole brain, pituitary, basal ganglia, and mesencephalon (Table 2). Mouse pituitary tissue shows no detectable expression of the 4 15-amino-acid form, even at long exposures. Therefore, like its rat and human homologs, the mouse D2, receptor is spliced into alternative forms. However, the smaller 4 l 5-amino-acid form of the receptor represents