Proc. Nati. Acad. Sci. USAVol. 88, pp. 7491-7495, September 1991Neurobiology

Cloning, molecular characterization, and chromosomal assignmentof a gene encoding a second D1 dopamine receptor subtype:Differential expression pattern in rat brain comparedwith the DiA receptor

(limbic system/adenylyl cyclase/guanine nucleotide-binding protein/intronless gene/['25I]SCH 23982 binding)

MARIO TIBERI*, KEITH R. JARVIE*, CHRIS SILVIA*, PIERRE FALARDEAU*t, JAY A. GINGRICH*,NATHALIE GODINOT*, LUCIE BERTRAND*, TERESA L. YANG-FENGt, ROBERT T. FREMEAU, JR.§,AND MARC G. CARON*1II**Departments of *Cell Biology, §Neurobiology, and TMedicine, and I"Howard Hughes Medical Institute Laboratories, Duke University Medical Center,Durham, NC 27710; and tDepartment of Human Genetics, Yale University School of Medicine, New Haven, CT 06510

Communicated by Robert J. Lejkowitz, May 21, 1991 (received for review April 18, 1991)

ABSTRACT Multiple DI dopaminergic receptor subtypeshave been postulated on the basis of pharmacological, bio-chemical, and genetic studies. We describe the isolation andcharacterization of a rat gene encoding a dopamine receptorthat is structurally and functionally similar to the D1 dopaminereceptor. The coding region, which is intronless, encodes aprotein of 475 amino acids (Mr 52,834) with structural featuresthat are consistent with receptors coupled to guanine nucle-otide-binding regulatory proteins. The expressed protein bindsdopaminergic ligands and mediates stimulation of adenylylcyclase with pharmacological properties similar to those of theDI dopamine receptor. The gene encoding the human homo-logue of this receptor subtype is located to the short arm ofchromosome 4 (4p16.3), the same region as the Huntingtondisease gene. In striking contrast to the previously cloned DIreceptor, little or no mRNA for the receptor described here wasobserved in striatum, nucleus accumbens, olfactory tubercle,and frontal cortex. High levels ofmRNA for this receptor werefound in distinct layers of the hippocampus, the mammillarynuclei, and the anterior pretectal nuclei, brain regions thathave been shown to exhibit little or no DI dopamine receptorbinding. On the basis of its properties we propose that thisdopamine receptor subtype be called DIB1

The actions of dopamine were originally thought to bemediated by an interaction with two distinct receptor sub-types: D1 receptors, which were coupled to the stimulation ofadenylyl cyclase, and D2 receptors, which were either linkedor not linked to the inhibition of adenylyl cyclase (1). Re-cently, molecular biological approaches have supported andextended this pharmacological classification. At least threedifferent receptor genes code for dopamine receptor sub-types-namely, D1, D2, and D3 (2-9). These receptors belongto the large family of receptors coupled to guanine nucle-otide-binding regulatory protein (G protein) and are believedto contain seven membrane-spanning domains (10).

Traditionally, the central actions of dopaminergic com-pounds have been attributed to their interactions with D2dopamine receptors (11). However, recent studies have in-dicated that D1 dopamine receptors have important functionsin the central nervous system (12). The distribution of D1receptors in brain has been studied by using the selectiveligand SCH 23390. Although this ligand binds to a single classofreceptor sites, recent evidence has suggested the existenceof multiple D1 receptor subtypes (13). For instance, it has

been demonstrated (14) that injection of rat striatal mRNAinto Xenopus oocytes directs the expression of a D1 dopa-mine receptor coupled to activation of phospholipase C andphosphatidylinositol phosphate metabolism. Furthermore,dopamine does not stimulate adenylyl cyclase in theamygdala, a tissue known to contain specific binding sites forthe radiolabeled Dl-selective antagonist SCT{ 23390 (13). Inthe periphery, D1 receptors have been shown to stimulateadenylyl cyclase as well as phospholipase C (15-17). More-over, peripheral D1 receptors differ pharmacologically fromtheir central nervous system counterparts (13). Using thecloned human D1 receptor as a probe, we have reported thatmultiple hybridizing bands on Southern blot analysis at lowstringency could be observed. This finding is consistent withthe presence of other closely related receptors (2). We nowreport the cloning of a D1 receptor subtypett that haspharmacological and biochemical properties similar to thoseof the previously cloned D1 receptor but differs strikingly inits mRNA distribution.

MATERIALS AND METHODSCloning Strategy. Degenerate primers corresponding to the

fifth (5'-AACCATGGATCCTACATCCCTGTGGCCAT-CATGATTGTCACNTA-3') and sixth (5'-CCNCACAAA-CACACGACAACCGATGGAAAGAAGCTTAAGAT-CAAT-3') transmembrane (TM) regions of the human D1dopamine receptor (2) were used in the polymerase chainreaction (PCR) to amplify sheared human genomic DNA. ThePCR products were sequenced by using the dideoxy chain-termination method (18). One of these products [V-15; 230base pairs (bp)] displayed a significant homology with thehuman D1 receptor and corresponded to the fifth TM region,the third intracellular loop, and the sixthTM region. The V-15clone was used as a template for the synthesis ofa 32P-labeledprobe by PCR. The purified probe was used to screen 1.5 X106 recombinants of a rat testis genomic library in ADASH II(Stratagene). Duplicate nylon filters (Biotrans membranes,ICN) were hybridized in a buffer containing 5 x SSC (1x SSCis 0.15 M NaCl/0.015 M sodium citrate, pH 7.0), 5x Den-hardt's solution (0.1% Ficoll/0.1% polyvinylpyrrolidone/0.1% bovine serum albumin), 0.05M sodium phosphate at pH

Abbreviations: G protein, guanine nucleotide-binding protein; TM,transmembrane.tPresent address: Ontogdndse et Gdndtique Moldculaire, CHUL,Universitd Laval, Quebec, Canada G1V 4G2.**To whom reprint requests should be addressed.ttThe sequence reported in this paper has been deposited in theGenBank data base (accession no. M69118).

7491

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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6.5, 0.1% SDS, 50% (vol/vol) formamide, 200 ,ug of shearedsalmon sperm DNA per ml, and 32P-labeled V-15 probe (1 x106 cpm/ml) at 420C for 18-22 hr. At the end of the hybrid-ization period, filters were first washed in a solution con-taining 2x SSC and 0.1% SDS at room temperature and thenwashed at 500C in a solution containing O.1x SSC and 0.1%SDS. Nucleotide sequencing of both DNA strands was doneby the dideoxy chain-termination method (18).

Expression and Functional Characterization. African greenmonkey kidney (COS-7) cells were transiently transfectedwith the pCMV5-DR5 expression construct [a 4.2-kilobase(kb) EcoRI restriction fragment of DR5] by the DEAE-dextran procedure (2). Human embryonic kidney (293) cellswere transiently transfected by using a calcium phosphatetransfection system (Bethesda Research Laboratories/LifeTechnologies). Binding assays were performed on COS-7 cellmembranes and analyzed as previously described (2). Ade-nylyl cyclase activity in 293 cells was measured as described(2). For phosphatidylinositol turnover measurement, 48 hrafter transfection, COS-7 cells were labeled overnight withmyo-[3H]inositol (18.3 Ci/mmol; 1 Ci = 37 GBq). [3H]Inositolphosphates were measured as previously reported (19).Gene Localization. Both isotopic and nonisotopic in situ

chromosomal hybridizations were carried out to localize thehuman homologue of the DR5 clone by using standardprocedures (20, 21). The full-length 4.2-kb EcoRI genomicDNA fragment was labeled with [3H]dCTP and [3H]dTTP, orwith biotin-11-dUTP (New England Nuclear) by nick-translation and was used at 25 ng/ml and 200 ng/ml forisotopic and fluorescent hybridization, respectively.mRNA Localization. PCR Northern. Poly(A)+ RNA was

isolated from Sprague-Dawley rat tissues according to themethod of Badley et al. (22). PCR ofRNA was performed bystandard techniques (2).mRNA size. Poly(A)+ RNA isolated from rat hippocampus

(22) was fractionated by electrophoresis on a 1.2% agarosegel containing formaldehyde, transferred onto nylon mem-branes, and then hybridized with a specific 32P-labeledBamHI/Sac I restriction fragment of the DR5 clone.

In situ hybridization. A 4.2-kb EcoRI restriction fragmentfrom the DR5 clone was subcloned in pBluescript II SK+(Stratagene). 35S-labeled antisense- or sense-strand RNAprobes were prepared by in vitro transcription, and rat brainsections were hybridized as previously described (23). Therat D1A receptor probe was a 2.1-kb EcoRI restriction frag-ment and the D2 receptor probe was a 1.5-kb BamHI/Sac Irestriction fragment (2, 23).

RESULTS AND DISCUSSIONTo isolate potential members of the D1 receptor family, weused PCR amplification of human genomic DNA in conjunc-tion with the screening of a rat genomic library. Severalclones were isolated and processed for plaque purification byusing this procedure. A 4.2-kb EcoRI restriction fragmentfrom one clone (DR5) had an open reading frame of 1425 bpcoding for a protein (475 amino acids) with a calculatedmolecular weight of 52,834. The restriction map of the DR5clone and the nucleotide and predicted amino acid sequencesare shown in Fig. 1. The putative initiator methionine wasselected on the basis of the best Kozak consensus sequence(24) found in frame with the remainder of the coding blockand preceded by a stop codon.

Hydropathicity analysis of DR5 revealed the presence ofseven stretches of hydrophobic amino acids (data not shown)that presumably correspond to the seven TM regions typicalof G protein-coupled receptors (10). At the amino acid level,this putative receptor has about 50%o overall identity with therat and human D1 dopamine receptors. Within TM regions,the DR5 clone has 80%o identity with the D1 receptor, whereas

AEco RI Bam HI Bgl I

I IBql I Sph I Sac I Sph I

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500 bp

B-693 GAATTCAAGGTCCTATGACCCAGAATAGGGGTTCGGGATACAGTTGTGACTTCG

AAGGCCACTCTCCTATCCTCTAAGTCTCTGGTTTGTCTAGAGGCCTCTGGATCTCCTCCACCCAGAAGTGTTCCACGAGAGACACCAAGAGAGGTGTTTGGGAGAAGCTAATTCATGGGTTTGGGGCMAGGGTGTGGCACTGGGTTCACTCTCGGACCTGTGTGGGCCTCTAAATTGGAAGAAGACATCAGAGACTCATGAAGCTAGGAAG

ACTCCCAACCAACACACJ4TGGCCCACTTCCTrTCTCTCCTCCCACAQTCCAGCCACTCCCCCGCCGCATCGGTCGTTTCGGTCCCATCTTGGGGACCAGAGGTGCGCAAGAGTGTTACCATTIAAGGATCCTAAGCGGTGCACGGTGAGCGCTCCTCGGGTCGGGGACGGTCAGCTGCACGGCCCGGACGAOCTCTGGGGTGCCCGATGGGGCCTTCCACGGGGCGCAGCGGGAAGTTGGGACCGCAAGCAGAGACCCGAGCTACTCAGCGCGAC

ATG CTG CCT CCT GGG CGC AAC CGC ACG GCT CAA CCG GCA AGG CTG GGA TTA CAGMET Lou Pro Pro Gly Arg An Arg Thr Ala Gln Pro Ala Arg Lou Gly Lou Gln

AGG CAA CTG GCT CAG GTG GAC GCC CCA GCG GGC TCT GCA ACC CCA CTG GGA CCCArg Gln Lau Ala Gin Val Asp Ala Pro Ala Gly Ser Ala Thr Pro Lou Cly Pro

GCG CAG GTG GTC ACC GCA GGC CTC CTG ACT CTC CTA ATC GTC TGG ACC TTG CTCAla GIn Val Val Thr Ala Gly Lou Leu Thr Leou Lou Ii. Va1 Trp Thr Lou Lou

GGG AAC GTG CTA GTG TGT GCT GCC ATC GTC CGC AGC CGC CAT CTG CGC GCC AAGGly Asn Val Leu Va1 Cys Ala Ala Ii. Val Arg SHr Arg His Lau Arg Ala Lys

ATG ACC AAC ATC TTC ATC GTA TCC CTA GCT GTC TCA GAC CTC TTC GTG GCA TTGMET Thr Ann Il. Ph. Il. Val Ser Lou Ala Val S-r AsD Lau Ph. Val Ala Lou

CTG GTC ATG CCC TGG AAG GCT GTG GCT GAG GTG GCT GGG TAC TGG CCC TTT GGGLeu Val MET Pro TrD Lys Ala Val Ala Glu Val Ala Gly Tyr Trp Pro Ph. Gly

ACA TTC TGC GAC ATC TGG GTG GCC TTT GAC ATC ATG TGC TCC ACT GCC TCC ATCThr Ph. Cys Aso Il. Tra Val Ala Ph. AD Ii. MET Cys Her Thr Ala SHr Ile

CTG AAT CTG TGT ATC ATC AGC GTG GAC CGT TAC TGG OCT ATT TCC AGA CCC TTCLou Ann Lau CYs Ile Ile Ser Val Aso Arg Tyr Trp Ala I1e SHr Arg Pro Ph.

COC TAC GAG COC AAG ATG ACC CAG CGA GTA GCC CTG GTC ATG GTG GGC CTG GCCArg Tyr Glu Arg Lys MET Thr Gln Arg Val Ala Leu Val MET Val G1Y Leu Ala

TGG ACC TTG TCC ATC CTC ATC TCC TTC ATC CCC GTC CAA CTC AAT TCO CAC AGATrD Thr Lou Ser Ile Lou Ile Her Ph. Ile Pro Val Gln Lou AMn TrD His Arg

GAC AAG GCA GGC TCC CAG GGC CMA GAG GGC CTG CTG TCC AAT OGC ACA CCC TGGAsp Lys Ala Gly Her Gln Gly Gln Glu Gly Leou Lou Ser Ann Gly Thr Pro Trp

GAG GM GOC TCO GAG CTA GAA GGG AGG ACG GAG AAC TGT GAG TCC AGC CTG AMCGlu Glu Gly Trp Glu Leu Glu Gly Arg Thr Glu Asn Cys Asp Ser Ser Lou Asn

CGA ACC TAT GCC ATC TCC TCG TCA CTC ATC AGC TTC TAC ATC CCG GTG CCC ATCArg Thr Tyr Ala Ile Ser Ser Her Lou Ile Ser Ph. Tyr Ie Pro Val Ala Ile

ATG ATC GTG ACC TAT ACG CGT ATC TAC CGC ATT GCG CAC GTG CAG ATC CGG CGGMET I1e Va1 Thr Tyr Thr Arg Ile Tyr Arg I1e Ala Gln Va1 Gln Ile Arq Arg

ATC TCC TCC CTA GAG AGG GCA GCT GAG CAT GCT CAG AGT TGC CGG AGT CGT GGAI1e Ser Ser Leu Glu Arg Ala Ala Glu His Ala Gln SHr Cys Arg Her Arg Gly

GCC TAT CAA CCT GAC CCC AGC CTG CGA GCG TCC ATC AMG AAG GAG ACC AAG GTCAla Tyr Glu Pro Asp Pro Ser Lau Arg Ala Ser Ile Lys Lys Glu Thr Lys Val

TTC AAA ACC CTG TCA ATC ATC ATG GGG CTC TTC GTG TGT TGC TGG TTC CCT TTCPh. Lye Thr Leu Ser MET Ile MET Gly Va1 Phe Val Cys CYs Try Leu Pro Ph-

TTC ATC CTG AAC TCT ATG GTT CCT TTC TGC AGT AGT GGG GAT GCC GAG GGC CCAPh. Ile Lau Ann Cys MET V.1 Pro Ph. Cys Ser SHr Gly Asp Ala Glu Gly Pro

-640-569-498-427-356-285-214-143-72-1

5419

10836

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21672

27090

324109

378126

432144

486162

540190

594198

648216

702234

756252

810270

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AAG ACT GGC TTC CCT TGT GTC AGC GAG ACC ACC TTC GAC ATA TTC GTC TGG TTT 1026Lys Thr Gly Ph. Pro CyH Val Ser Glu Thr Thr Ph. AMD Ile Ph. Va1 TrD Ph. 342

GGC TGG GCC AAC TCC TCT CTC AAT CCC ATC ATC TAT GCC TTT AAT GCA GAC TTCGIy TrD Ala Asn Ser Ser Leou AMn Pro Ile Ile Tyr Ala Ph AMn Ala Asp Phe

CGC AAG GTG TTT GCC CAG CTG CTG GGC TGC AGC CAC TTC TGC TTC CGG ACC CCAArg Lye Va1 Ph. Ala Gln Leu Lau Gly Cys Ser His Ph. Cys Ph. Arg Thr Pro

GTG CAG ACG GTA AAC ATC AGT AAT GAG CTC ATC TCC TAC AAC CAA GAC ACG GTCVal Gln Thr Val Asn Ile Ser Asn Glu Lou Ile Ser Tyr Asn Gln Asp Thr Val

TTC CAC AAG GAG ATC GCT ACT GCC TAT GTC CAC ATG ATA CCG AAT GCA GTA TCCPhe His Lys Glu Ile Ala Thr Ala Tyr Val His MET Ile Pro Asn Ala Val Ser

TCC GGA GAC AGG GAG GTG GGA GAG GAG GAG GAG GAG GGG CCT TTC GAT CAC ATGSer Gly Asp Arg Glu Val Gly Glu Glu Glu Glu Glu Gly Pro Phe Asp His MET

TCT CAA ATC TCT CCA ACG ACG CCA GAC GGT GAC CTG GCT GCT GAG TCT GTC TGGSer Gln Ile Ser Pro Thr Thr Pro Asp Gly Asp Leu Ala Ala Glu Ser Vai Trp

GAG CTT GAC TGT GAG GAA GAG GTT TCC TTA GGC AAA ATC TCA CCT CTC ACC CCCGlu Leu Asp Cys Glu Glu Glu Val Ser Leu Gly Lys Ile Ser Pro Leou Thr Pro

AAT TGT TTC GAT AAA ACT GCT TAG AAACATTCTCATGGGCATATACAATGGTGGCCATATTTCAsn Cys Phe Asp Lys Thr Ala *

1080360

1134378

1199396

1242414

1296432

1350450

1404468

1467475

15381609

FIG. 1. Restriction map (A) and nucleotide and deduced aminoacid sequences (B) of the DR5 rat genomic clone. Potential N-linkedglycosylation sites are indicated by double underlines. The putativemembrane-spanning domains are underlined. The stop codon isrepresented by an asterisk.

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Table 1. Equilibrium dissociation constant values (Kd) ofdopaminergic compounds measured by competition with[1251]SCH 23982 binding in COS-7 cell membranes

Kd, nM

Human D1A Rat D1BCompound receptor receptor D1B/D1A

AntagonistsSCH 23390 0.17 0.11 0.6cis-Piflutixol 0.65 2 3.1(+)-Butaclamol 1 6 6.0cis-Flupentixol 4 7 1.8SCH 23388 15 10 0.7Haloperidol 24 35 1.5cis-Teflutixol 24 37 1.5Fluperlapine 75 510 6.8Spiperone 450 2600 5.8

AgonistsFenoldopam 17 11 0.6SKF 38393 135 100 0.7Apomorphine 360 240 0.7NPA 1,540 1050 0.7Dopamine 12,000 3900 0.3

Binding parameters shown are the result of two independentexperiments conducted in triplicate determinations. For each drug,the two competition curves were coanalyzed and fitted to a one-sitemodel. NPA, N-propylnorapomorphine.

the amino- and carboxyl termini are the most divergentregions (<20%o identity). In contrast, within the TM regionsDR5 shares only 47% and 39o identity with the rat D2 and D3dopamine receptors, respectively. Furthermore, an asparticresidue (Asp-118) in the third TM region and two serineresidues (Ser-224 and Ser-227) in the fifth TM region arepresent in this putative receptor as they are in every clonedcatecholamine receptor. These residues have been postu-lated to be involved in the interaction with the amino andcatechol hydroxyl groups of catecholamines (25, 26). Puta-tive sites for N-linked glycosylation are found in the aminoterminus (Asn-7) and the second extracellular domain (Asn-194). A cysteine residue (at position 370) is found in thecarboxyl tail near the seventh TM region. This residue isconserved in most of the G protein-coupled receptors and is

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FIG. 2. Stimulation of adenylyl cyclase in membranes preparedfrom 293 cells transfected with pCMV5-DR5 expression construct.Results are the mean ± SEM of a representative example of threeindependent experiments done in triplicate determinations. Drugsand concentrations used are dopamine (DA), 100 ,uM; fenoldopam(FENOL), 1 AM; SCH 23390 (SCH), 1 &M; and alprenolol (ALP), 1AM.

palmitoylated in the 32-adrenergic and rhodopsin receptors(27, 28). In addition, several consensus sites for proteinkinase A and protein kinase C exist on the putative cytoplas-mic domains of the DR5 gene product. Phosphorylation of Gprotein-coupled receptors by kinases has been proposed to beimportant in the regulation of responsiveness of these recep-tor systems (29).To characterize the function of the DR5 clone, a pCMV5-

DR5 expression construct was transiently transfected inCOS-7 and 293 cells. In membranes prepared from COS-7cells transfected with pCMV5-DR5, the D1 receptor antago-nist [125I]SCH 23982 (New England Nuclear) bound specifi-cally to one homogeneous class of binding sites with adissociation constant (Kd) of 0.41 ± 0.01 nM (n = 3). Thisvalue is similar to the Kd value for this ligand reported formembranes from rat striatum (30) or cells transfected with thepreviously characterized D1 dopamine receptor clone(pCMV5-D1) (0.35 ± 0.02 nM, n = 2) (2). In untransfected ormock-transfected COS-7 cells, little or no specific bindingwas observed. Table 1 summarizes the binding affinities ofdopaminergic agonists and antagonists for the binding of[125I]SCH 23982 in membranes prepared from COS-7 cellstransfected with either pCMV5-DR5 or pCMV5-D1. Theresults show that the pharmacological profile of the rat

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FIG. 3. (A) PCR analysis of D1B dopamine receptor mRNAisolated from different rat tissues. A specific primer (5'-TTGTTTCTATGCAGCAGACTA-3') was used to initiate first-strand cDNA synthesis from 100 ng of poly(A)+ RNA by avianmyeloblastosis virus reverse transcriptase. After inactivation of thereverse transcriptase, specific primers (5'-GCCTATGTCCACAT-GATA-3' and 5'-AAAGCATATGCACACTGGAGT-3') were usedto amplify a 339-bp fragment ofthe D1B receptor. PCR was performedfor 25 cycles, and products were fractionated on a 1% agarose gel andblotted onto a nylon membrane. The membrane was hybridized withSph I restriction fragment of the D1B receptor, labeled with[32P]dCTP by nick-translation. The blot was then washed at 65°C ina solution containing 0.1x SSC and 0.1% SDS. PCR done in absenceof the reverse transcriptase did not result in any detectable product(data not shown). (B) Northern blot of rat hippocampal tissue probedwith the BamHI/Sac I restriction fragment of the rat DIB dopaminereceptor. The lane contains 10 ,ug of poly(A)' RNA from rathippocampus. After overnight hybridization, the nylon filter was firstwashed in 2x SSC/0.1% SDS at room temperature and then washedin 0.1x SSC/0.1% SDS at 55°C.

Neurobiology: Tiberi et al.

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receptor (DR5 clone) is comparable to that observed for thehuman D1 dopamine receptor. In general, antagonists wereslightly less potent, while the agonists displayed slightlyhigher affinities for the rat receptor.

In 293 cells transiently transfected with pCMV5-DR5, do-pamine and the D1-selective agonist fenoldopam stimulatedadenylyl cyclase activity by 2- to 3-fold (Fig. 2). This effect isblocked by the Dl-selective antagonist SCH 23990 but not bythe (B-adrenergic antagonist alprenolol or the D2-selectiveantagonist raclopride (1 ,.M; data not shown). The agonistSKF 38393 (1 ,uM) also increased the enzyme activity by about2-fold. Dopamine did not stimulate phosphatidylinositol phos-phate turnover in COS-7 cells transiently transfected with thepCMV5-DR5, whereas norepinephrine increased phosphati-dylinositol phosphate metabolism by about 200% in cellstransfected with the a1B-adrenergic receptor (31) (data notshown). These results demonstrate that the rat clone DR5encodes a distinct G protein-coupled receptor with pharma-cological and biochemical properties similar to those of thepreviously cloned D1 dopamine receptor. On this basis wepropose that this receptor be referred to as the D1B subtype,whereas the previously cloned D1 receptor would be referredto as the D1A subtype.To determine the chromosomal location of the human

homologue of the D1B receptor, a 3H-labeled 4.2-kb EcoRIfragment was hybridized to human metaphase chromosomes.Specific labeling was observed at three sites, the secondary

D1A mRNAAntisense AnI

BA

CPu,

E

constriction of chromosome 1 (lq12), the centromeric regionofchromosome 2 (2p 1.2-qll.2), and the terminal band oftheshort arm of chromosome 4 (4p16.3). Of 116 grains scoredfrom 50 cells, 16 (13.8%), 14 (12.2%), and 16 (13.8%) wereover chromosome bands 1q12, 2p11.1-q11.2, and 4p16.3,respectively. It is widely appreciated that the secondaryconstriction of chromosome 1 (1q12) and the centromericregion of chromosome 2 (2p11.2-q11.2) contain heterochro-matin mainly composed of repetitive sequences. Therefore,to further determine the specific site of localization, fluores-cence in situ hybridization was performed in the presence ofexcess human genomic DNA (21). Under these conditions,only the chromosome 4 (4pl6.3) location was specificallylabeled (data not shown). Interestingly, the chromosome 4location on which this receptor gene is located has beenimplicated as the region containing the Huntington diseasegene (also 4p16.3) (32). However, the significance of thislocalization remains to be determined.The tissue distribution of the rat D1B receptor mRNA was

examined by PCR analysis. A specific 339-bp fragmentspanning from nucleotide 18% (residue Ile-401 in the car-boxyl tail) and 114 nucleotides of the 3' untranslated regionof the D1B receptor message was amplified. The most prom-inent signal was observed in hippocampus and hypothala-mus; weak signals were present in striatum and cerebellum.No detectable products were observed from temporal cortex,kidney, lung, heart, and liver (Fig. 3A). Northern blot anal-

D, mRNAtisense Sense

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FIG. 4. Comparison of distribution of D1A, D1M, and D2 dopamine receptor mRNAs in rat brain coronal sections by in situ hybridization.(A, D, and G) Hybridization with 3"S-labeled antisense D1A receptor probe. (B, E, and H) Hybridization with 35S-labeled antisense D1B receptorprobe. (C and F) Hybridization with 35S-labeled sense D1B receptor probe. (I) Hybridization with 35S-labeled antisense D2 receptor probe. AcbC,nucleus accumbens; APT, anterior pretectal nucleus; CPu, caudate-putamen; Hippo, hippocampus; LM, lateral mammillary nucleus; RS,retrosplenial cortex; SNC, substantia nigra pars compacta; Tu, olfactory tubercle; VI, layer VI of the isocortex.

APT

-SNC\

DmRNA L

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ysis of hippocampal poly(A)+ RNA revealed a prominenthybridizing band at 3.0 kb and a less intense band at 3.7 kb(Fig. 3B). These two mRNAs are likely to be derived from theD1B receptor gene, since these two bands remained afterextensive high-stringency washings (0.1x SSC/0.1% SDS at650C). In contrast, the message for the rat D1A dopaminereceptor is about 4.0 kb (3, 5).

In situ hybridization was used to map more precisely theD1B receptor mRNA in rat brain. These results indicate thatthis D1 dopamine receptor has an mRNA distribution distinctin rat brain from that of the D1A receptor (Fig. 4). Prominentlabeling was found in the lateral mammillary nuclei, in theanterior pretectal nuclei, and in several layers of the hippo-campus (Fig. 4 B, E, and H). In contrast, little or no messagewas detected in striatum, nucleus accumbens, and olfactorytubercle, regions in which D1A receptor mRNA is abundant(Fig. 4 A, D, and G). Furthermore, dopaminergic neurons inthe substantia nigra that express abundant D2 receptormRNA (Fig. 41), did not express detectable D1A and D1Breceptor mRNAs (Fig. 4 G and H). However, there was an

overlapping expression of D1B and D2 receptor mRNA in thelateral mammillary nuclei (Fig. 4 H and I). Interestingly, themammillary bodies, anterior pretectal nuclei, and hippocam-pus, which express high levels of D1B mRNA, have beenshown to have very low levels ofD1 receptor binding (33, 34).These observations raise the possibility that this receptor issynthesized in cell bodies where the mRNA is found andtransported to nerve terminals elsewhere in the brain.

Heterogeneity within subfamilies of G protein-coupledreceptors has been documented for the adrenergic, seroto-nergic, and muscarinic receptors. This multiplicity has beenbased on distinct pharmacological properties, signal trans-duction mechanisms, and differences in tissue distribution(35). Although several lines of evidence have raised thepossibility of the existence of D1 dopamine receptor sub-types, the possibility of multiple central nervous systemadenylyl cyclase-coupled D1 subtypes had not been advanced(13). Indeed, with the dopaminergic ligands currently avail-able, this receptor subtype could not have been discriminatedpreviously on the basis of pharmacological properties. Themost interesting aspect of this receptor gene is its highlyrestricted expression when compared with the previouslycharacterized D1A receptor gene (23). This unique expressionpattern implies that this receptor might mediate a specificsubset of dopaminergic functions. In particular, the presence

ofmRNA in the anterior pretectal nucleus suggests a possiblerole for this receptor subtype in visual processes. Its specificexpression in the mammillary nuclei and hippocampus may

implicate this receptor in dopaminergic regulation of limbicphysiology. The use of cell lines expressing the D1B receptorsubtype should allow for the design of new selective phar-macological agents capable of elucidating the physiologicalfunctions of this receptor.While this paper was under review, a study reporting the

cloning of another human dopamine receptor of the D1receptor family was published by Sunahara et al. (36). At theamino acid level, this receptor has 83% overall identity withthe rat D1B receptor reported here. This value is lower thanthe 90% overall identity between the rat and human D1Areceptor homologues. In addition, by in situ hybridizationthis human receptor gene displays an mRNA distributionsimilar to that of the D1A receptor, whereas a totally differentdistribution is observed for the D1B receptor in rat brain.Moreover, the human D1-like receptor described by Suna-hara et al. binds dopamine with higher affinity than either theD1A or the D1B receptor. These data suggest that these twoclosely related genes encode distinct receptor proteins.

We thank J. Didsbury for the 293 cells, S. Cotecchia for assistancewith measurement of phosphatidylinositol phosphate metabolism,and U. Schambra for preparing the brain slices. M.T. and K.R.J. arefellows of the Medical Research Council of Canada. This work wassupported by Grants NS-159976 and IP53-MH44211 from the Na-tional Institutes of Health.

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