Molecular cloning of the rat A2 adenosine receptor: selective co-expression with D2 dopamine receptors in rat striatum

186

Molecuh, r Brain Re.search, I4 (1992) 186-195 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0169-328X/92/$05.00

BRESM 70436

Molecular cloning of the r a t A 2 adenosine receptor: selective co-expression with D 2 dopamine receptors in rat striatum J. Stephen Fink ~', David R. Weaver b, Scott A. Rivkees b, Robert A. Peterfreund ~', Alexia E. Pollack ~', E l i z a b e t h M. A d l e r ~' a n d S t e v e n M. R e p p e r t b "Laborato#T ol' Molecular Neurobiology and hLahoratoo' (~l' Developmental ETtronobiology, Mas.~achu,~etts General Howital aml Departments o[ Neurology, Pediatrics and Anesthesia, Harvard Medical School, Boston. MA 02114 (USA) (Accepted 4 February Ic)CJ21 Key words." Adenosine: Adenosine Az receptor: Basal ganglia: Dopatnine: Dopaminc D e receptor: cDNA

A cDNA fragment homologous to other G protein-coupled receptors was isolated from rat brain using the PCR method and demonstrated to be abundantly expressed in striatum. Using this fragment as a probe, a 2.1 kb full-length cDNA was isolated from a rat striatal cDNA library. This cDNA encodes a protein of 410 amino acids and is highly homologous to previously isolated adenosine receptor cDNAs. Expression of this eDNA in COS cells revealed high affinity ( K d = 38.6 nM) and saturable binding of the A. adenosine receptor-selective ligand [~H]CGS 21680. Agonist displacement profile of [3H]CGS 21680 binding was consistent with an adenosine receptor of the Ae subtype (NECA > (R)-PIA > CPA > (S)-PIA). In situ hybridization demonstrated that rat A~ adenosine receptor mRNA was co-expressed in the same striatal neurons as D e dopamine receptor mRNA, and never co-expressed with striatal D l dopamine receptor mRNA. Several lines of evidence have previously suggested that dopamine-induced changes in motor behavior can be modulated by adenosine analogs acting at the A 2 subtype of adenosine receptor in the forebrain. The co-expression of D 2 dopamine and A 2 adenosine receptors in a subset of striatal cells provides an anatomical basis for dopaminergic-adenosinergic interactions on motor behavior. forebrain li
INTRODUCTION

site of D A - a d e n o s i n e A , interaction. H o w e v e r , the anA m o n g the diverse physiological effects of adenosine and adenosine analogs are their ability to effect changes in motor behavior 71417"324°. Adenosinergic agonists inhibit and adenosine antagonists enhance dopamine-in,' duced h y p e r m o t-m t y~

11.12.15

atomical and cellular basis of the behavioral interaction of

adenosinergic

agonists

and

antagonists

with

the

dopaminergic system is poorly understood. The polymerase chain reaction (PCR) method can be

. The physiological effects of

used with degenerate oligonucleotide primers based on

adenosine appear to be mediated by interaction with 2

conserved amino acid sequences among G protein-linked

types of m e m b r a n e - b o u n d receptor proteins, A~ and A e,

receptors 3~ to amplify novel receptor fragments from rat

which are coupled to guanine nucleotide-binding pro-

bratn ~ . Using the P C R method we have isolated a e D N A encoding a m e m b e r of the G protein-linked receptor

teins (G proteins). The A~ and a 2 subtypes of adenosine receptors have been defined by their ability to inhibit

family which is abundantly expressed in the rat striatum.

or stimulate adenylyl cyclase, respectively, differences in

Expression of this e D N A demonstrated that it encodes

ligand binding profile and anatomical distribution in brain 1e542"4-~. Modulation of m o to r behavior by dopam-

a rat A , adenosine receptor. The predicted amino acid

ine ( D A ) agonists is the result of activation of the D~ and/or D~ subtype of D A receptor which are segregated

canine receptor RDC82), which has recently been shown to be an A~ adenosine receptor-~' . To begin to charac-

sequence of this receptor is highly homologous to the

in subsets of striatal neurons and in distinct striatal ef-

terize the cellular and anatomical basis for a d e n o s i n e - D A

ferent pathways ~. Adenosinergic interaction with the

interactions on m o t o r behavior we determined, using in

brain D A system appears to occur through the A . adenosine receptor 3s~~e. Ligand binding autoradiography

sire hybridization, whether the rat A 2 receptor m R N A

has demonstrated that D~. D, and A : receptors are

expressing D~ or D e D A receptor m R N A s .

is expressed within the same cells in rat striatum as those

abundantly expressed within the striatum and limbic Correspondence. J.S. Fink, Molecular Neurobiology Laboratory, Department of Neurology, Massachusetts General Hospital. I49 13th St., Charlestown, MA 021"~c/,-,USA. Fax: (1) 617-726-5677.

187 MATERIALS AND METHODS

PCR amplification of A , receptor Poly(A) + RNA from rat medial basal hypothalamus explants (containing arcuate nucleus, median eminence, and attached pars tuberalis 6) was isolated using a Fast Track mRNA isolation kit (Invitrogen). First-strand cDNA was synthesized using 2 ltg of poly(A) ÷ RNA primed with oligo(dT) and murine Moloney leukemia virus reverse transcriptase (Bethesda Research Labs.). This first strand cDNA was used as template for PCR with Ampli Taq DNA polymerase (Perkin Elmer Cetus) using degenerate primers corresponding to amino acids within conserved regions of several G protein-linked receptors33. Each degenerate primer included a restriction endonuclease site on the 5' end to facilitate subcloning of PCR amplification products. The first amplification used primers corresponding to the second (primer A; 4,096-fold degenerate) and third (primer B; 4,608-fold degenerate) transmembrane domains. The second round of PCR amplification used 5% of the first PCR amplification product and primers A and B. Primer A = 5'GCGTCTAGA(AC)NG(CT)NA(AC)NAA(CT)TA(CT)(CT)T-3', where N = ACTG. Primer B = 5'-TTCAAGCTT(CG)(AC) N(ACG) N(AG)TA(CGT)C(GT)(AG)TC-3'. The PCR amplifications were for 30 cycles using the following parameters: 94°C (1.5 min), 42°C (1 rain), 72°C (1.5 min). The products of the second PCR amplification were digested with EcoRI and HindlII, the digestion products separated by agarose gel electrophoresis and a DNA band of approximately 210 bp was transferred onto NA-45 paper (Schleicher and Schuell) and eluted into 1 M NaCI. The eluted DNA was extracted with phenol, precipitated with ethanol and subcloned into M13mpl8 or M13mpl9. Random M13 clones were sequenced by the Sanger method using Sequenase (United States Biochemical). Inserts from clones which, on the basis of predicted amino acid sequence, were homologous to the second and third transmembrane domains of G protein-linked receptors were isolated from the replicative form of M13. These putative G protein-linked receptor fragments were labeled with [32P]dCTP by the random priming method and used as a probe for in situ hybridization histochemistry to brain sections. One fragment (R8) hybridized exclusively to striatum. One reading frame of this putative G protein-linked receptor fragment was 82% homologous to the orphan receptor RDC823. At the time of the isolation of the rat R8 cDNA neither the distribution nor the identity of RDC8 had been reported.

cDNA library screening The PCR-generated receptor fragment R8 was used to screen a rat striatal cDNA library (gift of O. Civelli, University of Oregon) in 2 gtl0, generated from first-strand cDNA that had been primed with oligo(dT). The 210 bp R8 cDNA fragment was labeled with [a-32P]dCTP (2000 Ci/mmol) by the random priming method and used as a probe to screen the cDNA library using the following conditions: 50% formamide, 1 M NaCI, 1% SDS, 10% dextran sulfate and 100 tLg/ml denatured salmon sperm at 42°C. Colony Plaque Screen filters (New England Nuclear) were washed at 2 x SSC, 1% SDS at 65°C followed by 0.1 x SSC, 1% SDS at room temperature. To obtain a cDNA clone encoding the most 5' region of the R8 cDNA, a rat striatal cDNA 2 gtl0 library generated by random priming (Clontech) was screened using a 120 bp DNA fragment corresponding to the 5' end of the R8 coding region. Hybridization and washing conditions were identical to those used in the initial library screening. Bacteriophage inserts were subcloned into M13 or pBluescript (Stratagene) and sequenced by the Sanger method.

Expression studies One cDNA clone (DT-35), obtained from the oligo(dT)-primed striatal cDNA library, was excised from the bacteriophage by digestion with EcoRI and subcloned into the expression vector pcDNA I (Invitrogen). This 2,066 bp cDNA clone contained the entire coding region, 3 bp of 5'-untranslated sequence and 836 bp

of the 3'-untranslated sequence. Expression studies were performed in COS-6M cells as previously described35. Briefly, COS-6M cells (a gift of B. Seed, Massachusetts General Hospital) were grown as monolayers in DMEM supplemented with 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 ltg/ml) in 5% CO~ at 37°C. The DT-35 cDNA in pcDNA I was transfected by the DEAE-dextran method and cells were harvested 48 h after transfection. Intact cells were preincubated with adenosine deaminase (ADA, l0 U/ml, 30 min at 37°C) to degrade endogenous adenosine. Binding of the A~-selective ligand [3H]CGS 21680 (New England Nuclear, 42 Ci/mmmol) to intact cells (750-1000 l~g protein/ ml) was performed in the presence of 2 U/ml A D A . Binding reactions proceeded for 2 h at 25°C in a final volume of 200 !d. Non-specific binding was determined by the inclusion of unlabeled 5'-N-ethylcarboxamidoadenosine (NECA, 100 I~M). All determinations were done in triplicate. Protein was determined using the Bradford method with BSA standards. Binding data were analyzed by computer using an iterative non-linear regression program 3°.

Northern blot Total cellular RNA was isolated using guanidine thiocyanate and separation in cesium chloride 36. RNA was fractionated on an agarose-formaldehyde gel, transferred to GeneScreen (New England Nuclear) by electro-transfer and hybridized with cDNA clone DT-35 labeled with [32p]dCTP to a specific activity of >10 ~ cpm/~g by the random priming method. Hybridizations were performed in 50% formamide, 1 M sodium chloride, 1% SDS, 10% dextran sulfate and denatured salmon sperm (100 l~g/ml) at 42°C. Final washing conditions of membranes was 0.1 x SSC and 0.1% SDS at 22°C for 40 min.

In situ hybridization to brain In situ hybridization was performed as described previously35. The distribution of A 2 adenosine mRNA was examined in six Sprague-Dawley rats (four 50-day-old males, 150 g and 2 adult females, 250-300 g; Charles River). For each brain, 15 l~m coronal sections were examined at 180 ~m intervals throughout the entire brain. Additional serial sections (5 ~tm) from the striatum of two 50-day-old male rats were used for co-localization studies (see below). For hybridizations 40 id hybridization buffer containing 1.01.5 x 107 cpm probe/ml were applied to each slide. Hybridization buffer was 50% formamide, 10% w/v dextran sulfate, 2 x Denhardt's solution, 0.9 M NaCI, 50 mM NaHzPO 4, 5 mM EDTA, 0.1% SDS, 100 mM dithiothreitol, 500 itg/ml herring sperm DNA, 500 ~g/ml yeast total RNA. After overnight hybridization, coverslips were removed in 2 x SSC and the slides were washed in 2 changes (30 min each) 2 x SSC. Slides were then incubated in RNase (USB, 10 ttg/ml in 0.5 M NaCI, 10 mM Tris-HCl) for 1 h at 37°C. Final washing was 2 x SSC (30 min, room temperature), 0.1 x SSC (twice for 30 min at 53°C) and 0.1 x SSC (twice for 30 min at room temperature). Film autoradiograms were generated by apposing slides to Kodak SB-5 X-ray film for 8-15 days. Subsequently, selected slides were dipped in Kodak NTB-2 emulsion diluted 1:1 with 0.6 M ammonium acetate (0.3 M final), dried, and stored for 1 month at 4°C in light-tight, plastic boxes containing dessicant. After development sections were counterstained with Methylene blue or thionin.

35S-Riboprobes Antisense and sense RNA probes were labeled with [a-thio3SS]UTP (1100 Ci/mmol; New England Nuclear). Adenosine A e receptor sense and antisense RNA probes were generated from a 326 bp PstI fragment from the coding region of DT-35 encompassing transmembrane domains II, III and part of IV (nucleotides 100 to 426 according to the numbering of Fig. ib). Limited studies with two other antisense R8 probes confirmed the distribution observed with the 326 bp R8 fragment. These other probes were a 635 bp PstI fragment consisting entirely of 3'-untranslated sequence (nucleotides 1431 to 2066 according to the numbering of Fig. lb) and

188

a 02 Ib RP-21 DT-35

A

ATO

b GCTGCAGCTATGGACCGAGAGCTGGCCCAGGCCTGCATCCCTGCTGAGCCTGCCCAAGTGTGGCTGCTCCCACC ATG GGC Met Gly

TCC T C G GTG Ser S e t Val

AAC GTG CTC G T G TGC Asn Val Leu V a l Cys

TAC A T C ACG GTG GAG CTG G C C ATC GCT G T G CTG GCC ATC CTG G G C Tyr Ile Thr Val GIu Leu A l a Ile Ala V a l Lou Ala Ile Leu G l y TGG G C C GTG TGG ATC AAC A G T AAC Trp A l a Val Trp Ile ASh Ser Asn

CTG C A G AAC Leu G l n Ash

GTC ACC AAC T T C Val Thr Asn Phe

TTT GTG GTA T C G CTG GCG G C G GCT GAC A T T GCA GTG GGT GTG CTC GCC ATC CCC TTC G C T Phe Val Val Ser Leu Ala A l a Ala A s p Ile Ala Val Gly Val L e u Ala Ile Pro Phe A l a ATC ACC ATC A G C ACC GGC T T C TGC GCC GCC TGC C A C GGC Ile Thr Ile SeE ThE Gly P h e Cys Ala Ala Cys HIs Gly

TGC C T C TTC Cys Leu Phe

60

20

TTC GCC Phe Ala

180

60

TGT T T T Cys Phe

GTC CTG GTC C T C ACG CAG A G T TCC ATC TTT AGC C T C TTG GCT A T C GCC ATC GAC CGC TAC Val Leu Val Leu Thr Gln Set Set Ile Phe Set Leu Leu Ala Ile Ala Ile Asp A r g Tyr

300

100

ATC GCC ATC C G A ATT CCA C T C CGG TAC A A T GGC T T G GTG ACA G G T GTG AGG GCG AAG G G C Ile Ala Ile A r g Ile Pro Leu Arg Tyr Asn Gly Leu Val Thr G l y Val Arg Ala Lys G l y ATC ATT GCA A T T TGC Ile Ile A l a Ile Cys

TGG G T G CTG TCG TTT GCC A T T GGC CTG A C C CCC ATG CTG GGC T G G Trp V a l Leu Ser Phe Ala Ile Gly Leu T h r Pro Met Leu Gly T r p

AAC AAC Asn Asn

TGC A G T CAG A A A GAC Cys Ser Gln Lys A s p

TGT CTG Cys Leu

TTC G A G GAC GTG G T G CCC ATG AAT Phe G l u Asp Val V a l Pro Met Asn

CAG AAG Gln Lys

TAC A T G GTT TAC TAC AAC Tyr M e t Val Tyr T y r Asn

TTC TTT GCG TTC Phe Phe Ala Phe

GAG C G G ACT CGG TCC ACG C T G Glu A r g Thr Arg Ser Thr Leu

GAG GTC CAC GCT G C C AAG TCC CTG GCC A T C ATC GTC G G G CTC Glu Val His Ala A l a Lys Ser Leu Ala Ile Ile Val G l y Leu

TGG TTG CCG C T G CAC ATC A T C AAC TGT TTC ACC TTC TTC Trp Leu Pro L e u His Ile Ile Ash Cys Phe Thr Phe Phe

180

TGC T C C ACG Cys Ser Thr

660

220

TTT GCT CTG T G C Phe Ala Leu Cys TGC CGG CAC G C C Cys Arg His A l a

780

260

TAC C T G GCC ATC ATC CTC TCC CAC AGC A A C TCC GTC GTC AAC C C C Tyr Leu Ala Ile Ile Leu Set His Set A s n Ser Val Val Asn P r o

TTC ATC TAC GCC TAC AGG A T C CGG GAG TTC CGC C A G ACC Phe Ile Tyr A l a Tyr A r g Ile Arg Glu Phe Arg Gin Thr

TTC C G G AAG ATC ATC CGA A C C Phe A r g Lys Ile Ile A r g T h r

CTG A G G CGG Leu A r g Arg

CAG G A A tCC TTC CAG GCA G G G GGT TCC A G T GCC Gln Glu Pro Phe Gln Ala G l y Gly Ser Set Ala

TGG GCC Trp Ala

GCT CAC AGC A C T GAG Ala His Ser T h r Glu

GGA G A G CAG GTT AGC CTC CGC CTT AAT GGC CAC Gly G l u Gln Val Ser Leu A r g Leu Ash G l y His

CCC CTG GGG G T A Pro Leu Gly V a l

CAC GTC His Val

540

CTG GCC ATC TAC CTA CGG A T T TTT CTG GCG GCC C G G Leu Ala Ile T y r Leu Arg Ile Phe Leu Ala Ala A r g

CAG A T G GAG AGC CAG CCC CTG C C A GGG Gin Met Glu Ser Gln Pro Leu Pro Gly

CCT CCG TGG CTC ATG Pro Pro Trp Leu Met

140

GGG AAC TCC ACG A A G ACC TGC GGC GAG GGC CGG GTG A C C Gly Ash SeE Thr Lys Thr Cys G l y Glu Gly Arg Val T h r

GTG TTA CTG C C C CTT CTG CTC ATG Val Leu Leu P r o Leu Leu Leu Met AGA CAG CTG A A G Arg Gin Leu Lys

420

900

300

CTG G C A Leu A l a 1020

340

TGG GCC AAC GGC AGT GCC A C C CAT TCC GGA CGG CGG CCC AAT GGC TAC ACT CTG GGG C T G Trp Ala Asn Gly Ser Ala T h r His Ser GIy Arg A r g Pro Ash G l y Tyr Thr Leu Gly Leu GGG GGT GGA G G G AGT GCC C A A GGC TCT CCT CGG G A T GTG Gly Gly Gly G l y Ser Ala G i n Gly Ser Pro Arg A s p Val CAG GAA GGC C A A GAG Gln Glu Gly Gln Glu

CAC C C T GGC CTA AGG His Pro Gly Leu Arg

TCC TCA TGG T C T TCA GAG T T T GCC Ser Set Trp Set Ser Glu Phe Ala

CCT TCC Pro Ser

GAG C T T CCT ACC CAG GAG C G C Glu Leu Pro Thr Gln Glu A r g

1140

380

GGT C A T CTG GTC C A G GCT AGA GTA GGA G C T Gly His Leu Val G l n Ala Arg Val Gly A l a TGA G G G A A A G A C A T T T T A A T A T T T T T G G T T G G C T G G A C *

CAATCTCACTAAGGGAAGAGAAACCCAATGGGCGCGTGGCTCCCACTTTGAACTACAAAGAGGGGGCATGAAGTTG

GAGCAGCATGAAGCCCAGTAAGAAAGGCCTGGGGTGGAGGAAGCGATGCTTCTGCTTCGTGCTACGGGGCCCTGTG TTAGGTCAGGGCTGCAGTAGCATCTGCAAAGGCAGGGCCCAGTTCCCCTGCTCCAGAAGCGTCCAAAAAGCTGTCT TGTCTCTCTAGAGCGGTTGTGGCTTAGAGGACTGGCCTGGCCCGATGCTAGAATATAGGAGCTTCAGACCTCCTGC

TGTAGTACACTACTCTCCCCAGACTGTCTAGGGCTCAGGGAGCTGCTGGCCTAGAAGTGGCACTTGGCTATTTCTT TTTCAAGAAGATAAAAATGTGAGGAAACCCATTCTATTTTATTGACTTCCCCCCTCCCCTGCCTGCTGGGTCTGTG GTCGATCCTGCTGGGAACCCCCCCAACCAAGGAATTGAAGGTGGTCTTCTTGGGCTAGCCCAAGCTACCATGCACT

TAGTCACAGGGCTATCTCTGACCAACAAAGCTGGCCTGGAAGACAGCAACTATGAAGGGAAGGATTCCAGAGCATG GGCTCAGGTCCCATGAGAGAGATTAGAGATGTCAAGCCATGGACCTGAACCTGGGTAGTTCAGCGCTACCCTGTCT GAGGCCCTGACTACTGCCTTTCCTTCCAGAGGGACTTGTTTGTTTGTTTGTTTGTGGTTTTTTTTTTCCCTGAGGT AAAATAAAATGAGCCACACTGTGTTTTAAATTTAAAAAA

1268

410 1343 1419 1495 1571 1647 1723 1799 1875 1951 2027 2066

189

I

II

MGS;%~T%~L~LA I I ~ C ~ ]~SN~QN~N" r&tA2 dogA2 ]NVI.~LVS~ I]~/~RD/~F C~ ~ ratAl MPPYI SAF Q ~ dogAl MPPAI SAFQA~G~ V I ~ L V S V ~ I~K%~QA~RD;~F~Z III ratA2 B dogA2 ratAl dogAl ~

v

~

-

~

~

A IR ~ N G I ~ K G ~

R

~

T

~

~

V

GI ~ ~ F I ~ A V

I~PRTY~I~

H

~

"-KDG ~

A ~ M ~

T C ~ G R V ~

I ~ I ~ P K E G ~ C ~ Q V ~

Q

D

w

~

QD

VI I~

v

V

VfzI,II~m~G~V~s- S ~ P Q K - Y ~ K I ~ ~ ~ ~

SC-~S I ~

VII

~~

; ~ S ~

~

T~k~S~V~F ~ I % ~ H

~ ~ D

I I ~ F

R~VLRRQE~QAGG; SAWALAAHSTEGEQVSLRLNGHPLGVWANGSATHSGRRPNGY R

C

Q

RS~VLRRRE~FKAGGT SARALAAHGSD GEQ ISLRLNGHP P GVWANG SAPHP ERRPNGY P ~ IDED LP EEKAED 326

~I~~~RCQP~VD~.DPP~.~.APHD el

~

VI

ratA2 dogA2 ratAl dogAl

ratA2 dogA2 ratAl dogAl

I ] ~ QTY~

~ ~ % ~ ~

IV

S~I~IAI B~NGI~G ~ ~

MST ~ F C A A C ~ t F ~ M ~

•

el

e e e e e

ee

e e e e e

•

326 •

•

•

l i e

•

e e e e e e e e e

ratA2 TLGLGGGGSAQGSPRDVELPTQE ...... RQEGQEHPGLRGHLVQARVGASSWSSEFAPS 410 dogA2 TLGLVSGGIAPESHGDMGLPDVELLSHELKGACPESPGLEGPLAQDGAGVS 412 Fig. 2. Alignment of the amino acid sequences of the rat A 2, dog A 2, rat A 1 and dog A l receptors 23"24"27"35. To maximize alignment, gaps (indicated by hyphens) were introduced. Amino acids common to all 4 receptors are enclosed in shaded boxes. The putative transmembrane domains are indicated by lines above the amino acid sequences and labeled by roman numerals. Amino acids in the rat A2 receptor sequence which differ from the dog A 2 receptor are indicated by a dot above the rat sequence. Putative glycosylation sites are indicated by open boxes.

the full length cDNA (clone DT-35) in pcDNA I. Dopamine D 2 receptor cDNA sense and antisense RNA probes were generated from a 702 bp region of the D 2 cDNA from the Sac I site at nucleotide 652 to nucleotide -505; this fragment contained 652 bp of coding region and 50 bp of 5'-untranslated sequence. Dopamine DI receptor cDNA sense and antisense RNA probes were generated from a 180 bp fragment corresponding to a region of coding sequence of the rat D 1 receptor cDNA from nucleotides 506 to 68631. D1 and D 2 receptor cDNAs were generated by PCR using specific primers and first-strand rat striatal cDNA as template and identity confirmed by sequencing.

matched and had silver grains in both adjacent sections were scored 'co-labeled'; nuclei which were matched but had silver grains in one field but not in the other field were scored 'not co-labeled'. With our present method 42% of neuronal nuclei (stained by Methylene blue) in one field could be identified in the adjacent section. RESULTS

Cloning o f the rat adenosine A 2 receptor Following

sequential

PCR

amplifications

using

rat

Co-localization in situ hybridization

b r a i n first s t r a n d c D N A as t e m p l a t e , a 210 b p f r a g m e n t

For co-localization of receptor mRNAs, camera lucida projections of fields from adjacent sections were projected onto paper using a Leitz Wetzlar projection microscope. All nuclei which were stained with Methylene blue (or thionin) were drawn in outline and scored when overlayed with silver grains (reference field). The identical field from the adjacent section (comparison field) was projected onto the drawing from the reference field. Nuclei which were

w a s i s o l a t e d w h i c h h a d 84% a m i n o acid i d e n t i t y in o n e r e a d i n g f r a m e to t h e p r e v i o u s l y i s o l a t e d c a n i n e ' o r p h a n ' r e c e p t o r RDC823. T w o o v e r l a p p i n g c D N A c l o n e s , D T - 3 5 a n d R P - 2 1 , w e r e i s o l a t e d u s i n g t h i s 210 b p R 8 c D N A f r a g m e n t as a p r o b e (Fig. l a ) . T h e d e d u c e d a m i n o acid

Fig. i. a: two overlapping cDNA clones which provided sequence information for the rat A 2 cDNA, The coding regions are represented by filled rectangles and the Y-untranslated (Y-UT) and 5'-untranslated (5'-UT) sequence by solid lines. The approximate position of the codon ATG corresponding to the initial methionine residue of the longest open reading frame is shown by an arrow. The cDNA clone DT-35 contained 3 bp of 5'-untranslated sequences, b: nucleotide sequence of the rat A 2 receptor cDNA representing the sequence information obtained from the cDNA clones RP-21 and DT-35. The deduced amino acid sequence of the open reading frame is shown below the nucleotide sequence. The nucleotide and amino acid numbering begin with the first methionine residue of the open reading frame and are shown to the right of each line. The stop codon is indicated by an asterisk (*). The polyadenylation signal sequence is underlined.

190

1

sequence of the longest open reading frame of the cDNA

2

3

4

5

predicted a protein of 410 amino acids (Fig. la). As with other G protein-linked receptors 33, the rat DT-35 receptor c D N A predicted a protein with seven t r a n s m e m b r a n e domains (Fig. 2). The high homology

a

2000

-

%z x

1500

% E

I

1000

v

B(fm011mg p

g °

I

2000

r

~

1000

o uD

03 CO 0

Bma× = 1,866 fmol/mg 500

T

Fig. 4. Northern blot analysis of A2 adenosine receptor mRNA m rat brain areas. Each lane contained 20 yg of total cellular RNA. The locations of the 18S and 28S ribosomal subunits is indicated by arrows (left) . Lanes: 1, striatum: 2, cerebellum; 3, hypothalamus: 4, midbrain: 5, cortex. Exposure, 16 h with i intensifying screen.

I

I

0

I

40

[3H]CGS

gl00-o= -~

"

I

I

80

-

21680

-

I

I

120

(nM)

i ) b

80-

a

o CO qC)

mLO

I

160

• NECA

A (R)-PIA • CPA

60 -

to

a (S)-PIA

-r"

,o

.2

40

CO

20-

0

i

0

i 9

l 8

17

6

5

4

i 3

-Log drug concentration (M)

Fig. 3. Expression of the rat A2 receptor in COS-6M cells transfected with the rat A2 receptor cDNA in pcDNA I assayed by [sH]CGS 21680 binding, a: saturation study showing specific (filled squares, I ) and non-specific binding (filled triangles, A) to transfected COS-6M membranes. Inset: Scatchard transformation of the specific binding data. Data points shown are the means of 3 separate experiments: in each experiment, each point was performed in triplicate. Computer analysis revealed the presence of a single class of high affinity binding sites (Kd = 38.6 nM; B..... = 1,866 fmol/mg protein), b: competition analysis of various adenosine ligands for [sH]CGS 21680 binding in transfected COS-6M cells: filled squares (I), NECA; triangles (A), (R)-PIA: circles (e), CPA: open squares (El), (S)-PIA. Ki values are listed in the text. Results are the means of 3 separate determinations. Each data point of each experiment was performed in triplicate.

(Fig. 2) between the predicted amino acid sequence of the canine RDC8 and rat DT-35 receptor cDNAs (overall identity 82%, with 96% identity in the t r a n s m e m b r a n e domains) suggested that DT-35 encoded the rat homoIogue of the canine receptor RDC8. The canine RDC8 receptor has recently been identified as an A2 subtype of adenosine receptor > and expression studies of rat DT-35 c D N A confirmed its identity as a rat A= subtype of adenosine receptor (see below). The predicted amino acid sequence of the rat A 2 receptor is also highly homologous to the rat AE and canine A~ receptors (Fig. 2) both overall (52% in both) and within the transmembrane domains (64% in both23"2427'35). As with the canine A=, rat adenosine A~ and canine adenosine A~ receptor cDNAs, the rat A 2 receptor c D N A has a very short N-terminus without consensus sites for glycosylation and lacks an asparate residue in the third transmembrane domain which is present in receptors which interact with cationic amines (Fig. 2). Despite the overall similarity in amino acid sequence between the canine and rat A2 receptors, the rat amino acid sequence diverges considerably from the canine sequence in the C-terminus of the protein and somewhat in a region between the putative fourth and fifth t r a n s m e m b r a n e domains (Fig. 2). The nucleotide sequence of the rat c D N A also predicts a different initial methionine residue than in the canine sequence (Figs. lb and 2), although the second methionine residue in the canine sequence is also present in a context favorable for translation initiation >. Expression o f the rat A 2 receptor c D N A Because the rat DT-35 clone was highly homologous

191 TABLE I Co-localization of A2, D 1 and sections

D2

receptor mRNAs in adjacent

A

* Mean + S.E.M. based on multiple fields analyzed. For each comparison the following number of separate fields were compared: A2 vs D 2 and A 2 vs Dl, 3 in the sagittal plane and 3 in the coronal plane; A2 vs A2 and D2 vs D 2, 2 sagittal and 2 coronal fields; D L vs D j, 3 sagittal and 2 coronal fields. Number of cells Co-labeled

Not co-labeled Total

%*

A2 vs D 1

182 1

15 239

197 240

93_+2 0

D 2 vs D2 D 1 vs D 1 A2 v s A 2

152 156 117

5 7 6

157 163 123

97+1 95_+1 96_+3

A 2 vs D 2

to the canine RDC8, which had recently been demonstrated to be an A 2 adenosine receptor 26, we tested whether the rat DT-35 c D N A encodes an A2 adenosine receptor. W h e n expressed in COS-6M cells the rat DT-35 c D N A in p c D N A I produced specific and saturable binding of the Az-selective agonist [3H]CGS 21680 (Fig. 3a). No specific binding was detected in COS-6M cells transfected with the p c D N A I vector alone (data not shown). Scatchard analysis of the binding data (Fig. 3a) revealed a single class of [3H]CGS 21680 binding sites (Hill coefficient = 0.986), with a dissociation constant (K0) of 38.6 nM and a Bmax of 1,866 fmol/mg protein. The ability of several adenosine agonists to compete for [3H]CGS 21680 binding (Fig. 3b) demonstrated that the non-selective adenosine agonist N E C A was the most potent (K i = 8.07 + 2.0 x 10-8 M). The A~-selective agonists N6-(R)-phenylisopropyladenosine [(R)-PIA] and N6-cyclopentyladenosine (CPA) were less potent (K i = 3.1 + 0.26 × 10-6 M and 1.0 + 1.0 × 10-5 M, respectively). The [3H]CGS 21680 binding sites on transfected COS-6M cells demonstrated more than 100-fold stereoselectivity for the (R)P I A e n a n t i o m e r compared with the N6-(S)-phenylisopropyladenosine [(S)-PIA] e n a n t i o m e r (K i > 10 -4 M). The rank order of agonist potency ( N E C A > ( R ) - P I A > C P A > (S)-PIA) is characteristic of the A 2 adenosine receptor subtype 4'16'24'43. Regional brain dbtribution o f rat A 2 receptor m R N A

Northern blot analysis of A 2 receptor m R N A in several brain regions demonstrated a b u n d a n t expression of a specific 2.1 kb band in rat striatum (Fig. 4). A faint A 2 m R N A band was also detected at longer exposures in cortex and midbrain, but virtually no A 2 m R N A was detected in hypothalamus or cerebellum (Fig. 4). In situ hybridization to brain confirmed the a b u n d a n t and ex-

Fig. 5. Film autoradiograms showing the brain distribution of A 2 , D2 and D 1 receptor mRNAs by in situ hybridization using [3SS]antisense RNA probes. Sagittal sections of a rat brain are shown. Hybridizations using the sense A2 receptor probe produced no specific labeling, cp, caudate-putamen; Acb, nucleus accumbens; OT, olfactory tubercle; SN, substantia nigra; pit, pituitary; ic, inferior colliculus.

clusive expression of A 2 receptor m R N A in striatum (Fig. 5). A 2 m R N A was present throughout the entire caudate-putamen at all anteroposterior levels, in the nucleus accumbens and in the olfactory tubercle. Expression of A2 m R N A in caudate-putamen and nucleus accumbens/olfactory tubercle was virtually indistinguishable from D 1 and D 2 receptor m R N A s in topographical pattern determined by in situ hybridization (Fig. 5) and in a m o u n t determined by Northern Blot (Fink, J.S., un-

192

Fig. 6. Bright-field emulsion autoradiograms demonstrating co-expression of A 2 and D 2 receptor mRNAs in the same ceils in adjacent sections of rat caudate-putamen. Cells with nuclei visible in both sections are circled. Cells expressing both A 2 and D 2 receptor mRNA are indicated by arrows.

published). As has been observed previously 28, D 2 r e m R N A was present in other brain areas including the pituitary, ventral midbrain, tectum, globus pallidus, and cortex. No cells expressing A 2 receptor m R N A were detected in these brain areas. Hybridizations performed using a sense A 2 R N A probe showed no specific expression in brain (Fig. 5).

due to cross-hybridization.

ceptor

Cellular distribution of rat A2 receptor m R N A in caudate-putamen Expression of D 1 and D 2 receptor m R N A s within caudate-putamen is segregated in different cell populations 13'z°'22. Because the A 2 receptor m R N A was abundantly and exclusively expressed within the rat caudateputamen, we determined whether A 2 receptor m R N A was co-expressed with D~ and/or D2 receptor mRNAs. When adjacent sections were hybridized with A 2 and D~ c R N A probes, o r A 2 and D z c R N A probes, A 2 receptor m R N A was virtually exclusively co-expressed in cells expressing the D 2 receptor m R N A (Table I; Fig. 6). When pairs of adjacent sections were hybridized to D 2 and A2 probes, 93% of the sampled striatal neurons were labeled in both sections. However, D~ and A 2 probes never labeled the same neurons in adjacent sections (Table I). When pairs of adjacent sections were hybridized with the same probe, the same neurons were labeled in both sections 95-97% of the time (Table I). Colocalization of the A 2 and D 2 receptor m R N A s was not observed in other D2 receptor-expressing sites (pituitary, cortex, tectum, ventral midbrain) in the same sections, demonstrating that A2-D 2 m R N A colocalization is site-specific and not

DISCUSSION Several lines of evidence indicate that the rat DT-35 c D N A encodes an A 2 adenosine receptor. The overall predicted amino acid sequence of the rat DT-35 c D N A is highly homologous (80% identity) to the canine A 2 r ec e p t o r RDC8, particularly in the transmembrane domains (95% identity). DT-35 is also highly homologous to both the rat and canine A 1 receptors (Fig. 2). The brain distribution of the rat DT-35 receptor m R N A is similar to that of the canine A 2 receptor RDC8 m R N A 3v and to the distribution of A 2 receptors visualized in rat brain by receptor binding autoradiography using the A 2selective ligand [3H]CGS 21680 is. Expression of the rat DT-35 c D N A in COS-6M cells resulted in the transient expression of high affinity binding sites for [3H]CGS 21680 and an agonist potency profile at this binding site similar to the A 2 receptor in rat striatal membranes 1624. Finally, the rat DT-35 c D N A is coupled positively to the generation of c A M P when stably expressed in C H O cells (Pollack, A.E., Peterfreund, R.A. and Fink, J.S., unpublished observations). The rat A2 receptor shares structural features with the canine A2, rat A 1 and canine A~ receptors 23'24"27"35 suggesting that they are members of a subfamily of G protein-linked receptors with features different from those of monoamine, muscarinic or peptide receptors (Fig. 2). The rat A2 receptor has a short amino terminus lacking N-glycosylation acceptor sites, contains a short third cy-

193 toplasmic loop (which may participate in G protein interactions) and lacks an aspartic acid residue in the third transmembrane domain which is present in all adrenergic, dopaminergic, serotonergic and muscarinic receptors 33'41. Like the canine A2 receptor, the rat A 2 receptor has a longer C-terminus than the A 1 receptor from the same species 35 and contains multiple threonine and serine residues (a total of 19 in the rat A 2 receptor and 11 in the canine A2 receptor) in the C-terminus which are potential phosphorylation sites that could be important in receptor regulation 39'41. There are several differences between the rat A 2 and canine A2 cDNAs that deserve mention. First, the rat A 2 receptor cDNA contains only one potential initial methionine residue, whereas the canine A 2 receptor contains t w o 19. Second, the predicted amino acid sequence in the rat A2 receptor diverges from the canine A2 receptor in a region between the fourth and fifth transmembrane domains (second exofacial loop) and in the C-terminus (Fig. 2). We have confirmed these sequence differences in the rat A 2 cDNA in the N-terminus, the second exofacial loop and C-terminus in two independently isolated cDNAs (Fink, J.S., unpublished observations). The potential glycosylation sites in the second exofacial loop are preserved despite these inter-species sequence differences (Fig. 2). The species divergence in the C-terminus is particularly striking. However, many of the amino acid differences between canine and rat A 2 receptor in the C-terminus are conservative substitutions. Indeed, if conservative substitutions are included, the overall amino acid identity between canine and rat A 2 receptors is 95%, suggesting a single molecular form of A 2 striatal receptor. The exclusive expression of A 2 m R N A in the rat striatum is consistent with receptor binding autoradiography and lesion studies which have suggested synthesis of A 2 receptors within neurons intrinsic to the striatum 1'29. The brain distribution of r a t A 2 m R N A is also consistent with the distribution of canine A 2 r e c e p t o r m R N A 37. The localized expression of A2 m R N A and receptor protein within striatum provides an anatomical basis for the effects of adenosine agonists on behavior and the interactions between adenosinergic agonists and antagonists on dopaminergic stimulation of motor behavior. Ade-, nosine agonists act like neuroleptic drugs without interacting directly with dopamine receptors and attenuate the behavioral effects of D A agonists, while adenosine antagonists enhance the behavioral effects of dopamine agonists 3,7,8,9,14,17,32,40. Expression of rat A 2 receptor m R N A within D 2 receptor-expressing neurons is segregated to a subset of intrinsic striatal neurons in the rat caudate-putamen. Virtually all neurons within the rat caudate-putamen

which express A 2 receptor m R N A also express D 2 receptor m R N A , but never express D 1 m R N A (Table I). We are not able to exclude the possibility that there may be a small population of neurons which co-express low levels of A 2 and D~ m R N A which might be below the limits of detection in these cells using in situ hybridization. J While this manuscript was in preparation, Schiffman et a138 demonstrated that A 2 receptor m R N A in rat striatum, identified using oligonucleotides based on the canine A 2 receptor nucleotide sequence, was exclusively co-expressed in a subset of striatal neurons expressing enkephalin (ENK) m R N A , but never expressed in cells expressing substance P (SP) mRNA. Because all ENK mRNA-containing neurons express D 2 receptors 2°, and SP-expressing neurons are largely Dl-expressing 22, the observations of A2-ENK co-expression 38 are entirely consistent and complementary to our findings of DE-A 2 co-localization. Recent pharmacological evidence suggests that the interaction between A 2 adenosinergic analogs and dopaminergic agonists on locomotor behavior occurs by postsynaptic interaction with D E receptors 3'8'9. The coexpression of A 2 and D E mRNAs in the same striatal cells identifies a subset of striatal cells which may be particularly important for this adenosinergic-dopaminergic behavioral interaction. Expression of D E receptors is also segregated to a subpopulation of medium-sized striatal neurons which project to the globus pallidus in the rat la. In the rat this 'indirect' striatal efferent pathway involves the globus pallidus, subthalamic nucleus and substantia nigra reticulata/entopeduncular nucleus. Taken together with evidence for co-expression of A 2 adenosine receptors with both D E receptor m R N A (this report) and ENK as, these data suggest that A 2 receptors are co-expressed in an ENK-D 2 receptor-positive subset of medium-sized neurons projecting to the globus pallidus. This conclusion is supported by the presence of low levels of A 2 receptor binding in the globus p a l l i d u s 18'29. This striatopallidal pathway may be the striatal efferent system which is selectively regulated by A2-D 2 receptor interaction. However, segregation of A 2 receptor-expressing cells within the striatopallidal pathway (and their absence in the D~-containing striatonigral pathway) as well as regulation of the activity of the striatopallidal pathway by A 2 analogs awaits direct demonstration. Adenosine A 2 receptors do not appear to be expressed in the large, cholinergic neurons of the striatum as which are known to express D E receptors 2'21. This suggests that the large cholinergic striatal neurons are a subset of D 2p o s i t i v e neurons which do not express A 2 mRNA. Because the number of cholinergic neurons in the striatum is small, this population could account for the slightly

194 smaller percentage mRNA

of cells c o - e x p r e s s i n g A 2 a n d D 2

W h e t h e r t h e c e l l u l a r m e c h a n i s m of i n t e r a c t i o n b e t w e e n

(93 + 2%) t h a n t h e p e r c e n t a g e c o - e x p r e s s i n g D 2

A 2 a n d D 2 r e c e p t o r s w i t h i n single s t r i a t a l cells is by an-

o r A 2 w h e n t h e s a m e p r o b e is u s e d o n a d j a c e n t s e c t i o n s

t a g o n i s t i c effects o n a d e n y l y l cyclase, by t h e effects of

(97 _ 1% a n d 96 _+ 3%, r e s p e c t i v e l y ; T a b l e I). T h e i s o l a t i o n of t h e r a t A 2 r e c e p t o r c D N A

a d e n o s i n e r e c e p t o r a c t i v a t i o n o n t h e affinity of t h e D 2 h a s per-

mitted identification of the subtype of striatal neuron expressing A 2 receptor mRNA.

r e c e p t o r for D A m or by s o m e o t h e r m e c h a n i s m r e m a i n s to b e d e t e r m i n e d .

T h e exclusive co-local-

i z a t i o n of t h e A 2 r e c e p t o r w i t h i n t h e D2 r e c e p t o r - e x -

be achieved.

Acknowledgement,s. The authors thank Frederick Huntress, Jim Deeds and Monica Drozd for expert technical assistance. The authors also appreciate the willingness of Dr. G. Vassart to share data prior to publication. The studies were supported by grants from the National Institutes of Health (DK42125, HD00924), the National Science Foundation (BNS 89-08542), The Tourette Syndrome Association, The National Parkinson Foundation, The Scottish Rite Foundation for Research in Schizophrenia and a Post-doctoral Fellowship from the The United Parkinson Foundation (A.E.P.).

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pressing, enkephalinergic striatal neurons provides an a n a t o m i c b a s i s f o r t h e k n o w n b e h a v i o r a l i n t e r a c t i o n bet w e e n t h e a d e n o s i n e r g i c a n d d o p a m i n e r g i c s y s t e m s in the s t r i a t u m . T h i s i n f o r m a t i o n f u r t h e r s u g g e s t s t h a t t h e A2 r e c e p t o r is a site w h e r e s e l e c t i v e r e g u l a t i o n o f activity in t h e s t r i a t o p a l l i d a l

pathway

could

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