RFL9 Encodes Receptor

Scott

A. Rivkees*

and

Steven

an A*,-Adenosine

M. Reppert

Laboratory of Developmental Chronobiology Massachusetts General Hospital and Harvard Medical School Boston, Massachusetts 02114

We recently reported the cloning of a cDNA (designated RFLS) that encodes a novel A,-adenosine receptor subtype. We now fully characterize the pharmacological properties of RFL9 in stably transfected CHO cells by examining CAMP responses to drug treatments. The pharmacological profile of CAMP responses in RFLS-transfected cells was similar to that expected for A,,-adenosine receptors and distinct from that of CHO cells transfected with the A,,-adenosine receptor. When RFLS-transfected cells were compared with VA 13 fibroblasts, the human cell line in which endogenous A,,-adenosine receptors were originally characterized, the doseresponse curves of CAMP responses to drug treatments were highly correlated. Northern blot analysis of RNA prepared from VA 13 fibroblasts revealed specific hybridizing transcripts when probed for RFLS, but no hybridizing signal for Aa.-adenosine receptor mRNA. Using degenerate oligonucleotide primers designed to detect adenosine receptors by the polymerase chain reaction, only one cDNA fragment homologous to the rat A,,-adenosine receptor was isolated from VA 13 cells. These results strongly suggest that RFL9 encodes the proposed A,,-adenosine receptor subtype. The identification of the cDNA for an A,,-adenosine receptor will allow more rigorous characterization of its anatomical distribution and functional properties. (Molecular Endocrinology 6: 1596-1604,1992)

Since adenosine is present in all tissues (1, 2), understanding the receptor-mediated mechanisms by which adenosine acts has been difficult. However, with the cloning of the cDNAs encoding the A,- and Azareceptors (6-12), the importance of a molecular approach for dissecting adenosine’s action has become apparent. Sites of A,- and A,,-adenosine receptor gene expression have now been defined using molecular methods (9, 10, 12). The isolation of novel adenosine receptor subtypes is also more clearly feasible using sequence information of the cloned receptors (13). Recently, we cloned a novel adenosine receptor cDNA from rat brain, designated RFLS, that had a tissue distribution distinct from that of A,- or A*,-receptors (14). RFL9 encodes an A,-adenosine receptor subtype, because it is most highly homologous to the A,,-adenosine receptor (73% amino acid identity in the putative transmembrane regions), and when expressed, it couples positively to adenylyl cyclase (14). However, due to the presence of low levels of endogenous RFL9 in the acute expression system used in our initial studies (COS 6M cells), it was not possible to further characterize the pharmacological properties of the receptor (14). In this report, we functionally characterize RFL9 in stably transfected CHO cells by examining CAMP responses to drug treatments. Our results strongly suggest that RFL9 encodes the proposed Azb-adenosine receptor subtype.

INTRODUCTION

RESULTS Generation

The nucleoside adenosine acts through cell surface receptors to influence a wide variety of physiological processes (1, 2). Based on pharmacological and functional properties, adenosine receptors have been divided into two major types, A,-adenosine receptors which inhibit adenylyl cyclase and A,-receptors which stimulate adenylyl cyclase (2, 3, 4). A,-Adenosine receptors are further divided into Aza- and A,,-subtypes based on pharmacological criteria (5). 0888-8809/92/l 596-l 604$03.00/O Molecular Endocmology CopyrIght 0 1992 by The Endocrine

of Stable Cell Lines

CHO cells were identified as a favorable cell line for transfection with adenosine receptors for functional studies. Neither 5’Wethylcarboxamidoadenosine (NECA; 1 mM) nor adenosine (1 mM) induced CAMP accumulation in nontransfected CHO cells (n = 3). Furthermore, NECA (1 mM) did not inhibit forskolin (100 PM)-stimulated CAMP accumulation in CHO cells (n = 2). The mRNAs encoding RFL9 or the A,- and Apaadenosine receptors were also not detected in nontransfected cells by dot-blot analysis (n = 3). It is noteworthy that although CHO cells did not express

Society

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RFL9 is an A,,-Adenosine

Receptor

functional adenosine receptors, a significant level of [3H]NECA binding (100 + 23 fmol/mg) was present in nontransfected cells, precluding further ligand binding studies in transfected CHO cells with [3H]NECA. After transfection of CHO cells with RFLS, four clones expressing RFL9 mRNA were identified by dot-blot analysis; NECA (1 PM) stimulated CAMP accumulation in each clone. One clone was arbitrarily selected for subsequent CAMP studies. After transfection of CHO cells with the A2,-adenosine receptor cDNA, three clones expressing A,,-adenosine receptor mRNA were identified by dot-blot analysis; NECA (1 PM) stimulated CAMP accumulation in each of these clones. One clone was arbitrarily selected for subsequent CAMP studies. CAMP Studies: Comparison Adenosine Receptors

Comparison

60

of RFL9 and Aza60

To compare the pharmacological properties of RFL9 with A,,-adenosine receptors, CAMP responses to drug treatments were assessed in CHO cells transfected with either RFL9 or the rat A,,-adenosine receptor cDNA. Both cell lines were treated with drugs in parallel, and CAMP determinations were performed in the same assay run. Cells were used from passages l-l 2, and responses were constant for the duration of the study (Fig. 1 and Table 1). Dose-response curves were initially generated using eight adenosine agonists selected for their ability to distinguish among adenosine receptor subtypes (15). A different rank order of potency for drugs was clearly apparent between the two cell lines. Each agonist more potently stimulated CAMP accumulation in the cells expressing AZ,-adenosine receptors than in those expressing RFLS. The rank order of potency for stimulating CAMP accumulation in cells stably transfected with the Apaadenosine receptor was similar to that expected for Ap,-adenosine receptors (Fig. 1 and Table 1); the ECso values were similar to the inhibition constant values reported for ligand binding of A,,-adenosine receptors (5). The rank order of potency for stimulating CAMP accumulation in cells transfected with RFL9 was similar to that expected for endogenous A,,-adenosine receptors (5, 16, 17); the ECso values were also similar to those reported for A,,-adenosine receptors (5, 21, 22). Since A,,-adenosine receptors have higher affinity for adenosine antagonists than A2,-adenosine receptors (5) the potency of three antagonists to block adenosine-stimulated CAMP accumulation was examined next. In three separate studies, each antagonist more potently inhibited adenosine-stimulated CAMP accumulation in cells transfected with RFL9 than in those transfected with the AZ,-adenosine receptor (Fig. 2 and Table 2). CAMP Studies: Cells

1599

of RFL9 and VA 13

Azb-adenosine receptors were initially characterized in VA 13 cells by Bruns (16, 17) who examined CAMP

80 60 -

NCCA m ADO

CPA

_ .

CGS 4 ” IO

a

I. 6

I 4

“I 2

-log [Drug], M Fig. 1. CAMP Dose-Response Curves for Adenosine Agonists in CHO Cells Stably Transfected with the A,,-Adenosine Receptor (Upper), RFL9 (Middle), and VA 13 Cells (Lower) Each point represents the mean of two or three separate dose-response studies and is expressed as a percentage of the maximal CAMP response. The data in the upper panel are from studies in which A,,-receptor and RFLS-transfected cells were directly compared. The data in the lower two panels are from the studies in which RFL9 and VA 13 cells were directly compared. CGS, CGS-21680; CPA, NG-cyclopentyladenosine; ADO, adenosine; NECA, 5’-N-ethylcarboxamidoadenosine.

responses to adenosine analogs. We therefore directly compared CAMP responses to adenosine analogs in CHO cells stably expressing RFL9 with those in VA 13 cells. For each of the six adenosine agonists and three adenosine antagonists examined, the ECso and IGo values were very similar between CHO cells stably expressing RFL9 and VA 13 cells (Figs. 1 and 2; Tables 1 and 2). When the EC& and I& values obtained from cells transfected with RFL9 were compared by regres-

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MOL 1600

ENDO.

Table

1. Agonist

Vo16No.10

Studies:

E&

Values

for Stimulation

of CAMP

Accumulation Ratio

Cell line

Compound

CHO:FiFLS” ECsn

CHO:A,=

S-PIA NECA CPCA CPA R-PIA ADO CAD0 CGS-21680

8.8 6.3 9.9 1 .l 1 .l 1.3 5.5 8.0

f k f + + + + f

9.9 2.2 9.0 0.3 0.3 0.3 1.4 1.8

1 .l 1.2 6.4 6.6 9.1 1 .O 9.3 1.4

E-06 E-08 E-08 E-06 E-06 E-07 E-07 E-08

+ zk + + + + + +

0.3 1.7 4.7 2.0 7.0 0.6 3.5 0.1

VA 13’

E-05 E-06 E-06 E-05 E-04 E-05 E-05 E-04

6.7 + 8.7 E-05 7.8 + 2.6 E-06 1.3 + 2.0 E-04 1.5 f 1.4 E-04 1.4 f 0.6 E-05 6.4 f 0.4 E-05

RFL9 A,.”

RFL9 VA1 3’

10 16 64 90 106 132 170 18600

0.16 0.13 0.56 6.2 0.72 2.1

S-PIA, N6-(S)phenylisopropyladenosine; NECA, 5’-A!-ethylcarboxamidoadenosine; CPCA, 5’-cyclopropylcarboxamidoadenosine; CPA, cyclopentyladenosine; R-PIA, N’-(R)phenylisopropyladenosine; ADO, adenosine; CADO, 2-chloroadenosine; CGS-21680, 2[4-(2-carboxyethyl)phenylethylamino]-5’-N-ethylcarboxamidoadenosine. All values are mean f SEM. a From studies in which CHO cells stably transfected with RFL9 were compared with CHO cells stably transfected with the A,,adenosine receptor. b From studies in which CHO cells stably transfected with RFL9 were compared with VA 13 cells.

sion analysis with those of VA 13 cells, a highly significant correlation was apparent (Fig. 3). On the other hand, there was no correlation when the EC,, and lCsO values obtained from cells transfected with the A,,adenosine receptor were compared with those of VA 13 cells (Fig. 3). Importantly, the E& and I&,, values for RFLS-transfected cells in this series of studies were very similar to those obtained when RFLS-transfected cells were directly compared with CHO cells stably transfected with the A,,-adenosine receptor (see above).

RFL9 Gene Expression

in VA 13 Fibroblasts

RFL9 and A,,-adenosine receptor gene expression in VA 13 fibroblasts were assessed next. Northern blot analysis of 20 pg poly(A)’ RNA prepared from VA 13 fibroblasts revealed hybridizing transcripts of 1.8 and 2.4 kilobases when probed with the full-length RFL9 cDNA (Fig. 4). When the blot was stripped and reprobed with a full-length A,,-adenosine receptor probe, a hybridization signal was not apparent after a similar exposure time (data not shown). When the blot was reprobed with RFLS, clear hybridizing bands were again observed (data not shown). Polymerase Chain Reaction Analysis Receptor Subtypes in VA 13 Cells

of Adenosine

To examine if multiple RFL9 receptor subtypes were present in VA 13 cells the polymerase chain reaction (PCR) was used. First strand cDNA was prepared from mRNA obtained from VA 13 cells and subjected to PCR amplification using a set of degenerate oligonucleotide primers. The primers were based on regions of the fifth and sixth transmembrane domains that are conserved among the rat A,- and An,-adenosine receptor cDNAs and RFLS. A total of 30 recombinant clones were isolated and sequenced. Fragments of three adenosine receptor cDNAs were identified. One fragment [168

base pairs (bp)] was 93% identical (at the nucleotide level) to the corresponding segment of the rat A,adenosine receptor and thus appears to be a fragment of the human A,-adenosine receptor. A second fragment (174 bp) was 91% identical to the corresponding segment of the rat A,,-adenosine receptor and thus appears to be a fragment of the human A,,-adenosine receptor. A third fragment (162 bp) was 85% identical to the corresponding segment of the rat RFL9 and thus appears to be the human homolog of RFLS. No other adenosine receptor-like fragments were present among the clones. These data suggest that only one RFL9 receptor subtype is present in VA 13 cells.

DISCUSSION More than 10 yr have passed since Bruns (16, 17) provided evidence for a low affinity adenosine receptor subtype by characterizing the functional responses of VA 13 cells to adenosine analogs. Since those early observations, this receptor subtype, later designated as Apb (5) has received scant attention. Its direct characterization has been hampered considerably by the lack of selective radioligands. The molecular cloning of RFL9 (14) and its identification as an Anb-adenosine receptor now provides direct evidence for the existence of an An!,-adenosine receptor. Taken together, three lines of evidence suggest that RFL9 encodes an A,,-adenosine receptor. First, RFL9 displays lower affinity for adenosine agonists and higher affinity for adenosine antagonists than the A,,-adenosine receptor. The marked preference for antagonists and the lower affinity for agonists are fundamental features that distinguish A2a- from A,,-adenosine receptors (5). In addition, CHO cells stably transfected with RFL9 have very low affinity for the A,,-receptor-selective ligand CGS-21680 (18) and very high affinity for the adenosine antagonist 1,3-diethyl-8-phenylxanthine (DPX); DPX is relatively selective for A,,-receptors (5).

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RFL9

is an A,,-Adenosine

Receptor

1601

60

IO

8

6

4

2

IO

8

6

4

2

60

-log IDrug1.M Fig. 2. CAMP Dose-Response Curves for Adenosine Antagonists from CHO Cells Stably Transfected with the APa Adenosine Receptor (Upper), RFL9 (MicWe), and VA 13 Cells (Lower) Drugs were dissolved in medium containing 10 PM adenosine. Each point represents the mean of three separate doseresponse studies and is expressed as a percentage of the CAMP response induced by 10 PM adenosine. The data in the upper panel are from studies in which APa and RFL9 were directly compared. The data in the lower two panels are from the studies in which RFL9 and VA 13 cells were directly compared. AM, Aminophylline; DPX, 1,3-diethyl-8-phenylxanthine; XAC, xanthine amine congener.

The second line of evidence is that CHO cells stably transfected with RFL9 and nontransfected VA 13 fibroblasts exhibit very similar dose-response curves for several adenosine agonists and antagonists. The slight differences in drug potencies that were present between the two cell lines may reflect factors related to different cell types, or possibly species-related differences in A*,-receptors; RFL9 is a rat clone, whereas VA 13 cells are of human origin. It is also possible that

more than one A,,-like adenosine receptors are expressed in VA 13 cells. However, using PCR we did not find evidence of more than one RFLS-like receptor transcript in VA 13 cells. It is important to note that the necessity of a functional approach in characterizing RFL9 underscores the inherent difficulties in studying A,,-receptors. A,,-selective radioligands are not available. Thus we examined receptor binding using the nonselective adenosine agonist [3H]NECA. However, at the concentration needed to label AZb receptors, a high degree of apparent nonadenosine receptor [3H]NECA binding (19) was present in nontransfected CHO cells. The final line of evidence suggesting that RFL9 encodes an An,-receptor is that the RFL9 gene is expressed in VA 13 fibroblasts, as expected if it indeed encodes an A,,-adenosine receptor. We have also detected RFL9 mRNA in other cell lines, including murine NIH 3T3 cells and monkey kidney COS-GM cells (data not shown); it is believed that A,,-adenosine receptors are expressed in many fibroblast cell lines (20). When the structural features of Apa- and A,,-adenosine receptors (Fig. 5) are compared, several distinctive features are apparent. Both receptors have a short amino terminus, and glycosylation sites are only found in the second exofacial loop. The most prominent difference between the two receptors is the length of the carboxyl tail. The A,,-receptor carboxyl terminus is 100 amino acids long (12) whereas that of the A,,-receptor is 41 amino acids long (14). However, despite this marked difference in the length of the carboxyl tails, both receptors couple positively to adenylyl cyclase. The carboxyl terminus of each receptor is rich in serine and threonine residues, which may represent phosphorylation sites involved in desensitization mechanisms (21, 22). A,,-adenosine receptors have been shown to rapidly desensitize after agonist treatment (23). Before the examination of RFL9 mRNA expression by Northern blot analysis, the distribution of A,,-adenosine receptors was unclear. Functionally defined A2,,adenosine receptor had been identified in fibroblasts (16, 17), cardiac cells (24) and possibly in brain (25). Northern blot analysis, however, reveals that An!,-adenosine receptors are expressed in more tissues than previously appreciated. In rats, RFL9 mRNA is most heavily expressed in caecum, large bowel, and bladder (14). Lower levels of RFL9 mRNA are apparent in lung, thymus, spleen, epididymus plus vas deferens, and retroperitoneal white adipose tissue (14). This pattern of tissue expression is very distinct from that of A,- and A,,-adenosine receptors (10, 14). We anticipate that identification of the cDNA for an Anb-adenosine receptor will allow more rigorous characterization of its properties. The identification of tissue sources expressing high levels of An,,-receptor mRNA may now allow further pharmacological and functional characterization of endogenous A,,-adenosine receptors. The cellular localization of A,,-adenosine receptors

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MOL 1602

ENDO.

1992

Table

2. Antaaonist

Vo16No.10

Studies

I&

Values

for Inhibition

of Adenosine-Stimulated

CAMP

Accumulation

Cell line

DPX XAC AM

Ratio

CHO:A,,”

CHO:RFL9’ IC50

VA 13’

1.2 * 0.3 E-03 1 .O + 0.1 E-05 1 .S + 0.4 E-04

6.3 f 9.9 E-07 6.8 f 3.8 E-08 5.1 * 2.1 E-06

5.3 + 3.1 E-08 2.0 + 0.5 E-07 2.1 + 0.6 E-06

RFL9 AS,= 0.00024 0.00068 0.0026

DPX, 1,3-Diethyl-8-phenylxanthine; XAC, xanthine amine congener; AM, aminophylline. All values are mean c SEM. a From studies in which CHO cells stably transfected with RFLS were compared with CHO cells stably transfected receptor. b From studies in which CHO cells stably transfected with RFLS were compared with VA 13 cells.

within these the physiology

tissues should of adenosine

MATERIALS Cell

Culture

provide action.

new

insights

into

AND METHODS Techniques

Chinese hamster ovarian cells (ATCC, Bethesda, MD; CAL 61, batch F-91 77; referred to as CHO) were grown as monolayers in Ham’s F12 media supplemented with 10% fetal bovine serum (Sigma Chemical Co., St. Louis, MO), penicillin (100 U/ ml), and streptomycin (100 pg/ml). WI-38 VA 13 subline 2RCA cells (ATCC; CCL75.1, batch F-7346; referred to as VA 13) an SV40 virus-transfected human lung fibroblast cell line, were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented as above with serum and antibiotics. All cells were grown in a humidified atmosphere of 5% CO, at 37 C. Generation

of Stable

Cell Lines

The rat AZa (12) and RFLS cDNAs were cloned into the eukaryotic expression vector ~cDNA~/NEO (InVitrogen, San Diego, CA) and introduced into CHO cells using Lipofectin (GIBCO/BRL, Baltimore, MD) (26). Stably transfected cells were isolated and subsequently grown in the presence of Geneticin (0.5 mg/ml; GIBCO/BRL). Colonies expressing AZaadenosine receptor or RFLS mRNAs were identified by dotblot analysis (27) using full-length probes labeled with 32P by the method of random priming (28). Cytoplasmic RNA was extracted from cells after NP-40 lysis, as described (27). Studies

of CAMP

Accumulation

Cells were grown for 2 days in 24-well plates in tissue culture medium that did not contain Geneticin. When cells were confluent (50,000 cells per well), treatments were administered and CAMP accumulation assessed. Cells were washed once with DMEM and incubated with or without drugs (diluted in DMEM) for 10 min at 37 C. For antagonist studies, drugs were diluted in DMEM that contained 10 FM adenosine, similar to conditions used by Bruns et a/. (5) to assess the potency of adenosine antagonists. At the end of the treatment period, the medium was aspirated, and 0.25 ml 50 mM acetic acid was added. Cells were then scraped from the plates, transferred to an Eppendorf tube, and boiled for 5 min. After centrifugation (10,000 x g, 10 min), the supernatant was collected and stored at -80 C. The CAMP concentration in each sample was measured in duplicate by RIA (NEK-033, New England Nuclear, Boston, MA). At least two separate dose-response studies were performed for each drug per experiment. Each drug concentration was tested in duplicate in each dose-response study, and mean CAMP levels were calculated. For agonist studies, CAMP

RFL9 VA13’ 0.11 0.34 2.3 with the A,,-

values were expressed as a percentage of the maximal CAMP response, which was defined by a forskolin (100 PM) treatment group included in each run. For antagonist studies, CAMP values were expressed as a percentage of the CAMP responses to a lo-PM adenosine treatment group which was included in each run. The data (as percent values) from separate studies of the same drug were pooled, and ECso values (for agonists; half-maximally effective concentration) and I&,, values (for antagonists; half-maximally inhibitory concentration) were calculated using the EBDA/LIGAND nonlinear regression program which calculated mean + SEM values (29). Northern

Blot

Analysis

Total cellular RNA was isolated from 10’ cells using the guanidium-thiocyanate method, and poly(A)+ RNA was isolated using oligo-dT (30). Twenty micrograms of poly(A)+ RNA were fractionated on a 1% agarose-formaldehyde gel and transferred to a nylon membrane (Gene Screen, New England Nuclear). Blots were hybridized with RFLS or the rat AZareceptor cDNA 32P-labeled by the method of random priming (SA >l O9 cpm/ug). Hybridization reactions were performed at 42 C in 50% formamide, 1 M sodium chloride, 1% sodium dodecyl sulfate (SDS), 10% dextran sulfate, and denatured salmon sperm (100 pg/ml) at 42 C overnight. The final wash of blots was 0.2x SSC (1 x SSC = 0.15 M NaCI, 0.015 M Na citrate, pH 7.0) and 0.1% SDS at 65 C for 40 min. Blots were exposed to x-ray film with an intensifying screen at -80 C for three days. Before repeated probing, blots were boiled for 15 min in 0.1 x SSC containing 1% SDS. PCR Poly(A)+ RNA was prepared from VA 13 cells using established methods. Two micrograms of the RNA were primed with oligo(dT) and reverse transcribed with avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI). The first strand cDNA was subjected to two rounds of 30 cycles each of PCR amplification with 1 pg primer A [TCAGAATTCTA(TC)ATGGTlTACT(AT)(CT)AA(CT)TT(CT)TT] and primer B [TTCAAGCTTGGIA(AG)CCA(AG)(CG)(AT)IA(AG)IGC(AG)AA]. Each reaction cycle consisted of incubations at 94 C for 1.5 min, 45 C for 2 min, and 72 C for 2 min with Ampli Taq DNA polymerase (Perkin Elmer Cetus, Norwalk, CT). The amplified DNA was digested with HindIll and EcoRl and separated on an agarose gel. A prominent DNA band of approximately 220 bp was apparent which was electroeluted onto NA-45 paper (Schleicher & Schuell, Keene, NH) and eluted into 1 M sodium chloride. The digested DNA was then phenol-extracted, ethanol-precipitated, and subcloned into Ml 3 mp18 (GIBCO-BRL, Bethesda, MD). Recombinant clones were sequenced by the Sanger dideoxynucleotide chain termination method (31) using Sequenase (United States Biochemical, Cleveland, OH).

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RFL9

is an Apb-Adenosine

1603

Receptor

Kb 3

7.5

-

44 7

I

L

P

7

5

6

4

I

I

3

2

1.4 -

*

Fig. 4. Expression of the Poly(A)+ RNA (20 pg) RFL9 cDNA probe. Dark zation signals. RNA size the left column.

AM

*

X2

S-PIA

.CPA

8

7

0’ CPCA

6

eR-PIA

CADO’

ADO

WA0

RFL9 Gene in VA 13 Cells was hybridized with a 32P-labeled bands represent the specific hybridimarkers (GIBCO/BRL) are shown in

5

0 CGS

4

3

2

-log IDrugl, M RFL9 Fig. 3. Correlation between CAMP Dose-Response Studies Correlation between CAMP dose-response studies in which CHO ceils expressing RFL9 were compared with VA 13 cells (upper panel, r = 0.88, P < 0.001) and from studies in which cells expressing RFL9 or Aaa adenosine receptors were compared (lower panel, r = 0.11, P > 0.05). Values are E& and I& values for adenosine agonists and antagonists, respectively.

Acknowledgments The authors assistance.

thank

J. Deeds

and J. Lee for expert

Fig. 5. Structural comparison of the Rat Apa- and APb-Adenosine Receptors Depicted is the AZb-adenosine receptor. Y, Potential N-linked glycosylation sites. Amino acids that are identical between the two receptors are shaded.

technical

REFERENCES Received May 18, 1992. Revision received July 16, 1992. Accepted July 21, 1992. Address requests for reprints to: Dr. Steven M. Reppert, Laboratory of Developmental Chronobiology, Massachusetts General Hospital, Boston, Massachusetts 02114. This work was supported by NIH Grants DK-42125 (to S.M.R.) and K08D00924 (to S.A.R.). * Present address: James Whitcome Riley Hospital for Children, Room 5984, 702 Barnhill Drive, Indianapolis, Indiana

46202-5225.

1. Linden J 1991 Structure and function of the A, adenosine receptor. FASEB J 5:2668-2676 2. Jacobson KA, Van Galen PJM, Williams M 1992 Adenosine receptors: pharmacology, structure-activity relationships, and therapeutic potential. J Med Chem 35:407-

422 3. Van Calker regulates

D, Muller M, Hamprecht via two different receptors,

B 1979 Adenosine the accumulation of

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MOL 1604

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

END0.1992

cyclic AMP in cultured brain cells. J Neurochem 33:9991005 Londos C, Cooper DMF, Wolff J 1980 Subclasses of external adenosine receptors. Proc Natl Acad Sci USA 77:2551-2554 Bruns RF, Lu GH, Pugsley TA 1986 Characterization of the A2 adenosine receptor labeled by [3H]NECA in rat striatal membranes. Mol Pharmacol 29:331-346 Libert F, Parmentier M, LeFort A, Dinsart C, Van Sande J, Maenhaut C, Simons M-J, Dumont JE, Vassar? G 1989 Selective amplification and cloning of four new members of the G protein-coupled receptor family. Science 244:569-572 Maenhaut C, Van Sande J, Libert F, Abramwicz M, Parmentier M, Vanderhaegen J-J, Dumont JE, Vassar-t G, Schiffman S 1990 RDC8 codes for an adenosine AP receptor with physiological constitutive activity. Biochem Biophys Res Commun 173:1169-l 178 Libert F, Schiffman SN, LeFort A, Parmentier M, Gerard C, Dumont JE, Vanderhaeghen J-J, Vassart G 1991 The orphan receptor cDNA RDC7 encodes an A, adenosine receptor. EMBO J 10:1677-l 682 Mahan LC, McVittie LD, Smyk-Randall EM, Nakata H, Monsma FJ, Gerfen CR, Sibley DA 1991 Cloning and expression of an A, adenosine receptor from rat brain. Mol Pharmacol48:1-7 Reppert SM, Weaver DR, Stehle JH, Rivkees SA 1991 Molecular cloning and characterization of a rat A,-adenosine receptor that is widely expressed in brain and spinal cord. Mol Endocrinol 5:1037-l 048 Fink JS, Weaver DR, Rivkees SA, Peterfreund RA, Pollack AE, Adler EM, Reppert SM 1992 Molecular cloning of the rat AP adenosine receptor: selective co-expression with D2 dopamine receptors in rat striatum. Mol Brain Res 14:186-195 Tucker AL, Linden J, Robeva AS, D’Anqelo DD, Lynch KR 1992 Cloning and expression of a bovine adendsine Al receotor cDNA. FEBS Lett 297:107-l 11 Linden J, Tucker AL, Lynch KR 1991 Molecular cloning of adenosine Al and A2 receptors. Trends Pharmacol Sci 12:326-328 Stehle JH, Rivkees SA, Lee JJ, Weaver DR, Deeds JO, Reppert SM 1992 Molecular cloning and expression of the cDNA for a novel As-adenosine receptor subtype. Mol Endocrinol 6:384-393 Trivedi BK, Bridges AJ, Bruns RF 1990 Structure-activity relationships of adenosine A, and A2 receptors. In: Williams M (ed) Adenosine and adenosine receptors. Humana Press, Clifton, pp 57-l 03 Bruns RF 1980 Adenosine receptor activation in human

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