Proc. Natl. Acad. Sci. USAVol. 87, pp. 3102-3106, April 1990Biochemistry

Molecular characterization of a rat a2B-adrenergic receptor(catecholamine/cDNA/chlorpromazine)

DEWAN ZENG*, JEFFREY K. HARRISONt, DREW D. D'ANGELOt, CYNTHIA M. BARBERt, AMY L. TUCKERt,ZHIHONG Lut, AND KEVIN R. LYNCH*tDepartments of tPharmacology, *Biochemistry, and tCardiology, University of Virginia School of Medicine, Charlottesville, VA 22908

Communicated by Oscar L. Miller, Jr., February 2, 1990 (received for review December 21, 1989)

ABSTRACT a2-Adrenergic receptors comprise a hetero-geneous population based on pharmacologic and molecularevidence. We have isolated a cDNA clone (pRNGa2) encodinga rat a2-adrenergic receptor. A rat kidney cDNA library wasscreened with an oligonucleotide complementary to a highlyconserved region found in all biogenic amine receptors de-scribed to date. The deduced amino acid sequence displaysmany features of guanyl nucleotide-binding protein-coupledreceptors except it does not have a consensus N-linked glyco-sylation site near the amino terminus. Membranes preparedfrom COS cells transfected with pRNGa2 DNA display highaffinity and saturable binding to [3H]rauwolscine (Kd = 2 nM).Competition curve data analysis shows that RNGa2 proteinbinds to a variety of adrenergic drugs with the following rankorder of potency: yohimbine 2 chlorpromazine > prazosin 2clonidine > norepinephrine 2 oxymetazoline. RNGa2 RNAaccumulates in both rat kidney and neonatal rat lung (pre-dominant species is 4000 nucleotides). When a cysteine residue(Cys-169) that is conserved among all members of the seven-transmembrane-region superfamily is changed to phenylala-nine, the RNGa2 protein fails to bind [3H]rauwolscine afterexpression in COS cells. We conclude that pRNGa2 likelyrepresents a cDNA for a rat a2B-adrenergic receptor.

a2-Adrenergic receptors mediate a variety of tissue-specificresponses. Prominent among these are central and peripheralcontrol of circulation, aggregation of platelets, and control ofsecretion. There is pharmacologic, biochemical, and molecu-lar evidence that a2-adrenergic receptors are heterogeneousstructures. For example, Bylund et al. (1) have suggestedclassifying a2-adrenergic receptors into a2A and a2B subtypesbased on differential binding affinities of a-adrenergic drugs incompetition with [3H]rauwolscine binding. The a2A subtype,which is prominent in platelets, exhibits a low affinity forprazosin and a high affinity for oxymetazoline, whereas thea2B subtype, which is prominent in neonatal rat lung, exhibitsthe reverse order of affinities for these two drugs. The a2A anda2B subtypes coexist in tissues such as rat brain. Graham andcolleagues (2) have provided a biochemical correlate of By-lund's classification scheme. They reported that the pharma-cologic profiles of a2-adrenergic receptors in human plateletand neonatal rat lung were maintained after partial receptorpurification and that the a2B (i.e., neonatal lung) subtype wasnot glycosylated, an unusual property for a receptor that iscoupled with guanyl nucleotide-binding proteins. The differ-ences in binding between the subtypes were not due todifferential glycosylation, however, since the A subtype (par-tially purified from human platelets) maintained its bindingproperties following enzymatic deglycosylation.

Lefkowitz and colleagues (3, 4) have isolated two DNAclones that encode closely related proteins with bindingproperties expected of a2-adrenergic receptors. One clone

(a2-C10), isolated from a human genomic library, encodes ana2A subtype (3), whereas a second clone (a2-C4), isolatedfrom a human kidney cDNA library, encodes a subtype withsome of the properties expected of Bylund's a2B subtype (4).For example, the a2-C4 protein has a greater affinity forprazosin than oxymetazoline but is glycosylated followingexpression of the cDNA in COS cells. Venter and colleagues(5) have further shown that the a2A-adrenergic clone isfunctional in that Chinese hamster ovary cells transfectedwith this DNA respond to a2-adrenergic agonists by inhibit-ing forskolin-stimulated increases in cAMP. Southern blotsof human genomic DNA hybridized to a2A-adrenergic recep-tor DNA showed the existence of three sets of bands, andonly two of these were accounted for by known (i.e., a2-C10and a2-C4) adrenergic receptor genes (3, 4). Thus it appearsthat there exists a third, closely related gene in the a2-adrenergic subfamily.To obtain the full set of a2-adrenergic clones for our studies

on the role of a2-adrenergic receptors in blood pressurecontrol, we screened a rat kidney cDNA library with anoligonucleotide derived from a consensus nucleotide se-quence of known biogenic amine receptors. In this report wedescribe a cDNA encoding a molecule with the ligand bind-ing, tissue distribution, and structural properties expected ofan a2B-adrenergic receptor. §

METHODSCloning and Sequence Analysis. Two degenerate oligonu-

cleotides (5'-CTNGAYGTGCTGTKCTGCACSKCSTC-CATCPTGMACCTGTGCG-3' and 5'-CAGSSYGATGPCG-CACAGGT-3'; N = G, A, T, or C; Y = T or C; K = G or T;S = G or C; P = A or G; and M = A or C) were synthesizedon a Biosearch 8600 syfnthesizer and purified. The 3' terminalnonamers of these oligonucleotides are complementary; la-beled, double-stranded DNA was prepared by the action oftheKlenow fragment of DNA polymerase I on the annealedoligonucleotide templates in the presence of radiolabeled (32p)deoxynucleoside triphosphates. The resulting 54-residue,8192-fold degenerate probe, which had a specific activity of 5x 106 dpm/pmol, is targeted to the putative transmembrane IIIregions of biogenic amine receptors. Approximately 1 millionrecombinants from a rat kidney AgtlO cDNA library (6) werescreened with this probe. The filters were hybridized at 50°Cin a hybridization solution as previously described (6); the finalwash was at 42°C in 50 mM Na+ for 1 hr. The hybridizingcDNAs were subcloned into a pGEM series plasmid forrestriction mapping and nucleotide sequencing.

Expression and Binding Studies. The cDNAs were furthersubcloned into the vector pRjb2 (7) for expression in COS-1cells. The Rjb2 plasmid (a gift from Derk Bergsma, SmithKline & French) contains a Rous sarcoma virus long terminalrepeat promoter and bovine growth hormone polyadenyly-lation site. COS-1 cells were transfected (10 ,g of plasmid

§The sequence reported in this paper has been deposited in theGenBank data base (accession no. M32061).

3102

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Proc. Nati. Acad. Sci. USA 87 (1990) 3103

DNA per 6 x 105 cells in a 100-mm dish) by the DEAE-dextran method as described by Cullen (8). Forty-eight hoursafter transfection, cell monolayers were rinsed three timeswith 5 ml of cold phosphate-buffered saline and then scrapedinto 5 ml of phosphate-buffered saline. The cells were pel-leted by centrifugation at 6500 x g for 20 min. The cell pelletwas resuspended in TME buffer (20 mM Tris Cl/5 mMMgCI2/1 mM EDTA, pH 7.4), homogenized in a Douncehomogenizer, and centrifuged at 100,000 x g for 30 min. Theresulting membrane pellet was resuspended in TME buffer ata protein concentration of 1-2 mg/ml, aliquoted, and frozenat -70TC until use.For binding studies, samples were thawed and diluted to a

concentration of 50 ,ug of protein per ml with TME buffer. A100-pl sample of the membrane suspension was added to 100pl ofTME buffer containing [3H]rauwolscine (87 Ci/mmol; 1Ci = 37 GBq) with or without the indicated concentration ofunlabeled drug. Binding reaction mixtures were incubated at250C for 60 min, stopped by the addition of 4 ml of ice-cold20 mM Tris Cl at pH 7.4, filtered through glass fiber (What-man GF/C) filters, and then rinsed twice with 4 ml of cold 20mM Tris Cl at pH 7.4. A final concentration 0.9 nM[3H]rauwolscine was used for competition analysis. Nonspe-cific binding was determined in the presence of 10 ,AMyohimbine. Protein concentrations were measured by themethod of Bradford (9).Data Analysis. Saturation data were analyzed by using a

nonlinear least-squares curve-fitting program (single-sitemodel). Competition curve data were analyzed by nonlinearregression analysis with the GraphPAD program (GraphPADSoftware, San Diego, CA). Data from competition experi-ments were fitted to a sigmoid curve with a variable slopefactor. IC50 values obtained from competition curves wereconverted to K, values according to the equation K, =IC50/(L/Kd + 1), where L is the radioligand concentrationand Kd is the dissociation constant of the radioligand (10).RNA Purification and Analysis. Neonatal rat lung was

removed from 1-day-old Sprague-Dawley rats, and kidneyswere from adult Sprague-Dawley rats. The tissues weresnap-frozen in liquid nitrogen and stored at -140°C until use.RNA was extracted from the frozen tissues as described (11)and poly(A)+ RNA was isolated by oligo(dT)-cellulose affin-ity chromatography (12). RNA was dissolved in sterile water,its concentration was measured spectrometrically, and it wasstored at -70°C until use. RNA was denatured by heating in50% (vol/vol) formamide/4.4 M formaldehyde, electropho-resed through a 1% agarose gel containing 2.2 M formalde-hyde, transferred to a nylon membrane, and hybridized toradiolabeled DNA by standard methods.Two oligonucleotides (5'-AAGCTTGACCACGGCCAG-

CACAAAGG-3' and 5'-GGATCCKCCCTGCCTCATCAT-GPKCC-3') were synthesized, purified, and used to amplifya 576-base-pair fragment of clone dz6a (see text and Fig. 1)located between nucleotides 930 and 1505 (refer to Fig. 1) byusing the polymerase chain reaction (13). This fragment waslabeled with [a-32P]dCTP to a specific activity of -8 x 107dpm/,ug by using the random priming method (14) and wasused as a hybridization probe for the Northern blot. This blotwas hybridized, washed, and exposed to x-ray film in thepresence of an image-intensifying screen.

Drugs. Yohimbine, prazosin, clonidine, norepinephrine,and oxymetazoline were from Sigma; chlorpromazine wasobtained from Smith Kline & French; tritiated rauwolscinewas purchased from New England Nuclear; and tritiatedidazoxan was from Amersham.

RESULTSOne highly conserved region of the biogenic amine family ofthe guanyl nucleotide-binding protein-coupled receptor su-perfamily is the third putative transmembrane region. To

identify heretofore undescribed genes in this family, wescreened a complex rat kidney cDNA library with a 54-residue degenerate oligonucleotide. The sequence of thisprobe was chosen from a consensus of the sequences imme-diately surrounding the Asp-113 [f2-adrenergic receptornumbering (15)] residue of known biogenic amine receptors,including catecholamine, muscarinic acetylcholine, and ser-otonin receptors. Three independent clones, dz6, dz5, anddz3, were isolated and subcloned into the EcoRI site of theplasmid vector pGEM7Zf(+) for sequence analysis. ThecDNA clone dz6 contained the largest insert (-2.9 kilobasepairs); the nucleotide sequence of the larger of two EcoRIfragments (dz6a, 2.2 kilobase pairs) present in this cDNA wasdetermined as was the sequence of two other cDNAs, dz3and dzW. All three cDNAs were found to represent the samemRNA population. Fig. 1 shows the nucleotide sequencederived from these clones and the conceptualized amino acidsequence. We have named the nucleotide sequence derivedfrom these cDNAs pRNGa2 (for Rat Non-Glycosylated). Theonly difference among these three cDNAs in their regions ofoverlap is that both dz3 and dz5 contain the codon TGT(encoding cysteine) at amino acid position 169 while dz6contains the codon TTT (encoding phenylalanine) at thisposition.pRNGa2 contains a single extended translational reading

frame that encodes a protein of 453 amino acids (50,265 Da).This reading frame begins with a methionine, but its codon isnot in the context of an optimal translation initiator consen-sus sequence (17). In contrast, the methionine codon atposition 6 (nucleotides 383-385, Fig. 1) is in an optimalinitiator codon context. Thus the RNGa2 mRNA is possiblyan example of a "bifunctional mRNA" (18); i.e., it mightencode two proteins, one of 453 amino acids and another,translated from the second methionine, of 447 amino acids.The long open reading frame is preceded by 368 nucleotides;this region contains multiple in-frame termination codons, asdoes the region following the reading frame terminationcodon at positions 1727-1729 (Fig. 1). The 5' noncodingregion also contains a small open reading frame (minicistron)extending from nucleotide 203 to 235.Hydropathy analysis (19) of the RNGa2 protein revealed

seven clusters of hydrophobic residues 21-28 amino acids inlength that could represent membrane-spanning regions (datanot shown). Separating these hydrophobic domains are vari-able-length stretches that contain polar amino acids. Align-ment of the pRNGa2 protein sequence with that of the82-adrenergic receptor (15) sequence (data not shown)revealed that Leu-59, Asp-63, and Arg-115 in RNGa2 corre-spond to the Leu-75, Asp-79, and Arg-131 residues (132-adrenergic receptor numbering) that are known to be con-served in all mammalian members of the guanyl nucleotide-binding protein interactive receptor superfamily described todate. Furthermore, the RNGa2 protein has an aspartic resi-due at position 97; this is equivalent to the aspartic residue atposition 113 that has been implicated in ,82-adrenergic ligandbinding (20) and that is conserved at this position in all knownbiogenic amine receptors.A query of all known members of this superfamily with the

RNGa2 sequence using the algorithm of Lipman and Pearson(21) revealed that RNGa2 is most likely related to the knowna2-adrenergic receptors (47% identity overall to either a2-C1Oor a2-C4), followed by the other catecholamine receptors,and the serotonin-lA receptor (all at <30% identity). Theoptimized alignment of RNGa2 with the other a2-adrenergicreceptor subtypes is shown in Fig. 2; alignment of theputative third cytoplasmic loop is not shown because of thelow level of similarity in this region. Clearly, the mostconserved regions are the putative transmembrane domains.Within these regions, RNGa2 is 77% identical to either thea2-C1O or a2-C4 (3, 4) proteins, the C10 and C4 subtypes are

Biochemistry: Zeng et al.

3104 Biochemistry: Zeng et al. Proc. Natl. Acad. Sci. USA 87 (1990)

PSI I PS(Il1 CTGCAGGCGCGGCTCCGGGAGAGAGAGCGCCTTTTCGGAGCGCAGGCTGCCGTTCCCCAGTCAGTC TGTTGCGTCCTGCAGCTGTGCGTGCGACGGCGCAACTTCCCTCTA

1 12 GTCCCGGGACAGCAGTGGGACAACTTGGAAAACTTCTCTGGGACAGACTGTAGGGACGCTGGGCACCGGTGGAAGAGGATATAAGAGACCGATGGCTGGTCCTGGGTATCC223 CCGGACCTCCTAGGCCGCTTTCCGTCAGGCTACGGTGCTTCAGAGCCATCTGCACCTCCTGGGGGACGACCCCCAACAGTAGCGGAGCCAGAGTGGCGCTATCTCCTGCGT

334 ACCGTGAGTTTGGCCAGCATCGTTCCCTCTGGACATGTCCGGCCCCACCATGGACCATCAGGAGCCCTACTCGGTGCAGGCCACCGCCGCCATCGCGTCGGCCATCACC TT* ~~~~MS G P T M D H Q E P Y S V Q A T A A I A S A I T F

4 45 TCTCATCCTTTTCACCATTTTCGGCAATGCGCTGGTCATTCTGGCTGTGTTGACCAGCCGCTCACTCCGTGCACCACAAAACCTGTTCCTGGTGTCACTGGCAGCAGCCGAL I L F T I F G N A L V I L A V L T S R S L R A P Q N L F L V S L A A A D

556 CATCCTAGTGGCTACTCTCATCATCCCTTTCTCTCTGGCCAACGAGCTGCTGGGCTACTGGTACTTCTGGCGTGCGTGGTGCGAGGTCTACCTGGCGCTAGACGTGCTCTTI L V A T L I I P F S L A N E L L G Y W Y F W R A W C E V Y L A L D V L F

667 CTGTACCTCCTCCATCGTGCACCTGTGTGCCATCAGCCTGGACAGGTACTGGGCAGTGAGCCGAGCATTGGAGTACAAC TCCAAGCGCACTCCGTGCCGCATCAAGTGCATC T S S I V H L C A I S L D R Y W A V S R A L E Y N S K R T P C R I K C I

(T)7 78 CATCCTCACTGTGTGGCTCATTGCAGCTGTCATTTCTCTACCGCCCCTCATCTACAAGGGCGACCAACGCCCCGACGCCCGCGGGCTCCCCCAGTGTGAGCTCAACCAGGA

889

1000

1111

1222

1333

1444

1555

1666

1777

18881999

I L T V W L I A A V I S L P P L I Y K G D Q R P D A R G L P Q C E L N Q E(F) Petl

GGCCTGGTACATCTTGGCTTCCAGCATCGGATCTTTTTTTGCTCCCTGCCTCATCATGATCCTCGTCTACCTGCGAATCTACGTGATTGCCAAACGCAGCCACTGCAGAGGA W Y I L A S S I G S F F A P C L I M I L V Y L R I Y V I A K R S H C R GTCTCGGAGCCAAGAGGGGCTCTGGAGAAGGTGAGTCCAAGAAGCCCCAGCCGGTTGCTGGGGGAGTGCCAACCTCAGCTAAGGTGCCCACCCTGGTCTCTCCTCTATCTTCL G A K R G S G E G E S K K P Q P V A G G V P T S A K V P T L V S P L S STGTTGGAGAGGCCAATGGACACCCCAAGCCTCCAAGAGAGAAGGAGGAGGGGGAGACCCCTGAAGATCCTGAGGCCAGGGCTTTGCCCCCAACTTGGTCTGCCCTTCCCAGV G E A N G H P K P P R E K E E G E T P E D P E A R A L P P T W S A L P R

ATCAGGCCAAGGCCAGAAGAAGGGGACTAGTGGGGCGACTGCAGAGGAGGGAGATGAAGAGGATGAGGAAGAGGTGGAAGAATGTGAACCCCAAACACTGCCAGCATCTCCS G Q G Q X K G T S G A T A E E G D E E D E E E V E E C E P Q T L P A S P

TGCCTCGGTATGCAACCCACCCTTGCAGCAGCCTCAGACTTCTCGGGTACTGGCCACACTTCGTGGCCAGGTGCTTC TGGGCAAGAATGTGGGAGTTGCCAGTGGGCAGTGA S V C N P P L Q Q P Q T S R V L A T L R G Q V L L G K N V G V A S G Q W

GTGGCGCAGACGGACACAGCTGAGCCGGGAGAAGAGGTTCACCTTTGTGCTGGCCGTGGTCATTGGAGTTTTCGTGGTCTGCTGGTTTCCTTTCTTCTTCAGCTACAGCCTW R R R T Q L S R E K R F T F V L A V V I G V F V V C W F P F F F S Y S L

GGGGGCCATCTGCCCACAGCACTGCAAGGTACCGCATGGCCTCTTCCAGTTCTTCTTCTGGATCGGCTACTGCAACAGCTCTTTGAACCCTGTCATCTACACCGTCTTCAAG A I C P Q H C K V P H G L F Q F F F W I G Y C N S S L N P V I Y T V F NCCAGGACTTCCGCCGTGCCTTTCGAAGGATCCTTTGCCGGCCGTGGACCCAGACTGGCTGGTGAGGCTGCCTGTTCGCTGTCTATGCCAGGGTTGGTGCCAGGACCACCCGQ D F R R A F R R I L C R P W T Q T G W *

GTGGTAGTGCCCCACCTCCACAGTCTCTATGGCCATTTCTCTGGGGCCCACTTGCAGACCTCATCCTCAGACTCTCCTGAAGGGGAAAGGACAAAAATGCTGTTCCTAGGG

26

63

100

137

174

211

248

285

322

359

396

433

453

TCCTCCGTAA

FIG. 1. Nucleotide and amino acid sequence of RNGa2. The nucleotide sequence is from three independent rat cDNAs; sequence analysis(16) of dz3 yielded nucleotides 36-928, dz5 yielded nucleotides 1-1544, and dz6 yielded nucleotides 135-2008. Clones dz3 and dz5 thus do notcontain the full reading frame. The sequences were identical at all positions of overlap except at nucleotide 874, where dz3 and dz5 had aguanosine and dz6 had a thymidine (see text). In-frame termination codons are indicated by asterisks; nucleotide and amino acid numbers areon the left and right margins, respectively.

76% similar to one another. Conversely, in the region that liesbetween putative transmembrane regions V and VI, RNGa2is -22% identical to either C10 or C4 proteins, whereas theseproteins are 28% identical to one another over this region.The feature common to members of this receptor superfam-

ily that is absent in RNGa2 is a consensus site for N-linkedglycosylation near the amino terminus ofthe protein. The onlyglycosylation consensus site (Asn-Xaa-Ser/Thr) in RNGa2occurs at position 421. If the seven-transmembrane-regionmodel (23) of this receptor family is correct. Asn-421 isnormally buried in the membrane (transmembrane region VII)and thus is presumably not glycosylated.To determine if RNGa2 has the binding characteristics

expected of an a2-adrenergic receptor, we expressed cDNA

clone dz6a in COS-1 cells and attempted to bind [3H]-rauwolscine to membranes prepared from these cells. How-ever, repeated attempts to show specific [3H]rauwolscinebinding failed regardless of the transfection method (DEAE-dextran, electroporation, or calcium phosphate precipitation)or the expression vector [Rjb2 (7) orCDM8 (24)] used, nor didother biogenic amine ligands (e.g., '25I-labeled cyanopin-dolol, [3H]prazosin, [3H]idazoxan, [3H]SCH23390, etc.) bindto membranes from the transfected cells. Among the possibleexplanations for these failures are the lack of a potentialamino-terminal glycosylation consensus site, the small openreading frame in the 5' noncoding region, the presence of thefirst methionine codon embedded in an unusual (i.e., asregards initiator methionine codons) sequence, and the lack

MGSLQPDAGNASWNGTEAPGGGARATPYSLQVTLTLVCLAGLLMLLTVFGNVLVIIAVFTSRALKAPQNLFLVSLASADILVATLVIPFSLANEVMG 97

MSGPTMD-HQEPYSVQATAAIASAITFLILFTIFGNALVILAVLTSRSLRAPQNLFLVSLAAADILVATLIIPFSLANELLG 81

:: .. . .....::. :.::::.:.:.:...........:::::.::::::::.:::::......................::::

115

III IV V

a2-C10 YWYFGKTWCEIYLALDVLFCTSSIVHLCAISLDRYWSITQAIEYNLKRTPRRIKAI IITCWVISAVISFPPLISIEKKGGGGGPQPAEPRCEINDQKWYVISSCIGSFFAPCLIM 212

:::: ..:.::::::::::::.... :.::: :::: ::: ::.: :.:::::... :.::.::...:.:::::

RNGa2 YWYFWRAWCEVYLALDVLFCTSSIVHLCAISLDRYWAVSRALEYNSKRTPCRIKCIILTVWLIAAVISLPP93IYGDQ--RPDARGLPQCELNQEAWYILASSIGSFFAPCLIM 193

.C 22...6W2-C 4 YWYFGQVWCGVYLALDVLFCTS SIVHLCAISLDRYWSVTQAVEYNLKRTPRRVKAT IVAVWLISAVISFPPLVSLYRQ---- -PDGAAYPQCGLNDETWYILSSCIGSFFAPCLIM 22 6

a2-C1O ILVYVRIYQIAKR

RNGaL2 ILVYLRIYVIAKR

a2-C4 GLVYARIYRVAKR

(143 aa)

(162 aa)

450

453

VLAVV::V:VLC:.::::I::L:::C::C.:V:...L.::I::.::::::L:::I::V:::::::::::::..:..

( 13 6 aa ) EKRFTFVLAVVMGVFVLCWFPFFF IYSLYGICREACQVPGPLFKFFFWIGYCNSSLNPVI YTVFNQDFRPSFKHILFRRRRRGFRQ 4 61

FIG. 2. Comparison of the RNGa2 amino acid sequence with those of other a2-adrenergic receptors (3, 4). The putative third cytoplasmicdomain of the receptors is not shown because of lack of meaningful similarity in this region. Putative transmembrane regions are overlined andconsensus sequences for N-linked glycosylation occurring near the amino terminus are indicated by carets. Sequences were optimally alignedusing the ALIGN algorithm (22). Two dots (:) indicate amino acid identity; a single dot (.) indicates a conservative amino acid substitution; and

gaps are indicated by dashes.

a2-C1O

RNGa2

a.2-C4 MASPALAAALAVAAAAGPiigGAGERGSGGVAiigGASWGPPRGQYSAGAVAGLAAVVGFLIVFTVVGNVLVVIAVLTSRALRAPQNLFLVSLASADILVATLVMPFSLANELMA

Proc. NatL. Acad. Sci. USA 87 (1990) 3105

of a cysteine residue at position 169. We tested each possi-bility, but the insertion of a potential glycosylation site(Met-Ala-Gly-Pro-Thr-Asn-Arg-Ser-Met, residues 1-9) andthe elimination of the entire 5' noncoding region of dz6a (upto the Nco I site at nucleotides 381-386) did not result indetectable binding (data not shown). Therefore, the phenyl-alanine residue encoded at position 169 in dz6a (see Fig. 1)was changed to a cysteine as is present in both the dz3 anddzS cDNAs. This was accomplished by melding the dz5 anddz6a cDNAs at their internal Pst I sites. The nucleotidesequence of the resulting construct, dz5.6a (Cys-169), wasdetermined and found to be identical to dz6a at all positionsexcept the thymidine -- guanosine change at nucleotide 873and an additional 45 nucleotides derived from dz5 at thebeginning of the 5' noncoding region (refer to Fig. 1). Thedz5.6a construct was placed in a Rjb2 vector and expressedby transfection into COS cells.Membranes from cells transfected with the dz5.6a con-

struct specifically bound [3H]rauwolscine (Fig. 3). This bind-ing was both saturable and of high affinity (Kd = 2 nM);another a2-adrenergic selective drug, [3H]idazoxan, also spe-cifically bound to the transfected cell membranes (Kd = 20nM, data not shown). To further characterize the ligandbinding properties of the RNGa2 protein, the binding com-petition of [3H]rauwolscine with a set of drugs known todiscriminate among a2-adrenergic receptor subtypes wasstudied. Fig. 4 shows the competition of yohimbine, chlor-promazine, prazosin, clonidine, norepinephrine, andoxymetazoline for the [3H]rauwolscine binding sites on trans-fected COS cell membranes. Analysis of the competitioncurve data revealed the following Ki values: yohimbine, 5nM; chlorpromazine, 7 nM; prasozin, 27 nM; clonidine, 56nM; norepinephrine, 374 nM; and oxymetazoline, 613 nM.The slope factors of the yohimbine, chlorpromazine, andprasozin competition curves were all 0.95, whereas thecompetition curves of the three lower affinity compoundswere slightly more shallow, with slope factors ranging be-tween 0.75 and 0.85.To determine the size ofthe RNGa2 mRNA(s) and whether

RNGa2 RNA accumulates in neonatal lung [as expected ofana2B-adrenergic receptor subtype (2)], we hybridized aRNGa2-specific probe to RNA extracts from these tissues.

c._

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0~-a

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10 20 3[3H]Rauwolscine, nM

The specific probe, which consists ofnucleotides 929-1505 ofthe dz6 cDNA (see Fig. 1), was generated by the polymerasechain reaction (14). This region ofthe cDNA encodes a regionof the RNGa2 protein that is least similar to other knowna2-adrenergic receptors (refer to Fig. 2). When applied to aNorthern blot of rat kidney and neonatal lung RNAs (Fig. 5),this probe annealed toRNA species of -4000 nucleotides. Anadditional band at =1300 nucleotides is also evident inneonatal lung RNA extracts.

DISCUSSIONThe ligand-binding profile of the RNGa2 protein correlateswell with that reported for a2-adrenergic receptors of the Bsubtype. Rauwolscine is approximately 3-fold and 10-foldmore potent at this site than yohimbine and idazoxan, re-spectively. Oxymetazoline has a relatively low affinity forRNGa2 site while prazosin and chlorpromazine exhibitedrelatively high affinities. The ratio ofKi values ofthe subtype-selective drugs oxymetazoline and prazosin is 0.05. Addi-tionally, RNGa2 RNA accumulates in neonatal lung, theprototypical tissue for an a2B-adrenergic receptor. The re-sults of the binding studies, together with the apparent lackofglycosylation of the RNGa2 protein and the presence of itscognate mRNA in neonatal lung, lead us to believe that thisprotein is an a2B-adrenergic receptor.The finding that there is no potential N-linked glycosylation

site in the RNGa2 protein sets it apart from other knowna2-adrenergic receptors and from members of the seven-transmembrane-region superfamily as a whole. There are atleast two other members of this family, however, that also donot contain amino-terminal glycosylation consensus sites (26),although the ligands for these putative receptors are unknown.Our pRNGa2 clones are clearly not the rat analog of eitherpreviously described (human) a2-adrenergic receptor clone(a2-C10, ref. 3; a2-C4, ref. 4) because the RNGa2 amino acidsequence is as divergent from either of the human clones asthey are from one another. This is in contrast to the sequenceof the human a2-adrenergic receptor genomic clone describedby R. Weinshank, H. M. Lichtblau, and P. R. Hartig (personalcommunication), which is very similar to our RNGa2 through-out its entire length, including the divergent putative V-VI

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FIG. 3. Representative saturation isotherm and Scatchard plot(Inset) of [3H]rauwolscine binding to membranes prepared fromdz5.6a-transfected COS-1 cells. Total (A), specific (A), and nonspe-cific (e) binding are shown. Nonspecific binding was defined asresidual counts bound in the presence of 10 ,uM yohimbine. Datafrom each experiment were expressed as a percent of their respectiveBmax, and then all points (from four experiments) were plotted on asingle graph. The composite data were analyzed with a nonlinearleast-squares curve-fitting computer program (single-site model). AKd of2.0 + 0.2 nM (95% confidence limits) was determined. TheB,,values of the experiments ranged from 3 to 10 pmol/mg of protein.

11 10 9 8 7 6-log[competitorl (M)

FIG. 4. Representative experiment showing adrenergic drugs incompetition for the binding of [3H]rauwolscine (0.9 nM) to mem-branes prepared from dz5.6a-transfected COS-1 cells. Data fromthree individual experiments were analyzed on GraphPAD (nonlin-ear regression fit to a sigmoidal curve). The specific binding is shown.K, values were calculated from IC50 values. The following K; valueswere determined (mean ± SE, n = 3): yohimbine, 5.2 ± 0.7 nM;chlorpromazine, 7.5 ± 2.0 nM; prazosin, 27.2 ± 1.2 nM; clonidine,56.5 + 10.2 nM; norepinephrine, 374 ± 31 nM; and oxymetazoline,613 ± 44 nM.

Biochemistry: Zeng et aL

Proc. Natl. Acad Sci. USA 87 (1990)

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1 A1 .

0.24-

FIG. 5. Northern blot analysis of RNGa2 RNA accumulation.Two micrograms of rat kidney or neonatal rat lung poly(A)+ RNAwas denatured and electrophoresed through a 1% agarose gel underdenaturing conditions (25). After transfer to a nylon membrane,specific sequences were detected by hybridization to a radiolabeledfragment of dz6 cDNA (specific activity = 8 x 107 dpm/,tg). Thisfragment, which contains nucleotides 929-1505 of dz6 cDNA, isspecific for this a2B-adrenergic receptor subtype and was generatedby the polymerase chain reaction. Lane A, adult rat kidney; lane B,neonatal rat lung; markers were a poly(A)+ ladder (BRL). Exposuretime was 3 days in the presence of an image-intensifying screen.

intracellular loop region. This human protein is also similar inthat it possesses ligand binding properties that are indistin-guishable from those of the RNGa2 and contains no amino-terminal N-linked glycosylation sites (27). These similaritieslead us and R. Weinshank to believe that our rat RNGa2 cloneand his human genomic clone are analogs.We conceptualized two forms of RNGa2 from our cDNA

clones; one form, represented by cDNAs dz3 and dz5, has acysteine residue at position 169 while a second form, encodedby clone dz6, contains a phenylalanine at this position. Onlyexpression of a hybrid clone, dz5.6a, that is full length andencodes a Cys-169 exhibits any specific binding after expres-sion in COS cells. Assuming that the seven-transmembrane-region model of this receptor family is correct, then position169 lies in the extracellular domain between transmembraneregions IV and V. All members of the seven-transmembrane-region gene superfamily described to date from vertebratespecies contain at least one cysteine in this region. Further-more, Dixon et al. (28) have shown in site-directed muta-genesis studies of the /82-adrenergic receptor that this cys-teine (Cys-184) is essential for correct (i.e., ligand binding)presentation of this receptor when expressed in COS-7 cells.Our results confirm and extend to an a2-adrenergic receptorthe results of these studies in that the substitution of aphenylalanine for cysteine at the equivalent position inRNGa2 results in the loss of detectable specific ligand bind-ing. We do not know whether the dz6 cDNA (RNGa2Phe-169) represents an inactive allele in the Sprague-Dawleyrat genome, a functional protein when presented in its naturalcellular environment, or a fortuitous cloning artifact.

In summary, we have isolated rat cDNAs encoding aprotein, RNGa2, that is most similar to a pair of recentlydescribed a2-adrenergic receptor subtypes. The absence of aconsensus signal for N-linked glycosylation at the aminoterminus, the binding characteristics, and its apparent syn-thesis in neonatal lung convinces us that RNGa2 is ana2-adrenergic receptor of the B subtype.

We thank Drs. Joel Linden and Patrice Guyenet (University ofVirginia) for advice during the course ofthese studies. In addition, wethank Dr. Joel Linden and Dr. Richard Neubig (University of Mich-igan) for their gifts ofhuman platelet membranes that were used to testligands and Dr. Derk Bergsma (Smith Kline & French) for his gift ofthe expression vector Rjb2. We also thank Dr. Richard Weinshank(Neurogenetic Corp.) for communicating results regarding the humana2B-adrenergic receptor prior to their publication. During the courseof this work J.K.H. was supported by a National Research ServiceAward postdoctoral fellowship (F32 HL08223), D.D.D. was sup-ported by a National Research Service Award predoctoral trainingaward (T32 GM08136), and A.L.T. was supported by a NationalResearch Service Award postdoctoral training award (T32 HL07355).This work was supported in large part by grants from the NationalInstitutes of Health (RO1 DK37494 and R01 HL33513). K.R.L. is anEstablished Investigator of the American Heart Association, withpartial funding supplied by their Virginia Affiliate.

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