THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 266, No. 10, Issue of April 5, pp. 636556369,1991 Printed in U. S. A.

Molecular Cloning and Expression of the cDNA for the cula-Adrenergic Receptor THE GENE FOR WHICH IS LOCATED ON HUMAN CHROMOSOME 5*

(Received for publication, October 19, 1990)

Jon W. Lomasney," Susanna Cotecchia,' Wulfing Lorerqb Wah-Ying Leung," Debra A. Schwinn,d Teresa L. Yang-Feng,' Michael Brownstein," Robert J. Lefkowitz,'.'* and Marc G. Caronb*f*h From the Departments of "Pathology, dAnesthesiology, bMedicine, hCell Biology, and 8Biochemistry, and the /Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710; the 'National Institute of Mental Health, Bethesda, Maryland 20892; and 'Department of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06510

Pharmacological and molecular cloning studies have demonstrated heterogeneity of al-adrenergic recep- tors. We have now cloned two al-adrenergic receptors from a rat cerebral cortex cDNA library, using the hamster cxlB-adrenergic receptor as a probe. The de- duced amino acid sequence of clone RA42 encodes a protein of 560 amino acids whose putative topology is similar to that of the family of G-protein-coupled receptors. The primary structure though most closely resembles that of an al-adrenergic receptor, having approximately 73% amino acid identity in the putative transmembrane domains with the previously isolated hamster ( Y ~ B receptor. Analysis of the ligand binding properties of RA42 expressed in COS-7 cells with a variety of adrenergic ligands demonstrates a unique al-adrenergic receptor pharmacology. High affinity for the antagonist WB4101 and agonists phenyleph- rine and methoxamine suggests that cDNA RA42 en- codes the alA receptor subtype. Northern blot analysis of various rat tissues also shows the distribution ex- pected of the alA receptor subtype with abundant expression in vas deferens followed by hippocampus, cerebral cortex, aorta, brainstem, heart and spleen. The second al-adrenergic receptor cloned represents the rat homolog of the hamster alB subtype. Expression of mRNA for this receptor is strongly detected in liver followed by heart, cerebral cortex, brain stem, kidney, lung, and spleen. This study provides definitive evi- dence for the existence of three al-adrenergic receptor subtypes.

Receptors that mediate effects via guanine nucleotide-bind- ing regulatory proteins or G-proteins constitute a large and diverse family. Although these receptors bind different classes of ligands and mediate a wide variety of responses, the primary structure of these proteins is remarkably similar. Perhaps most characteristic is the presence of seven stretches of hy- drophobic amino acid residues which are thought to span the plasma membrane (1-3). The adrenergic receptors are among

* This work was supported in part by National Institutes of Health Grant HL16037. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank with accession number($ M60654 and M60655.

the most thoroughly characterized of this important super- family of closely related proteins. Although adrenergic recep- tors have historically been identified and classified by phar- macological and functional criteria, recent cloning of multiple adrenergic receptor cDNAs/genes has not only expanded the number of subtypes identified but also allowed for a compar- ison of the primary structure of these proteins, leading to a more sensitive and accurate classification. In addition, the availability of the nucleic acid sequences encoding for these receptors has made it possible to explore the molecular basis of receptor function (4-6).

Recently, the subfamily of a-adrenergic receptors has grown to include at least six distinct yet closely related members. Pharmacological studies suggest the presence of possibly four a2-adrenergic receptors (7-lo), of which the genes/cDNAs for three have been isolated to date (11-14). Two subtypes of al- adrenergic receptor (alA and alB) have been demonstrated pharmacologically by showing differential affinity of al-ad- renergic receptor binding sites for the antagonists WB4101 and phentolamine and agonists oxymetazoline and methox- amine. In addition, differential sensitivities of a,-mediated effects to the alkylating agent chlorethylclonidine (CEC),' and the calcium channel blocker nifedipine have served as distinguishing criteria (15-17). Our laboratory has previously isolated the cDNAs encoding for an aIB-adrenergic receptor from hamster and for a third subtype from bovine brain which fits neither the a l A nor a I B subtype definition and which is designated as the a I c (18, 19). We now report the cloning of a receptor which displays all the essential characteristics of the pharmacologically defined aIA-adrenergic receptor sub- type.

EXPERIMENTAL PROCEDURES

cDNA and Genomic Library Screening-A rat cerebral cortex cDNA library in the Okayama-Berg plasmid vector pcDVl (20) (7.5 X lo6 recombinants) was screened by Southern blot analysis with a probe from the cDNA encoding the hamster al"AR. Twelve aliquots of the library each containing 400,000 bacteria were grown in Luria Broth with 100 Fg/ml ampicillin. Plasmid DNA was isolated by the alkaline lysis method (21) and approximately 15 pg from each culture was digested with the endonuclease BamHI and loaded onto a 1% agarose gel. After electrophoresis, the DNA was transferred to a nitrocellulose membrane with 20 X SSC (1 X SSC = 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0) according to the method of

The abbreviations used are: CEC, chlorethylclonidine; kb, kilo- base(s); bp, base pair(s); ['251]-HEAT, 2-(~-(4-hydro~y-3-['~~I]iodo- phenyl)-ethylaminomethyl]-tetralone; AR, adrenergic receptor, G- protein, guanine nucleotide-binding protein.

6365

cDNA for alA-Adrenergic Receptor Southern (22). The Southern blot was incubated with 1 X lo6 cpm/ ml of probe in 5 X SSC, 50% formamide, 1 X Denhardt's solution, 10% dextran sulfate, 20 mM sodium phosphate, pH 6.5, 100 pg of sheared salmon sperm DNA/ml at 42 "C for 12 h. The blot was then washed in 0.5 X SSC, 0.1% SDS at 55 "C and exposed a t -70 "C with Kodak X-Omat film with Cronex enhancing screens. The probe consisted of the BamHI-XhoI 0.7-kb restriction fragment of the hamster aleAR cDNA (nucleotides 150-825) (18) and was labeled with "P by nick translation. A rat genomic library in EMBL 3 (2.3 X lo6 recombinants; Clontech) was screened to isolate a full length clone. 2 pg of phage DNA isolated from pools (200,000 pfu each) of the genomic library was amplified by polymerase chain reaction with 1 p M each of specific oligonucleotide primers (CTGGTCATCCTTTCGGTGGCC and CACCCGGCGCCACCTG) in 10 mM Tris-HC1, pH 8.3,50 mM KC1,1.5 mM MgCl?, 0.01% gelatin, 200 p M each dATP, dCTP, dGTP, dTTP, 2.5 units of Thermus aquaticus DNA polymerase (Taq DNA polymerase; Perkin-Elmer- Cetus). The amplification profile was run for 25 cycles: 2 min a t 92 "C, 2 min a t 45 "C, and 3 min a t 72 "C. The primers were from the amino-terminal region of the partial cDNA clone RAL. A single positive pool was then plated out and duplicate nitrocellulose filters were hybridized with 1 X lo6 cpm of probe in 6 X SSC, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin, 0.1% sodium pyrophosphate, 0.1% SDS, 100 pg of sheared salmon sperm DNA/ml a t 42 "C for 12 h and washed in 0.2 X SSC, 0.1% SDS at 55 "C. The probe consisted of the A~QI/SQCI 1.2-kb restriction frag- ment (nucleotides 666-1833 of Fig. 1) of the partial cDNA clone RAL.

Expression-To facilitate construction of the expression vector for the ala-adrenergic receptor, a PstI-StuI 1019-kb restriction fragment from the cDNA clone RAL was ligated with a PstI-StuI 1254-kb restriction fragment from an overlapping genomic clone. In the 700- bp region of overlap the nucleotide sequence for the genomic and cDNA clones were identical demonstrating that both sequences are derived from the same gene. This PstI 2273-bp fragment was ligated into the PstI-digested mammalian expression vector pCMV5 (23). The resulting construct pCMV5alRA42, contained 140 bp of 5'- untranslated region, 1680 bp of coding region, and 790 bp of 3'- untranslated region. The cDNA isolated from bovine brain encoding

expression vector pBC12B1, as described previously (19). The cDNA for the ale-adrenergic receptor was placed into the mammalian

isolated from the rat cerebral cortex library which encodes for the mlB-adrenergic receptor, was expressed unaltered in the Okayama-

by the DEAE-dextran method (24). Berg pcDV1 vector. The constructs were transfected into COS-7 cells

DNA Sequencing-Single and double stranded DNA template was made from pTZ18R and pTZ19R (Pharmacia LKB Biotechnology Inc.). Nucleotide sequence analysis was performed using overlapping templates by the dideoxynucleotide chain-termination method and by primer extension with T7 DNA polymerase (Sequenase; United States Biochemical Corp.) (25, 26) for both complete strands.

Northern Blot Analysis-Total cellular RNA was isolated from fresh Sprague-Dawley rat tissues by the guanidinium isothiocyanate/ cesium chloride method (27). Poly(A)+ RNA was selected by using 2 cycles of oligo(dt)-cellulose chromatography. After denaturation by glycoxylation, the RNA was fractionated by agarose gel electropho- resis and transferred to Biotrans membranes (ICN Biochemicals). The cDNA probes from RA42 (0.85-kb BamHI restriction fragment), Rale (0.7-kb BarnHI fragment) and Bale (1.3-kb NcoI-Hind111 frag- ment) were self-ligated with T 4 DNA ligase and labeled by nick translation.

Ligand Binding-COS-7 cell membranes were prepared as de- scribed previously (19). Approximately 10 fmol of receptor/0.25 ml of assay mixture was incubated in TNE buffer (50 mM Tris-HC1, 150 mM NaC1, 5 mM EDTA, pH 7.4) a t 25 "C for 1 h with ["'I]HEAT (2200 Ci/mmol). Assays were placed on ice, filtered onto Whatman GF/C membranes, and washed with ice-cold buffer. For competition curve analysis, each assay contained 100 PM [Iz5I]HEAT. Nonspecific binding was determined with 1 PM prazosin. Data were analyzed by computer with an iterative nonlinear regression program (28).

CEC Inactiuation-10 p~ CEC was incubated in 1-ml volumes of hypotonic buffer (5 mM Tris, 5 mM EDTA, pH 7.6) with membranes made from COS cells transfected with pCMV5alRA42 or pcDV1Ralw The protein content was the same for each receptor during CEC treatment (0.5 mg). After treatment for 10-30 min a t 37 "C, the reactions were stopped by adding 16 ml of ice-cold buffer, and centrifuged at 40,000 X g for 15 min at 4 "C. The membranes were washed three times with 8 ml of ice-cold buffer. They were then resuspended in TNE buffer and residual alAR binding was assessed

by incubation with 0.5 nM ["'I]HEAT. Chromosomal Localization-Zn situ hybridization was performed

with nick-translated cDNA and genomic probes labeled with [3H] dATP, [3H]dCTP, and [3H]dTTP. Hybridization to human chromo- some preparations, post-hybridization wash, emulsion autoradiogra- phy, and silver grain analysis were performed according to previously published procedures (29).

Materials-Sources of drugs were as follows: phentolamine (CIBA- Geigy); prazosin (Pfizer Diagnostics); WB4101, CEC (Research Bio- chemicals, Natick, MA); corynanthine, oxymetazoline, norepineph- rine, phenylephrine, epinephrine, methoxamine (Sigma); indoramine, (Ahbott); [lZ51]HEAT (Du Pont-New England Nuclear).

RESULTS AND DISCUSSION

Screening of a rat cerebral cortex cDNA library at moder- ately low stringency with a cDNA probe derived from the hamster alB-adrenergiC receptor, identified two distinct clones. The first clone contains a 1545-bp open reading frame encoding for a protein of 515 amino acids. Hydropathy analy- sis of the protein sequence indicates the presence of seven hydrophobic clusters of 20-25 residues, each separated by stretches of hydrophilic residues (30). This pattern is similar to that observed with various members of the G-protein- coupled receptor family ( 2 ) . The putative topography of this superfamily of receptors predicts that the hydrophobic clus- ters span the plasma membrane and that the hydrophilic stretches project from the membrane. Thus the amino ter- minus and three loops would extend into the extracellular space, and the carboxyl terminus and the three remaining loops would extend into the cytoplasm. The deduced amino acid sequence of this clone is 98.1% identical with that of the hamster alB-adrenergiC receptor and 98.5% identical with a previously reported rat alB sequence (31). It will herein be referred to as RalB.

The second clone, RAL contained a 1160-bp open reading frame which encoded for the carboxyl portion of a protein with five hydrophobic clusters of amino acids. These five putative transmembrane domains when compared with the sequences of known G-protein coupled receptors, had the highest similarity (72% amino acid identity) with transmem- brane regions 111-VI1 of the alB-adrenergic receptor.

In order to obtain a full length clone, aliquots totaling lo6 recombinants of a rat genomic library were screened by po- lymerase chain reaction using specific oligonucleotide primers from the 5' region (bp 974 to 989 from the sense strand and bp 1254 to 1237 from the antisense strand, Fig. 1) of the partial cDNA clone RAL. A positive pool was then plated and screened with a 1.2-kb ApaI/SacI restriction fragment from RAL. An 8-kb genomic clone was isolated. A 1.4-kb BamHI restriction fragment from the genomic clone was obtained which overlapped with the cDNA clone by -700 bp. The complete gene was constructed by splicing together two re- striction fragments one each from the cDNA and genomic clones, using a unique endonuclease restriction site (Stul) , present in the overlapping region. An open reading frame of 1680 bp from the genomic/cDNA construct encodes for a protein of 560 amino acids (Fig. 1). Hydropathy analysis confirms the presence of seven hydrophobic regions which have the following percent amino acid identities with corre- sponding regions of other adrenergic receptors: rat a l ~ 73%, bovine a l C 65%, rat f i 2 43%, and rat a 2 B 45% (19,32, 14). The amino acid identities between the transmembrane regions of adrenergic receptors in the same subfamily (subtypes) are typically in the 70% range. For instance, the transmembrane amino acid identities among the three human wadrenergic receptor subtypes ranges from 74 to 75% (13). Therefore, it appears that this receptor RA42 is an a1 subtype.

The primary structure of RA42, as shown in Fig. 2 , dem-

cDNA for alA-Adrenergic Receptor 6367

- P a l

[Apa;S*C1 SlUl [BarnHl IAPB;Apall [Sac1 rApal

BamHl

II

500 1 0 0 15DO XKK) 2yx)

5" OPEN READING FRAME "3 gonomlc clono ( Barn HI )

RAL cDNA clono

r.r.lrrrrrr.rPI6CC6CT0r~CGCACGGAGCCCCTTCGC 3 8 ~~~ ~~ ~

CCTCCCTCTCGACCGCCCCTCCTCCGTC 'CTGCACTGCCACTCCATGAGCC

T~ACTCCGCAGCCACCGCCGGAGTCGGTTCCCTCA

AACTTCGGCAGGACCGACGGGACTGGUICACGCGG GCCCGGTGACCTCTGTCCTGCTCTACCTGCAGCACTGTCC

GGGCTCTCTCCCCTCCACTTGCTCGCCCTGTGCCTGGGCTC

1 0 1 164 2 2 1 2 9 0 353 416 419

ATMCTTTCCGCGCATCCTGAGCGTCACTTTCG~UGGACCCCGGTCGAGCAGTAGCACTGGG 542 M T F R D I L S V T F E G P R S S S S T G 2 1 GGCTCCGGCGCCGGCCGCGGGCTGGCACGGTTGGCCCCGAGGGTGGGGCGGTGGGGGCGTG 605 G S G A G G G A G T V G P E G G A V G G V 4 2

CCCGGCGCTACAGGCCCCGCCGCTGTGGTGGGMCCGCCAGCGGCGACGACMCCAGAGCTCC 668 P 6 A T G C G A V V G T G S G E D N O S S 63

ACGCGAGMCCGGGAGCCCCGGCGAGCGGCGMGT~TGGCTCCCCGGCCGTCGGCGGGCTA 7 3 1

GTGGTGAGTGCTCAGGCCCTGGGAGTGGGTGTCTTCCTGGCGGCCTTCATCCTCACCGCTGTG 794 T G E P G A A A S G E V N G S A A V G C L 84

GCGGGCMCCTGCTGGTCATCCTTTCGGTGGCCTGCMCCGCCACCTACAGACGGTCACCMC 857 V V S A O C V G V G V F L A A F I L T A V 105

A G N L L V I L S V A C N R H L O T V T N 126

Y F I V N L A V A O L L L S A A V L P F S 1 4 7

GTCCACGTGCTGTGCTCCACTGCCTCCATCCTTAGCCTCTGCACCATCTCTGTGGACCGGTAC 1 0 4 6 A T M E V L G F W A F G R T F C D V W A A 168

CTCCGTGTGCGCUCTCGCTCMGTACCCAGCCATTATGCAGAGCGCMGGCCGCTGCCATT 1109 V D V L C C T A S I L S L C T I S V D R Y 1 8 9

. "" . . ~~

TATTTCATCGTGMCCTGGCCGTGGCTGACCTGCTTTTGAGTGCAGCTGTGTTGCCCTTCTCA 920

GCCACTATCGAGGTTCTACC~TTCTGGGCCTTCGGCCCMCCTTCTGCGACGTCTGG~C~~CC 983

CTCGCTCTCCTTTCGGCGGTGGCCCTGGTGGTATCTGTGGGACCGCTACTAGGTTGGMGGAG 1 1 7 2 V G V R H S L K Y P A I M T E R K A A A I 210

CCAGTGCCCCCGGATCAGCCTTTCTGCGCUITCACCGAGCAGCTGGGCTATCCMTCTTCTCT 1235 L A L L W A V A L V V S V G P L L G W K E 2 3 1

TCCGTGTGCTCCTTCTACCTACCCATGGCAGTGATCGTGGTCATGTACTCCCGCCTCTACCTC 1298 P V P P D E R F C G I T E E V C Y A I F S 252

GTCGCACGCAGCACTACGCGCAGCCTCGAGGCAGGCATCMGAGGGAGCCCGGCMGGCCTCC 1 3 6 1 S V C S F Y L P M A V I V V M Y C R V Y V 273

GAGGTGCTTCTGAGGATCCACTCTCGCGCCGCAGCGACCAGCGCC~GGATATCCCG~CA 1424 V A R S T T R S L E A G I K R E P C K A S 294

CAGAGTAGCMGGGCCACACCTTGCGCAGCTCGCTTTCCCTGAGGCTGCTCMGTTTTCCCGC 1 4 8 7 E V V L R I H C R G A A T S A K G Y P G T 315

GAG~GGCTGCCMGACCTTCGCCATCGTCGTGGGTGTCTTCGTCCTGTGCTGGTTCCCC 1 5 5 0 O S S K C H T L R S S L S V R L L K F S R 336

TTCTTCTTCGTCCTGCCTCTG~CTCTCTGTTCCCGCAGCT~CCGTCAGAGGGTGTCTTC 1613 E K K A A K T L A I V V C V F V L C W F P 357

MGGTCATCTTCTGGCTGGGTACTTCMTAGtTCTGTGMCCCCCTCATCTACCCCTGCTCC 1 6 7 6 F F F V L P L G S L F P O L K P S E G V F 378

AGTCGCCAGTTCMGCGCGCCTTCCTCCGCCTCCTGCGC~GCCAGTGTCGCCGCCGCCGCCGC 1739 K V I F W L G Y F N S C V N P L I Y P C S 399

S R E F K R A F L R L L R C O C R R R R R 420 CGCCTCTGG~TCTACGGCCACCACTGGCGAGCCTCGCCGGC~CGCCCCTTCCGACTGCGC 1 8 0 2 R L W S L R P P L A S L D R R R A F R L R 4 4 1

CCCCAGCCCTCGCATCGCTCCCCCCGGGGCCCCTCTAGCCCTCACTGCACACCCGGGTGGGG 1865 P O P S H R S P R G P S S P H C T P G C G 462

CTCGGCAGACACGCCGGAGACGCAGGATTCGGTCTCCAGCAGTCCAMGCCAGCCTCCGCCTC 1928 L G R H A G D A G F G L O O S K A S L R L 483

CCGCAGTGGAGACTGCTCGGGCCGCTACA~GACCCACGACCCAGCTGCGTGC~GGTGTCC 1 9 9 1 R E W R L L C P L O R P T T O L R A K V S 504

AGCCTGTCCCATMGATTCGTCTGGGGCCCGGCGCGCGGAGACTGCGTGTGCCCTGCGCTCC 2054

GACGTAGMGCAGTGTCCCTAMTGTTCCCCMGATGGGGCAGMGCTGTCATCTGCCAGGCT S L S H K I R S C A R R A E T A C A L R S

TATCAGCCGGGCGACTACAGCMCCTCCGAGAGACTGACATTTMGGACC~CTCAGGCTG E V E A V S L N V P O D G A E A V I C O A

52 5 2117

54 6 2180

560 2 2 4 3 2306 2369 2432

ACAMTTCTTCTTTTGCTGCTCCCAGGG~GATCCGTGGACAGTTTCTACTCCTTACTGCC 2 5 5 8 2495

ACCTGGAGGMCTCMTCGACCTACCATAGGACCTGCCATCCGCtMGTTTTGGTGGCGTATT 2 6 2 1

CCTMCTCTATGTTCCCCTCACCATACCCCCCGCCCCGGCCCTTACTCTTTACACTGTGTACA 2747 TAGTTCATTGTGCCTGTCTGTGCCCATCACTTCTCACTGGGATTCCAGGCTGCCAMGGGWG 2816 AGCCTTATTTATCATTTTC~CCTATTTATTGGTMGAGTACTTATATTTTGTTCAGACTGT 2873 - GC~CTCTCCATCTTTGCCTTGTMTGCATATAGAGCCTCCTTCTTCTG~TCCTTAGACA 2936

2948

CGATGCCCGCCGMCAMTCTCTTGCTCTTCCGCTCTGGGCCCACTGCCCTCTTCCCCACACT 2 6 8 9

FIG. 1. Restriction map, nucleotide sequence, and deduced amino acid sequence of the rat A42 alAR clone. 5'-Untranslated region = 480 bp, open reading frame = 1680 bp, 3'-untranslated region = 789 bp. The single-letter amino acid code is used.

onstrates the putative topology and the amino acid identities with the rat a l B receptor. There are two potential sites for N- linked glycosylation in the amino terminus (Asn-60 and Asn- 76). Several serine and threonine residues present in the intracellular loops and carboxyl terminus may represent po- tential sites for protein kinase C phosphorylation. In uitro phosphorylation of the alR-adrenergic receptor by protein kinase C and A has been reported and may represent a

mechanism for receptor regulation (33,34). Interestingly, this receptor lacks a consensus site for protein kinase A phos- phorylation, while both the a l ~ - and alc-adrenergic receptors contain a conserved consensus protein kinase A site in the third cytoplasmic loop.

In order to examine the ligand binding characteristics of RA42 a PstI restriction fragment containing the entire 1680- bp coding region and 140 bp of 5'-untranslated region was inserted into the mammalian expression vector pCMV5 (23). The resulting construct pCMV5alRA42 along with the con- structs for the rat a l B receptor PcDV~IB, and the bovine alr receptor pBC12BIalc (19), were used to transiently transfect COS-7 cells. COS-7 cells transfected with any one of the three vectors were able to bind the a antagonist ['"I]HEAT in a saturable manner with high affinity (KO, 70-100 pM). The level of expression for RA42 (pCMV5alRA42) was approxi- mately 400 fmol/mg protein, while that of the a l ~ and alc was approximately 2 pmol/mg of protein. Nontransfected COS-7 cells exhibited no specific binding. Competition curves for the binding of ["'I]HEAT to membranes expressing RA42 using adrenergic agonists and antagonists showed appropriate cyl-

adrenergic rank orders of potency with stereoselectivity. Table I shows the K; values for various adrenergic com-

pounds. It is apparent that the three receptors display unique rank orders of potency for adrenergic ligands. The RA42 receptor is distinguished from both the a l B and a l C subtypes by having approximately 10-fold higher affinity for the ago- nists norepinephrine, epinephrine, and phenylephrine. I t is further distinguished from the a l B subtype by methoxamine and WB4101 and from the alc subtype by oxymetazoline and phentolamine. The alB and alr subtypes are distinguished from each other by methoxamine, oxymetazoline, phentola- mine, and WB4101 as described previously (19).

Some of the most compelling pharmacological evidence to suggest heterogeneity of alAR comes from competition exper- iments which show a 10-fold difference in affinity for the antagonist WB4101 at a1 binding sites in hippocampus and cerebral cortex. In these brain regions, the high affinity alAR binding site (KIl = 1 nM) has been designated as the a1AAR as suggested by Morrow and Creese (15) while the low affinity binding site (KD = 10 nM) typified by the hamster alAR has been designated as the alRAR. The high affinity of the RA42 receptor for WB4101 (Kn = 2.1 nM) suggests that it represents the a l ~ subtype. In addition, it has been reported that the alAAR subtype has a higher affinity for the agonists phenyl- ephrine, methoxamine, and oxymetazoline, and antagonist phentolamine than does the alRAR subtype (15, 17, 35). The RA42 receptor has a higher affinity for phenylephrine, meth- oxamine, and phentolamine than does the RaIH receptor, again suggesting that it encodes for the pharmacologically defined a 1 A subtype.

To further characterize the RA42 receptor, Northern blot analysis was performed with cDNA probes from all three cyI -

adrenergic receptors to map receptor distribution in various rat tissues. Previous pharmacological studies have shown the ~ I A receptor to be expressed in the following rat tissues: cerebral cortex, hippocampus, and vas deferens, while the alB subtype is expressed in cerebral cortex and liver (15, 17). As shown in Fig. 3, at high stringency the RA42 probe detects a 3.0-kb mRNA species which is most abundant in vas deferens followed by hippocampus, cerebral cortex, aorta, brain stem, heart, and spleen. Therefore, the tissue distribution of RA42 further confirms that it represents the alA receptor. The alB probe detects two mRNA species (2.4 and 3.0 kb) in liver, followed by a single 2.4-kb species in heart, cerebral cortex, brain stem, kidney, lung, and spleen. This distribution fits

6368

FIG. 2. Seven transmembrane- spanning model of the new rat al- adrenergic receptor subtype. Solid circles indicate amino acids identical to the corresponding position in the rat a I B - adrenergic receptor. Transmembrane domains are defined by hydropathicity analysis (30). Potential sites of N-linked glycosylation are indicated by crosses.

TABLE I Pharmacological ligand binding profile

of various expressed a lAR subtypes Membranes from COS-7 cells transfected with either

pCMV5alRA42, pcDVIRalR, or pBC12BIalc were incubated with the nlAR antagonist ["'I]HEAT in the absence or presence of increasing concentrations of various adrenergic ligands. From the competition curves generated K,, values for each competing ligand was estimated using an iterative nonlinear regression program (28). The results shown are representative of at least two experiments. ND, not deter- mined.

Rat 0 1 A Rat e I ~ Bovine mIc

Agonists (-)Epinephrine 546 4,690 6,250 (-)Norepinephrine 100 10,500 9,730 (+)Epinephrine 18,100 ND ND Methoxamine 110,000 1,610,000 203,000 Phenylephrine 1,440 23,900 47,800 Oxymetazoline 2,140 824 114

Antagonists Prazosin 0.33 0.56 0.37 Phentolamine 111 340 15.3 Indoramine 611 226 ND Corynanthine 253 517 142 WB4101 2.1 28.6 0.68

that obtained from pharmacological studies for the aIBAR. The presence of two mRNA species in rat liver has been previously detected with a probe from the hamster a I R cDNA (37). The two species are most likely the result of differential polyadenylation of the a I B subtype mRNA. The fact that both species are detected equally at high stringency with the a I B

probe, and not a t all with the aIA or a1c probes, makes it unlikely that the two species actually represent different a1 receptor subtypes. The probe from the bovine aIc receptor did not detect a species of mRNA in any of the rat tissues tested (data not shown), consistent with previous results (19), even though high stringency Southern blots (not shown) revealed that the rat contains an analogous gene to the bovine a l c receptor gene. The fact that the bovine receptor is seemingly not expressed in any of the rat tissues known to contain the alA subtype is one of the reasons that though this receptor has a high affinity for WB4101, it cannot represent the a l ~ receptor subtype as described in the literature.

A further characteristic of the a1AAR subtype is that i t appears to be insensitive to treatment in hypotonic buffer

RA42

a l B

- 7.5 -4.4 -2.4 -1.4

- 7.5 - 4.4 - 2.4 - 1.4

FIG. 3. Northern blot of rat tissues probed with RA42 and Rale. Each lane contains 10 pg of poly(A)'-selected RNA. After hybridization, the filters were washed successively in 2 X SSC, 0.1% SDS at room temperature and then 0.1 X SSC, 0.1% SDS a t 55 "C, and exposed a t -70 "C for 5 days.

with micromolar concentrations of the alkylating agent CEC, while the a I ~ subtype is 95%+ inactivated (17). To assess the effect of CEC on the putative alA receptor RA42, transfected COS-7 cell membranes were incubated with 10 p~ CEC for 10 min a t 37 "C, washed extensively, and assessed for aIAR binding with [1251]HEAT. The results show that while 10 pbi CEC inactivates 75% of the c q ~ receptor, only 15% of the RA42 receptor is inactivated. These results demonstrate that the RA42 receptor is less susceptible to inactivation by CEC than the a l~AR.

Previous work has localized the gene encoding for the hamster ~ R A R to human chromosome 5q2R.qR2 (38), while the gene encoding for the bovine alcAR localized to human chro- mosome 8 (19). The gene encoding for the a I ~ receptor RA42 was also localized to human chromosome 5 by in situ hybrid- ization of a 0.85-kb BumHI fragment from the partial RAL cDNA clone to human metaphase chromosomes. Of 61 grains over 50 cells analyzed, 11 (18%) were over the chromosomal region 5, ,2~-~2, the same region which contains the a l ~ A R gene. No other chromosomal sites were labeled above background. To further confirm the chromosomal localization of RA42, in

cDNA for alA-Adrenergic Receptor 6369

receptors, their regulation, and molecular mechanisms of sig- nal transduction.

IC. 7I 1.

Acknowledgments-We would like to thank Donna Addison for expert assistance in preparation of the manuscript and Pam Szklut and Sabrina Exum for superb technical assistance.

1 2 REFERENCES 1. 2.

Lefkowitz, R. J., and Caron, M. G. (1988) J. Biol. Chem. 263,4993-4996 Dohlman, H. G., Caron, M. G., and Lefkowitz, R. J. (1987) Biochemistry

Wang, H., Lipfert, L., Malbon, C. C., and Bahouth, S. (1989) J. Bid. Chem.

Kobilka, B. K. Kobilka T. S. Daniel K. Regan J. W., Caron, M. G., and

O'Dowd, B. F.. Lefkowltz. R. J.. and Caron. M. G. (1989) Annu. Reu.

26,2657-2664

264,14424-14431

Lefkowitz, R. J. (1988) Scie'nce 2 4 0 , 1hO-131'6

3.

4.

5.

6.

7.

Neurosci. 12; 67-83

Sym Q u a d Biol. 53,487-497

Exp. Ther. 245,600-607

Dixon, R. A. F. Sigal, I. S., and Strader, C. D. (1988) Cold Spring Harbor

By1undl.D. B., Ray-Prenger, C., and Murphy, T. J. (1988) J . Pharmacol.

Bylund, D. B. (1985) Pharmacol. Biochem. Behau. 22,835-843 Murphy, T. J., and Bylund, D. B. (1987) J. Pharmacol. Exp. Ther. 2 4 4 ,

671 -67Q

31 I............. 8. 9.

10. 11.

Bylund, D. B. (1988) Trends Pharmacol. Sci. 9 , 356-361 Kobilka, B. K., Matsui, H., Kobilka, T. S., Yang-Fen , T. L., Franke, U.,

Caron, M. G., Lefkowitz, R. J., and Regan, J. W. r1987) Science 238 ,

Re an, J W Kobilka, T. S. Yang-Feng, T. L. Caron, M. G. Lefkowitz, 650-656

6305 h. J., and Kobilka, B. K. (i988) Proc. Natl. A&. Sci. U. S. A. 85,6301-

Lomasney, J. L., Lorenz, W., Allen, L. F., King, K., Regan, J. W., Yang- Feng, T. L., Caron, M. G., and Lefkowitz, R. J. (1990) Proc. Natl. Acad.

Zeng, D., Harrison J. K., D'Angelo, D. D., Barber, C. M., Tucker, A. L., Sci. U. S. A. 87,5094-5098

Lu, Z., and Lynch, K. R. (1990) Proc. Natl. Acad. Sci. U. S. A. 8 7 , 3102- 3106

Morrow, A. L., and Creese, I. (1986) Mol. Pharmacol. 2 9 , 321-330 Han, C., Abel, P. W., and Minneman, K. P. (1987) Nature 329,333-335 Minneman, K. P., Han, C., and Abel, P. W. (1988) Mol. Pharmacol. 33 ,

509-514 Cotecchia, S., Schwinn, D. A,, Randall, R. R., Lefkowitz, R. J., Caron, M.

$;:,and Kobilka, B. K. (1988) Proc. Natl. Acad. Sci. U. S. A. 8 5 , 7159-

".l V . V

5 FIG. 4. Autoradiographic silver grain distribution on hu-

man chromosome 5 after in situ hybridizations with cDNA and genomic DNA probes from the RA42 alA-adrenergic receptor.

12.

13.

14. situ chromosomal hybridization was performed with a ge- nomic RA42 probe which consisted of 450 bp of 5"untrans- lated region. Of 91 grains scored from 51 cells, 12 (13.2%) were found to be at chromosome bands 5q23-31 (Fig. 4). This makes the alAR the third adrenergic receptor to map to this position along with the a1BAR and &AR. The close proximity of three adrenergic receptors on the same chromosome sug- gests that this family of proteins may have arisen by gene duplication (38).

The genes encoding for the hamster alB and bovine alC receptors both contain introns. This gene structure makes these two receptors unique among members of the adrenergic receptor family since the genes encoding for the other six receptors (Dl, B2, p3, ale, a2cl0, a2c4) are intronless (11-14, 18, 19,39-41). The introns in the aIB- and alc-adrenergic receptor genes are located in an analogous region of the alA receptor gene which we have isolated from a cDNA clone. Therefore, it is not known whether or not the alA-adrenergic receptor gene contains introns.

The present study demonstrates unequivocally the exist- ence of three alAR subtypes. The rat alAR RA42 has higher affinity for WB4101, phenylephrine, methoxamine, and phen- tolamine and is less sensitive to inactivation by the alkylating agent CEC than the RalBAR subtype. The unique pharma- cology of this receptor, coupled with the expression of RA42 in rat tissues such as vas deferens, cerebral cortex, hippocam- pus, and aorta, demonstrate that this receptor represents the alAAR subtype.

The techniques of molecular biology have made it possible to identify genes encoding for adrenergic receptors before characterization of the receptor protein. The availability of three alAR with distinct molecular properties may provide a basis for the development of new drugs. The availability of the cDNAs encoding for three distinct alAR subtypes will also facilitate studies of the various structural domains of the

15. 16. 17.

18.

Schwinn, D. A., Lomasney, J. W., Lorenz, W., Szklut, P. J., Fremeau, R. T., Jr., Yang-Feng, T. L., Caron, M. G., Lefkowitz, R. J., and Cotecchia, S. (1990) J. Biol. Chem. 265,8183-8189

Okayama, H., Kawaichi, M., Brownstein, M., Lee, F., Yokota, T., and Arai, K. (1987) Methods Enz mol 154,3-28

Maniatis, T., Fritsch, E. J., and Sambrook, J. (1982) Molecular C h i n A Laboratory Manual, pp. 365-370, Cold Spring Harbor Laboratory, t o ld Spring Harbor NY

Andersson S. Davis D. L. Dahlback H., Jornvall, H., and Russell, D. W. Southern, E. M. (1975) J. Mol. Biol. 9 8 , 503-517

(1989) Jy B$I. Chek. 26k, 8222-8$29 Cullen, B. R. (1987) Methods Enzymol. 152,684-704 Vieira, J., and Messing, J. (1982) Gene (Amst.) 19 , 259-268 Sanger F. Nicklen S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U.

Chirgwin, J. M. Przybyla A. E. MacDonald, R. J., and Rutter, W. J.

DeL-ea?, A:, Hancock, A. A., and Lefkowitz, R. J. (1982) Mol. Pharmacol.

IIOJ

S. A.'74,5463-5A67

(1979) Biochemistry 18,'5294-5'299

19.

20.

21.

22. 23.

24. 25. 26.

27.

28.

29. Yang-Few, T. L., Landau. N. R.. Baltimore. D.. and Franke, U. (1986) Zl, 3-lti

Cyto end Cell Genet. 43,121-126 I ,

Kyte, f , and Doolittle, R. F. (1982) J. Mol. Biol. 157 , 105-132 Voigt, M. M., Kispert, J., and Chin, H. M. (1990) Nucleic Acrds Res. 18,

1 nc'3

30. 31.

Buckland. P. R.. Hill. R. M.. Tidmarsh. S. F.. and McGuffin, P. (1990) L"d0

32.

33.

34.

NucleicAcids Res. is, 682 '

Lefkowltz, 8. J.'(1987)'>. Biol. Chim. 262,3098-3105

I L Biol. Chem. 262 3106-3113

~ ~~ ~ ~~~ , -

Leeb-Lundber , L M F Cotecchia S. DeBlasi A,, Caron, M. G.,

Bouvier, M.. Leeb-Lundberg, L. M. F., Benovic, J. L., Caron, M. G.,

Minneman,K.-P.(1988) Pharmacol. Res. 40,'87-119 Han. C.. Abel. P. W.. and Minneman, K. P. (1987) Mol. Pharmacol.

and

and Lefkowitz. R. .J. (1987) I

35. 36.

37. Rossh , s. P., Lumkin, C. K., Taylor J. M. McGehee R. E. and Cornett L. 8. (1989) 71st Annual Meeting'of the'Edocrinh Socidy, p. 40, The'

Yang-Feng, T. L. g u e F. Zhon W Cotecchia, S., Frielle, T., Caron, M. Endocrine Societ , Bethesda, M D

G., Lefkowitz, R. J.,'add Frante, d. (1990) Proc. Natl. Acad. Sci. U. S. A. 8 7 , 1516-1520

Emorine, L. J., Marullo, S., Briend-Sutren, M.-M., Patey, G., Tate, R., Delavier-Klutchko, C., and Strosberg, A. D. (1989) Science 2 4 5 , 1118-

Kobilka, B. K., Frielle, T., Dohlman, H. G., Bolanowski, M. A., Dixon, R. 1121

A. F., Keller, P., Caron, M. G., and Lefkowitz, R. J. (1987) J. Biol. Chem. 2 6 2 , 7321-7327

Frielle, T., Collins, S., Daniel, K., Caron, M . G., Lefkowitz, R. J., and Kobilka, B. K. (1987) Proc. Natl. Acad. Scr. U. S. A. 84. 7920-7924

38.

39.

40.

41.