THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 35, Issue of December 15, pp. 24053-24058 1991 Printed in C? S.A.

An Adipose Tissue-specific @-Adrenergic Receptor MOLECULAR CLONING AND DOWN-REGULATION IN OBESITY*

(Received for publication, July 15, 1991, and in revised form, September 4,1991)

Patrick MuzzinS, Jean-Pierre RevelliS, Francoise KuhneS, Jeannine D. GocayneQ, W. Richard McCombiep, J. Craig Venters, Jean-Paul GiacobinoS, and Claire M. Frasern)) From the SDepartement de Biochimie Medicale, Centre Medical Universitaire, I21 I Geneve 4, Switzerland, the §Section of Receptor Biochemistry and Molecular Biology, Natwnul Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Marylnnd 20892, and the llSection of Molecular Neurobiology, Laboratory of Physiologic and Pharmacologic Studies, National Institute on Alcohol Abuse and Alcoholism, Rockville, Maryland 20852

Clones encoding an atypical &adrenergic receptor were isolated from a rat brown adipose tissue cDNA library. This receptor expressed in Chinese hamster ovary (CHO) cells displays a low affinity for @-adre- nergic antagonists and a high affinity for BRL 37344, an agonist that selectively stimulates lipolysis in adi- pose tissue. The rank order of potency for agonist- mediated increases in intracellular cAMP in trans- fected cells correlates with that for agonist-mediated stimulation of lipolysis in brown adipocytes. Northern blot analysis demonstrates that this receptor subtype is expressed only in brown and white adipose tissue where it represents the predominant &receptor sub- type. The amount of atypical &adrenergic receptor present in adipose tissue of obese (fa/fa) Zucker rats is reduced by up to 71% as compared with lean (Fa/Fa) control animals. These findings suggest that a change in the expression of this &adrenergic receptor subtype may play a role in obesity.

Brown adipose tissue is the main effector of cold- and diet- induced thermogenesis in rodents (1, 2), a process that can represent a major expenditure of energy and play an impor- tant role in overall energy balance (3, 4). Because brown adipose tissue has been demonstrated in humans of all ages (5, 6) and is often atrophied and quiescent in obese animals (7), much interest has recently been directed toward devel- opment of agents that stimulate this metabolic response as possible anti-obesity agents.

Brown adipose tissue metabolism is primarily controlled by norepinephrine released from sympathetic nerve terminals that acts through @-adrenergic receptors (0-ARs)’ (€49). Both PI- and &AR subtypes are present in rat brown adipose tissue (10, 11); however, pharmacological studies with novel ther- mogenic @-adrenergic agonists have suggested the existence

* This work was supported, in part, by Grant 32-26331-89 from the Swiss National Science Foundation and by grants-in-aid from the Ingeborg Naegeli, Ernst and Lucie Schmidheiny, Geigy-Jubilaums, and Roche Research Foundations. 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 accord- ance 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(s) M74716.

dressed. 11 To whom correspondence and reprint requests should be ad-

’ The abbreviations used are: B-AR, 8-adrenergic receptor; CHO, Chinese hamster ovary; IBAT, interscapular brown adipose tissue; bp, base pair(s); ICYP, iodocyanopindolol.

of an atypical @-AR in this tissue which mediates lipolysis (12). Parallel studies have also suggested the presence of atypical @ARs that exhibit similar pharmacological proper- ties in white adipose tissue (13, 14), digestive tract (15, 16), and skeletal muscle (17, 18).

Recently, a human gene was isolated that encodes for an atypical @-AR distinct from PI- and &-AR subtypes (19). This receptor, referred to as the P3-AR, displays a rank order of agonist potency for stimulation of cAMP accumulation dis- similar to that of PI- or 0,-AR subtypes. Northern blot analysis of poly(A)+ RNA from rats suggested that the &AR is ex- pressed in adipocytes, liver, skeletal muscle, and ileum (19). In part, because the reported pharmacology of the P3-AR was inconsistent with that of the atypical P-AR in brown adipose tissue (12), we constructed and screened a rat interscapular brown adipose tissue (IBAT) cDNA library with DNA probes encoding human &- and P2-ARs under conditions of low stringency. Several cDNA clones were isolated that encode a receptor which displays greater amino acid sequence similar- ity with the &AR than with either &- or P2-AR subtypes.

Characterization of the rat &-AR stably expressed in CHO cells demonstrates that the order of agonist potency for ac- cumulation of intracellular cAMP correlates with that for stimulation of lipolysis in brown adipocytes (12). The tissue distribution of the rat P3-AR mRNA suggests that this may represent a fat cell-specific P-AR subtype. Furthermore, the levels of P3-AR are markedly reduced in obese rats, and this finding may, in part, provide a molecular basis for the decrease in the responsiveness of adipose tissue to catecholamines in obesity.

MATERIALS AND METHODS

All organic and inorganic chemicals were of analytical or molecular biology grade. Restriction endonucleases were from New England Biolabs or Biofinex (Praroman, Switzerland). Prepacked oligo(dT)- cellulose columns and oligo(dT)12-18 were obtained from Pharmacia LKB Biotechnology Inc. Hybond N membranes were purchased from Amersham. Genescreen Plus membrane filters, [Y-~~PI-ATP (1000- 3000 Ci/mmol), and [’251]iodocyanopindolol (2200 Ci/mmol) were from Du Pont New England Nuclear. CHO-K1 cells were from the American Type Culture Collection. Tissue culture reagents were from GIBCO/Bethesda Research Laboratories. IC1 118,551 was a gift from IC1 (Macclesfield, U. K.), betaxolol was a gift from Synthelabo (Tours, France), BRL 37344 was a gift from Beecham Pharmaceuticals (Ep- son, U. K.), and alprenolol was gift from Hassle (Malndal, Sweden).

Rat ZBAT cDNA Library Preparation and Screening-Interscapu- lar brown adipose tissue from 5-6 male Sprague-Dawley rats was excised and rapidly frozen in liquid nitrogen. Total RNA was isolated by the cesium trifluoroacetic acid gradient method of Okayama et al. (20). Poly(A)+ RNA was isolated from total RNA using prepacked oligo(dT)-cellulose columns (21). The rat IBAT cDNA library was constructed in X Zap I vector (Clontech, Palo Alto, CA). The library

24053

24054 Fat Cell P-Adrenergic Receptor was screened under conditions of low stringency using a human genomic P1-AR probe' and a rat heart j3,-AR cDNA probe (22) (hybridization in 30% formamide, 6 X SSC, 5 X Denhardt's, 50 mM sodium phosphate (pH 7.4), 50 pg/ml sheared salmon sperm DNA at 42 "C for 18 h; washing in 2 X SSC, 0.1% sodium dodecyl sulfate a t room temperature, 0.5 X SSC at room temperature and 0.5 X SSC at 42 "C for 30 min each). Nine positive cDNA clones were isolated from >los recombinants screened. For sequence analysis, positive clones were excised from A ZAP, subcloned into pBluescript, and subjected to exonuclease I11 digestions as described previously (23). Plasmid DNA from deletion time points was isolated and purified using Qiagen columns. DNA was sequenced using Toq polymerase cycle sequencing kits and Applied Biosystems 373A DNA sequencers (Applied Biosys- tems, Inc., Foster City, CA). Greater than 90% of the final sequence was obtained from templates from both strands with at least a 2-fold redundancy in each orientation.

Stable Expression of the Rat &AR in CHO-Kl Cell.+" 1.5 kilobase fragment was excised from pBluescript using Sac1 (present in the multiple cloning site of the vector) and BumHI (contained within the cDNA clone at bp 1665) and inserted into the SacI/BamHI sites of the expression vector, pSVL (Pharmacia). CHO-K1 cells were co- transfected with pSVL containing the j3-AR and pMSVneo (24) using the calcium phosphate precipitation technique. Stable transfectants were obtained by growth of the cells in culture medium containing Geneticin (500 pg/ml). Colonies derived from single cells were isolated and expanded as previously described (24). Because the atypical p - ARs in rat adipose tissue display a low affinity for &adrenergic antagonists such as [1261]ICYP, cell lines were screened for the expres- sion of j3-ARs by measuring isoproterenol-mediated increases in in- tracellular CAMP.

Radioligand Binding Assuys-Membrane-associated @ - A b (3-6 pg of protein) were labeled with increasing concentrations of ['251]ICYP in the presence and absence of 10 p~ IC1 118,551 by incubation in Hank's buffer or with i3H]CGP 12177 in the presence and absence of 10 pM BRL 37344 in 50 mM Tris-HC1, pH 7.4, 10 mM MgC12, 50 p~ GTP at 37 'C for 30 min. Incubations were terminated by filtration over Whatman GF/C glass fiber filters using a Brandel cell harvester. Scatchard analysis of saturation isotherms was performed to yield estimates of K d (equilibrium dissociation constant) and Elrnax (total number of binding sites). The Kd value was utilized in computer analysis of competition displacement curves.

For competition studies, membranes (containing 3-4 fmol of ['''I] ICYP binding sites) were incubated with ['251]1CYP (-1 X K d con- centration) plus the indicated concentrations of competing ligands. Competition curves were analyzed according to a mass action model for receptor-ligand interactions using a computerized iterative non- linear least squares curve-fitting program (GraphPAD INPLOT, San Diego, CA). Competition experiments were performed two to three times in triplicate. Triplicate values from each experiment were averaged, and nonlinear regression was performed on data averaged from all curves for a given ligand.

cAMP Determinations-Confluent cultures of cells were incubated in Hank's solution containing 1 mM 3-isobutyl-1-methylxanthine plus the indicated concentrations of agonists for 20 min at 37 "C. Cyclic AMP concentrations were measured using a cAMP scintillation prox- imity assay (Amersham). Data were analyzed using a nonlinear least squares curve-fitting program (GraphPAD INPLOT, San Diego, CA).

Northern Blot Analysis-Poly(A)+ RNA was isolated from IBAT, epididymal white adipose tissue, brain, heart, ileum, liver, and lungs of male Sprague-Dawley rats. Fifteen pg of poly(A)+ RNA were electrophoresed in a formaldehyde-agarose gel as described (25) and transferred to GeneScreen Plus membranes by capillary blotting. A rat genomic &-AR DNA, a rat heart &-AR cDNA, and the rat h-AR cDNA were labeled by random priming with [ C Y - ~ ~ P ] ~ C T P t o specific activities of approximately 1 X lo9 dpm/pg of DNA. RNA blots were hybridized and washed under high stringency conditions. As a control, an oligo(dT)lz_18 probe was labeled with [y3'P]ATP using T4 poly- nucleotide kinase. Densitometric analysis of autoradiograms was performed with a high resolution densitometer. The Student's un- paired t test was used to determine statistical significance. To study the effects of obesity, mRNA from adipose tissues of male lean (Fa/ Fa) and obese (fa/fa) Zucker rats (9 weeks old) was isolated, and Northern blot analysis was performed as described above.

J. C. Venter, D. A. Robinson, and C. M. Fraser, unpublished results.

RESULTS

Cloning and Sequence Analysis of the Rat B3-AR-Several clones isolated from a rat brown adipose tissue cDNA library were demonstrated by restriction mapping to be different from P1- and &-AR cDNAs. Sequence analysis of a 2.3- kilobase pair cDNA clone revealed the presence of a single open reading frame of 1200 bp. This region encodes a poly- peptide of 400 amino acids with a predicted size of 43,169 daltons (Fig. 1). The nucleotide sequence contained within the open reading frame of the rat IBAT 0-AR was most similar to that of the human P3-AR (19) with 79% sequence identity (Fig. 2A). The deduced amino acid sequence of the rat IBAT B-AR is 49% and 40% identical with that of rat PI (26) and &ARs (27), respectively, and 80% identical with the human &.-AR described by Emorine et aE. (19) (Fig. 2B). Because of the high sequence similarity between this receptor and the human &AR, we defined this receptor as a rat 03- adrenergic subtype.

The rat P3-AR exhibits several structural features common to G protein-coupled receptors. It contains seven regions of hydrophobic sequence that are presumed to represent trans- membrane spanning domains and two putative sites of N- linked glycosylation sites (N-X-S/T) in the amino terminus (Fig. 2B) . Furthermore, the P3-AR contains several conserved amino acids that have been demonstrated to play important roles in B-AR-ligand interactions and receptor activation by agonists including Aspso, Asp114, Asp131, Cyslo7, Cyslffl, Cys"*, CYS"~, SerZw, and SerzW (28-31).

Permanent Expression of the Rat P3-AR in CHO Cells- Using a protocol for co-transfection of CHO cells, we obtained several clonal cell lines derived from single cells that perma- nently express the rat P3-AR. We chose one cell line that exhibited an approximate 20-fold isoproterenol-induced

Fat Cell P-Adrenergic Receptor 24055

- 3 1 CTggqqqq.aa.CTtCCcatCC~~G~=g=g~C~=G~GATGCCTCCG~GCCCTCAC~A~MC -37 tTCCttCtttCCCT.CCgCECC~~G~q=g~~C~q~GATGCCTCCGTGCCCTCACgAgMC

-T--"-"---CT-CC---CC--G------C--G-GATGCCTCCGTGCCCTCAC-A-MC

25 gGCTCTCTgGCtttCTGGtC=GACgCCCCCACCtTGG.CCC~gTgC~GCCMCACCAGTG 2 5 ~GCTCTCTtGCEC~~TGG~CgGAc~tCCCcAcc~TGG~gCccAaT~c~GccMCACCAGTG

-GCTCTCT-GC----TGG-C-GAC--CCCCACC-T~~~--CCCA-T-C-GCCMCACCAGTG

16 GGtTGCCAGGGGGT9CCaTGG==GCGCC~tTqGCtGC~GC~tTGCTGGC~TGGC g6 GGsTGCCAGGCOTtCCqTG~gGC~~~T~GCcGG9GC~~TGCTGGCGCTGGCggtq~t

GG-TGCCAGGCGT-CC-TGGC--GCGCC--T-GC-GC-GC--TGCTGGCGCTGGCggtg~t

1 4 1 CACpGTGGMGCCMCCTGCTGGT~ATC~~~Gc~ATCGCC~G~ACgCCGAGACT~CAG I41 qgCCACCGTGGAGCCMCCTGCTGGT=ATCgtgGCChTCGCCtGgACtCCGAGACT~CAG

qqc~C-GTGCGAGGCMCCTGCTGGT-ATC---GC-ATCGCC-G-AC-CCGAGACT-CAG

1 0 8 ACCATgACCMCGIGTTCGTGAC~TCGCTGGCCqCAGC~~C~TGGTg~TGGGACTCCTgG 199 ACCAT~CCMCGTGTTCGTGACTTCGCTGCCCaCIGCtMCtTGGT~qTGGGACTCCT=G

ACCAT-ACCMCGTGTTCGTGACTTCGCTGCCC-CAGC-GAC-TGGT"TGGGACTCCT-G

260 T~~TGCC~CC~~~GCCCACC~TTGGCGCTGACTGGCCACTGGCC~TTGGCCGC=ACTGGCTG 269 TqgTGCCqCCqGcGCCCAcCTTGGcGCTGACTGGCCACTGGCCgTTGGGCGC~ACTGGCTG

T--TGCC-CC-G-GGCCAC-TTGGCGCTGACTGGCCI)CTGGCC-TTGGGCGC-ACTGGCTG

330 CGIGCTGTGGIC~TC~GTGMCGTGCT~TGTGT~AC~GCCAGCATCG~ACCCTGTGCGCC

CGAGCTGTGGAC-TC-GTGGACGTGCT-TGTGT-AC-GCCAGCATCGA-ACCCTGTGCGCC

3 2 1 C G A G C T G T G M C g T C a G T G C C G T G C T ~ T G T a l C r C C A C C C T G T G C G C C

382 CTGGCtGTaMCCGCtACCTaGC=GT~ACCMCCCtCTGCGTTACGCC~CgCTGGTtACCA 391 CTGGC=GTqMCCGCTACCTg~tGTgACCMCCC~TGCGTTACGCCgC~CTGGT~ACCA

CTGCC-GT-GACCGCTACCT-GC-GT-AC~CCC-CTGCGTTACGCC-C-CTGGT-ACCA

4 4 3 AGCGCEGCGCCCGG~C'IGC~GTCCIGGICTGGTGT~~TCGTGTC=GCC~C=GTGTC~TTTGC 4 5 1 AGCGCtGCGCCCGGaC~GCtGTg~CCTGGT~GC~CGTGTCgGCCqCqGTGTCgTTTGC

AGCGC-GCGCCCGG-C-GC-GT-GTCCTGGTGTGC-TCGTGTC-GCC-C-GTGTC-TTTGC

504 GCCCATCATGAGCCAGTGCTIGGCGtGT~GC~GACGCtGAGGCGCMq~qTGtCACTCC 5 1 3 GCCCA~CATGAGCCAGTGCTGGCG~GTAGGCGC~GACGC~GAGGCGCAq~q~TG~CACTCC

GCCCATCATGAGCCAGTGGTGGCG-GTffi~-GACGC-GAGGCGCA----TG-CACTCC

545 MtCCGCGCTGCTGTtCCTTtGCCTCCMtATGCCCTACG=GCTGCT~TCCTCCTCCGTCT 5 5 4 McCCGCGCTGCTGTqCCTTeGCCTCCM=ATGCCCTACGtGCTGCTgTCCTCCTCCGTCT

M-CCGCGCTGCTGT-CCTT-GCCTCCM-ATGCCCTACG-GCTGCT-TCCTCCTCCGTCT

1 n 9qFGNgSaFLL ~NrShAPdHDvTQqRDEuWWD19IVnSLI nEP hGNdSdFLL A P N g S r A P g H D i T Q e R C E a ~ ~ ~ I l M S ~ l 1

1 MGAGaLsLGASEPcNLSSMPLPDGRAT~LLVlASPPASLLPPASEq~~PLS~WTA~GLLlALI 1 MGAG"LVICIASEP~NLSSALPDGMT~LLVDASPPASLLPPAS~~D~PLSQOWTA~GL~LI 1 MAPWPHENSSIAPWPD 1PTLabntANTSGLP GVPWeRALAG ALL ALA 1 MAPWPHkNgSLAfWSD aFTLdPPaANTSGLP GVPWaAALAG ALL ALA -

I

. ~.

I 4 VLAIYFGNVLVITAIMFERLOTVTN'IFITSLACRDLVNGLAWPFGAaHIlllXnWtFGNfWCEFWTS I4 VLRIWGNVLVITAI~U(~ERLQT~NYFITSLACADLVNGLAWPFGA~HI~~~~W~FGNFWCEFWTS

Hula. 11 69 VLLIVaGNVLVIV41AKTPRMTLTNLFIE(5LASADLVNGLLWPFGATII"*IGRWEYGSfFCELWTS HYm.8 13 48 YL~TVGGNLLV~VAIA~TPRLQ~T~VTSLA~DLVNGLLWPP~ATLALTGH~LGATGCELWTS Rat 111 4 8 TVGGNLLVItA1ArTPRLQTiTNVF~SLAtRDLV"GLLV~PgATLALTGH~LGATGCELWTS

" - Re1 II2 112 IDVLCVTASIETLCVIAWRY~AITSPFKYQSLLTKNXARV~I~lVSGLTSfLPlQMHWYR ATH Human D2 112 lDVLCVTASIETLCVIAVDRYfAITSPFK~QSLLT~KARViIIENWIVSGLTSfLPIQMHWYR ATH

R.I n1 137 MVLCVTASIETLCVIALDRYLAITIPFRYOSLLTRI\RI~SALVSFLPIIIH-~,EZD Humam R I iil M V L C V T A ~ I E T ~ C V I A L D R Y L A ~ T S P F R Y ~ ~ L ~ ~ ~ ~ ~ V C T ~ A ~ S A ~ V ~ F ~ P I L H H W W R A E S D Hum.. 83 116 W V L C ~ A S I E T L C A L A V D R Y L A V T N P L R Y G = L ~ K R ~ A R t A W L V W " V S A ~ V S f ~ I M S Q W W R V G ~ R.1 hl 1 1 3 WVLClrTASIETLCALAWRYLAVTNPLRYGtL~KRr~~WLVWIVSAtVSfAPIMSQWWRVGRD -

Ill IV H ~ ~ . ~ )) 179 q e A I n C Y R D E T C C D f f T N Q A Y A I A S s l V S F Y v P L V ~ ~ F V Y S R V ~ Q ~ A ~ Q L Q K I D K S E G R f 11.1 12 179 k q A I d C Y A k E T C C D F f T N Q A Y A I A S S I V S f Y V P L V V M I L Q K I D K S E G R ~ m., 111 7 0 1 ~ E A ~ R C Y N O P K C C D F V T N R Y A I A S S W S F Y V P L C I P U P F L t G P ~ R

Human 63 184 AEAQRCHSNPRCCaFASNY~LLSSSVSFYLPLLVNLFVYARVfWAtRQlRllRgELGRf PPEES R.1 13 181 A E A Q e C H S NPUCCsFASNHPY~LLSSSVSFYLPLLVIIIF~RfvRrELGRF PPEES -

V

606 CCTTCTACCTTCCCCTCCTtGT~ATGCTCTTCGTCTAtGCtCG~GTgTTCGT~GT~GCT~ 615 CCTTCTACCTTCCtCTtCTcGTgATGCTCTTCGTCTA~GCgCG~TtTTCGTqGTgGCTAc

CCITCTACCTTCC-CT-CT-GT-ATGCTCTTCGTCTA-GC-CG-GT-TTCGT-GT-GCTA-

667 GCGCCAGCgGCGtTTcqTGCGCcGGGMGCTGGCCCGt~TCCGCCCGAGM~CTCCGC9~ 67.5 GCGCClGCtGCGCTTgCTGCGCqGCGAGCTGGGCCGcTTT~CCGCCCGffiGAGTCTCCGCCG

GCGCCAGC-GCG-TT--TGCGC-GGGAGCTGGCCCG-TTTCCGCCCGAGGAGTCTCCGC-G

728 tCtCCGTCGCGCTCTCC~tCCCCtGCC~C~GT~GCGACaccC~Cq9C~tC9~AtGG~G~~~ 737 qCgCCGICGCGCTCTCtggCCCCgGCCcCgGT9GGAC9t9C9Ct~C9CCCGA~~G~~~~~

-C-CCGTCGCGCTCTC---CCCC-GCC-C-GT-GCGAC---C-C--C"C-GA-~G-GT~C

189 CCtCCTGCGG~CGGCGCCCtGCGcGCCTCCT~CCgCTCgGCGMCACCG~GCCCTG~GCAC 198 C c q C C T G C G G e C G G C G G C C o G C G C G C C T C C T ~ C t C T C C C

CC-CCTGCGG-CGGCGCCC-GCGCGCCTCCT-CC-CTC-GGGMCACCG-GCCCTG-GCAC

850 CTTGGGTCTCATtATGGGCAtCTTCtCTCTgTGCTfficTGCCC~TCTT~~~~G~~~~~~~ 859 CTTGGGTCTCATcATGGGA~CTTCaCTCTcTGCTGCtTGCCCTTCTTTCTGG~~M~~~~

CTTGGGTCTCAT-ATGCGCA-CTTC-CTCT-TGCTU~-TGCCCTTCTTTCTGCCCMCGTG

9 1 I c T G C G C G C ~ C T c G t G G G q C C C T C c C T A G T t C C c a C C g 9 a G t T T T C a T C G C C C T G ~ ~ ~ G ~ ~ 92. CTGCGCGCcCTgGgGGCcCCCTCtCTAGT~CCggGC~cqGcTTTC~Tt~~CC~~~~~~~~

CTGCGCGC-CT-G-GGG-CCCTC-CTAGT-CC--GC---G-TTTC-T-GCCCTGMCTGG-

972 TqGGETATGCCMFTCTGCCTTCMCCCGCTCATCTACTGCCGCAGCCCGCACTTTCGCg~ 981 TaGGtTATGCCMtTCTGCCTTCMCCCGCTCATCTACTGCCGCAGCCCGCACTTTCGC~g

T-GG-TATGCCM-TCTGCCTTCMCCCGCTCATCTACTGCCGCAGCCCGGACTTTCGC--

1033 CGCCTTCCGrCGTCTTCTGTGC~GC~~CGGtgG=CGt9g~CCg9a*GAGCCa=GCGt9GtC 1042 CGCCTTCCGcCGTCTTCTGTGCCGCTgCGG~~GtCGCctgCCtcc9GAGCCctGCG~cGCC

CGCCTTCCG-CGTCTTCTGTGC-GCT-CGG--G-CG----CC----GAGCC--GCG--G-C

1094 a C C t t C C C a G C t a g C e c t g t ~ g C G t ~ C ~ g q C ~ g ~ ~ = t C a C = G = t C M C ~ q q t t t q A t g q C t I 1 0 3 qcccgcccgGccctCttcscctcc4gcqttcctgcggCcCqGasCAqCccagcqcAgcccag~g~A~c~C~

~ ~ ~ " ~ ~ ~ ~ ~ ~ " . ~ " " " . ~ ~ " ~ . " ~ " " - - C - C " ~ c -

I155 ~tga~qGtgAgCGtCc~tt tcccacatgMgqaccatqGAG~t=taGcaagq.gcctga I164 gqctttGCCAaCG~CtCgacqgqt~gqtM==qggg=~GAGgg*=~Gg=g

" " " ~ " A - ~ ~ . ~ " " " " " - " " - " " - - G M -

Humam 11 241 Rat n2 241

QVEQHvQNLSQVEQDGRtGHGLRrS SnfCLKEHKAlXTLGIIMGTfTLCWLP

R.1 111 272 PPSPaPSP sPGPPRPA dSLANGRSSKRRPSRLVALREQtL4LKTLGIIMGVFTLCWLP HaQNLSQVEQDGRSGHGLRSS SkFCLKEHKALKTLGIIMGTFTLCWLP

~ ~ r , o . n nl 272 PPSP~PSPvpAPAPpPGPPRPAaaaatAPLANGRAgKRRPSRLVALREPMZKTLGIIMGVFTLCWLP Human 63 251 PPaPS RSlAPAP 11.1 u 248 PrsPS RSpsPA

VGTcAPpeGVPACGRRPmLLPLREHRALcTLGLIMGtFTLCWLP tVGTptaPdGVPsCGRRPARLLPLqEHPALrTLGLIMGiFsLCWLP

VI

Rat 81 289 FFIVN IVHVIrdNLIPKEVY1LLNWLGYVNSAMPLIYCRSPDFRIAFQELLC LRR 55 Human 12 289 FFIVN IVHVIqaNLIrKEVYILLNWIGYVNSqFNPLIYCRSPDFRIMOELLC LRR SS

R.1 nt 329 f F W VWIFHRdLVPDRLFVFFNWLGYANSAFNPIIYCRSPDFRKAFQrLLCCARRRhcRRR AaH Hummm I 1 340 F f W V W I F H R = L ~ D ~ f W ~ L G Y ~ S A F N P I I Y C R S P D f R ~ ~ q L L C C A R ~ R R R M t H

R.1 83 305 FFLAUVLRALVGPSLVPSqVFiALNWLGYANSAFNPLIYCRSPDFRdAFRRLLC syGgR Humm n3 308 F F L ~ R A L ~ G P S L Y P ~ ~ ~ F ~ I I I ~ I W L G Y A N S A F N P L I Y C R S P D F R ~ A F ~ L L C WZRR

m - VI1

FIG. 2. A, comparison of the nucleotide sequences encoding human and rat &-ARs. The DNA sequences of the coding regions plus 37 bp of 5'-untranslated and 33 bp of 3'-untranslated DNA from rat and human &ARs are shown. B, comparison of the deduced amino acid sequences of human and rat 0-AR subtypes. Putative transmem- brane domains are highlighted by solid bars. Putative sites for N-linked glycosylation in the rat &AR are highlighted by asterisks. Sequence data used in compilation of this figure were from 19, 26, 27, 36, 37. Nucleotide and deduced amino acid sequences of the indicated receptors were aligned using the Genalign program of the Intelligenetics Suite software. Gaps (represented by empty spaces) were introduced to maximize homologies.

crease in intracellular CAMP levels for more detailed phar- 0.4 nM, a value significantly greater than the Kd values for macological and biochemical characterization. Membranes ICYP binding to PI- (11 PM) (26) and &-adrenergic (30 pM) from transfected cells display saturable binding of the radi- (27) receptors, but comparable to that reported for ICYP oligand ['261]iodocyanopindolol (ICYP). The calculated equi- binding to the human &AR (0.5 nM) (19). The calculated Kd librium dissociation constant (Kd) for ICYP binding is 1.3 f for [3H]CGP 12177 binding is 43.7 f 4.1 nM. The density of

24056 Fat Cell 0-Adrenergic Receptor

Agonists produce dose-dependent increases in intracellular cAMP concentrations in transfected CHO cells with a rank order of potency of BRL 37344 > isoproterenol >> norepi- nephrine > epinephrine > zinterol > tazolol (Fig. 3, Table I). K,,, values for BRL 37344- and isoproterenol-mediated in- creases in intracellular cAMP in transfected CHO cells are in very good agreement with the EC,, values for increases in lipolysis in brown adipocytes as described by Arch et al. (12); i.e. 1.3 and 1.7 nM for BRL 37344 and 4.0 and 8.0 nM for isoproterenol in transfected cells and brown adipocytes, re- spectively.

In competition studies, @-adrenergic antagonists display a rank order of potency of alprenolol > propranolol > IC1 118,551 > betaxolol (Table I). The relatively low affinity of the rat &AR for antagonists is characteristic of the atypical &ARB in adipose tissue, ileum, and skeletal muscle (12-18). @-Adrenergic agonists display a rank order of potency of BRL 37344 (atypical agonist) > zinterol (&adrenergic agonist) > tazolol (&-adrenergic agonist) > (-)-isoproterenol > epineph- rine > norepinephrine > (+)-isoproterenol (Table I).

The pharmacological properties of the rat &AR differ

75

50

25

0 -10 -9 -8 -7 -6 -5 -4

Log Dose [Agonist1 IHI

FIG. 3. Agonist-mediated increases in intracellular cAMP in transfected CHO cells. Cells were exposed to increasing concen- trations of BRL 37344 (solid circles), (-)-isoproterenol (solid dia- monds), norepinephrine (open squares), epinephrine (open circles), zinterol (solid squares), or tazolol (solid triangles). Results are ex- pressed as the percent of forskolin-stimulated cAMP production. Basal and forskolin-stimulated levels of cAMP accumulation were 26 f 7 pmol and 620 f 42 pmol/106 cells, respectively. Data represent the mean L- S.E. of two separate experiments performed in duplicate.

significantly from those of the human B3-AR expressed in CHO cells (19). The rank order of agonist potency for inhi- bition of ['251]ICYP binding to the human &AR is BRL 37344 > norepinephrine > (-)-isoproterenol >> (+)-isoproter- enol > epinephrine (19) as compared with BRL 37344 >> (-)- isoproterenol > epinephrine > norepinephrine > (+)-isopro- terenol for the rat P3-AR. The pharmacological profile of the human &AR (19) does not agree with any known tissue pharmacology, nor is it consistent with the pharmacological definition of a &adrenergic receptor (isoproterenol more po- tent than epinephrine and norepinephrine). In addition, most of the classical nonselective ,%adrenergic antagonists do not inhibit ['251]ICYP binding to the human &-AR (19). There- fore, it appears that there are substantial differences between the rat D3-AR and the human O3-AR described by Emorine et al. (19).

Tissue Distribution of the Rat P3-AR-To investigate the tissue distribution of the P3-AR versus PI- and &ARs, poly(A)+ RNA from various tissues was isolated and fraction- ated on a formaldehyde-agarose gel (Fig. 4). BI-AR mRNA is present in brown and white adipose tissue, brain, heart, and lung. B2-AR mRNA is also present in those same tissues; however, with the exception of the lung, it is present at significantly lower levels. &AR mRNA is abundant in brown and white adipose tissue, with no &AR-specific mRNA de- tectable in brain, heart, ileum, liver, or lung. White adipose tissue from humans also contains an mRNA that hybridizes strongly with the rat (33-AR cDNA probe (data not shown).

Under identical conditions using probes of similar specific activities, it was estimated that D3-AR mRNA is present in a &fold and 4-fold excess over &-AR mRNA in brown and white adipose tissue, respectively, whereas the @TAR mRNA signal is virtually undetectable (data not shown). It has been difficult to quantitate the atypical P-AR in adipose tissue because available radiolabeled antagonists display up to 100- fold greater affinities for pl- and &AR subtypes than for atypical receptors. However, using high concentrations (100 nM) of 13H]CGP 12177 and the appropriate concentrations of propranolol and BRL 37344, it is possible to selectively meas- ure total @-AR binding and &-AR binding, respectively, in IBAT plasma membranes. Ligand binding analysis reveals the presence of two populations of [3H]CGP 12177 binding

TABLE I Comparison of pharmacological properties of rat and human B3-adrenergic receptors

Inhibition constants ( K ; ) of [1251]ICYP binding to the rat &AR were obtained from at least three competition assays performed in triplicate. Values for Kact and maximal levels of cAMP accumulation in transfected cells were obtained from two separate experiments performed in duplicate. Data are expressed as the mean f S. E. Values in parentheses are percentage forskolin (10 pM)-stimulated cAMP accumulation.

Inhibition of ICYP binding (Kt) Accumulation of cAMP ( R , )

Rat 8,-AR Human Br-AR Rat On-AR Human &-AR

Agonists BRL 37344 (-)-Isoproterenol (+)-Isoproterenol Norepinephrine Epinephrine Zinterol Tazolol

Antagonists Alprenolol Propranolol IC1 118,551 Betaxolol

0.25 f 0.04 57.0 -t 9.3 930 f 267 500 k 126 160 f 80 9.6 f 4.2

26.0 f 8.0

0.39 -t 0.03 1.5 f 1.8 5.6 f 1.3

16.7 & 12.2

WM nM

0.18 & 0.01" 1.3 k 1.0 (68) 5.9 f 1.3" 0.62 f 0.22" 4 f 1.5 (63) 3.9 -C 0.4"

0.48 f 0.80" 100 f 43 (58) 6.3 & 0.7"

3,800 f 280 (46) 10,000 f 4,600 (18)

19 f 3"

20 f 3" 210 f 150 (59) 49.2 * 5.3"

0.26 & 0.03"

"Data from Emorine et al. (19).

Fat Cell P-Adrenergic Receptor 24057

Kb

A 7.5 -

4.4 -

2.4 -

1.4 -

B

C

BAT WAT Bm Htl Ile Llv Lng BAT WAT

FIG. 4. Tissue distribution of 8-AR subtypes in the rat. Sam- ples represent 15 pg of poly(A)+ RNA from brown adipose tissue (BAT), white adipose tissue ( WAT), brain ( B m ) , heart (Hr t ) , ileum (Zle), liver (Lio), and lung (Lng). Northern blots were hybridized with ‘”P-labeled &-AR (A), /3,-AR ( B ) , or &-AR (C) specific DNA probes. The figure shows a representative autoradiogram. The positions of marker RNA are shown in kilobases (Kb).

sites with K d values of 0.59 and 29 nM that represent 30% and 70%, respectively, of the total number of @-AR in IBAT membranes, similar to data recently reported by Blum-Kaelin e t al. (32). The Kd value of the low affinity site, that is presumed to represent the &AR, is similar to that obtained for [3H]CGP 12177 binding to the &AR expressed in CHO cells (44 nM). The results from ligand binding data are con- sistent with those from Northern blot analysis and demon- strate that the &AR is the predominant @-AR subtype in adipose tissue.

Effect of Obesity on @-AR Levels in Brown and White Adipose Tissue-Numerous investigations have reported ab- normalities in brown adipose tissue of hereditary obese ani- mals (for review, see Ref. 7). In both obese (ob/ob) mice and (fa/fa) Zucker rats, the thermogenic response of brown adi- pose tissue to sympathetic stimulation is decreased as com- pared with lean controls (33,34). In obese (fa/fa) Zucker rats, @-adrenergic stimulation of adenylate cyclase is also reduced (35). Since it is possible that the decrease in tissue respon- siveness may reflect changes in @-AR expression, we examined the levels of @-ARs and receptor subtype-specific mRNA in obese (fa/fa) Zucker rats and lean control (Fa/Fa) animals. As shown in Fig. 5, PI- and &-AR mRNA levels are unchanged in brown and white fat of obese rats as compared to lean controls. In contrast, the level of &-AR mRNA is decreased by 60% and 71%, respectively, in brown and white fat in obese animals. Consistent with these data is the finding that the density of high affinity [3H]CGP 12177 binding sites is un- changed in obese animals as compared with controls, whereas

I I I

FIG. 5. Steady-state level of B-AR subtype mRNAs in rat brown (IBAT) and white adipose tissue (WAT). Fifteen pg of poly(A)+ RNA were electrophoresed, transferred to membrane filters, and hybridized with ‘*P-labeled &-AR (open columns), 02-AR (hatched columns), or &AR (solid columns) cDNA probes. The results are expressed as changes observed in the amount of B-AR subtype mRNAs in obese (fa/fa) Zucker rats as compared with lean controls (Fa/Fa). Lean rat values are expressed as 100%. The results are the mean f S.E. of three to five experiments. **, p < 0.01; ***, p < 0.001.

the number of low affinity binding sites is decreased by 56% in obese animals (data not shown).

DISCUSSION

The nature of the P-AR in brown adipose tissue that me- diates lipolysis has been a matter of dispute. Data have demonstrated that although PI- and P2-ARs are present in brown adipose tissue, a discrete receptor subtype that exhibits atypical pharmacological properties is most likely responsible for the thermogenic response observed in this tissue. This hypothesis is supported by the discovery of a novel group of @-adrenergic agonists that selectively stimulate lipolysis in adipose tissue (12). The rat brown adipose tissue @-AR cDNA isolated and characterized in this study displays a low affinity for classical @-adrenergic antagonists and a high affinity for the thermogenic agonist, BRL 37344. These properties are consistent with the biochemical properties of the @-AR in brown adipose tissue which mediates lipolysis (12).

The rat &AR is more similar in amino acid sequence to the human &-AR (19) than to rat Dl- (26) or @,-AR (27) subtypes. &-ARs from rats (26) and humans (36) exhibit 90% amino acid identity. Similarly, rat and human @,-ARs are 86% identical (27, 37). The human and rat &ARs display 80% sequence identity, suggesting that the Ps-AR described herein may be the rat homolog of the human &AR. However, the pharmacological properties of the rat &AR are markedly different from those of the human &-AR expressed in CHO cells. Moreover, Northern blot analysis demonstrates that rat &AR mRNA is expressed almost exclusively in brown and white adipose tissue, whereas the human Pa-AR mRNA is present not only in brown and white adipose tissue but also a t comparable levels in ileum, liver, and soleus muscle (19). We cannot explain the marked discrepancies in the pharma- cology and tissue distribution of the rat and human &-ARs. One possibility is that the rat @s-AR represents a novel atypical @-AR subtype preferentially expressed in adipose tissue.

It has been well documented that brown adipose tissue of obese animals responds poorly to catecholamine stimulation (33-35). Our results indicate that there is a decrease in the level of expression of the @,-AR in brown and white adipose tissue in obese rats, which most likely accounts for the de- creased response of adenylate cyclase following challenge with agonists (35). Identification of an adipose tissue-specific @- AR in rats and the finding that receptor levels are markedly reduced in an animal model of genetic obesity provide a basis for further studies into the regulation of this receptor in

24058 Fat Cell @-Adrenergic Receptor

physiological and pathological conditions in rodents and man. Tate, K., Delavier-Klutchko, C., and Strosberg, A. D. (1989) Science 245.1118-1121

Acknowledgments-We thank Jean-Pierre Giliberto and Pascal Villemin for excellent technical assistance, Dr. Doreen A. Robinson for help with the cDNA library screening, Prof. B. Jeanrenaud and Dr. F. Assimacopoulos-Jeannet for the Zucker rats, and Prof. G. Gabbiani’s laboratory for help with the densitometric analysis of Northern blots.

REFERENCES 1. Foster, D. O., and Frydman, M. L. (1978) Can. J. Physiol. 66,

2. bothwell, N. J., and Stock, M. J. (1979) Nature 281, 31-35 3. Trayhurn, P. (1989) Proc. Nutr. Soc. 48,165-175 4. Stock, M. J. (1989) Proc. Nutr. SOC. 48, 189-196 5. Bouillaud, F., Villarroya, F., Hentz, E., Raimbault, S., Cassard,

A.-M., and Ricquier, D. (1988) Clin. Sci. 75, 21-27 6. Lean, M. E. J. (1989) Proc. Nutr. SOC. 48, 243-256 7. Himms-Hagen, J. (1989) Prog. Lipid Res. 28,67-115 8. Seydoux, J., and Girardier, L. (1978) Experientiu Suppl. 32,153-

9. Bukowiecki, L., Follea, N., Paradis, A., and Collet, A. (1980) Am.

10. Rothwell, N. J., Stock, M. J., and Sudera, D. K. (1985) Am. J.

il. Levin, B. E., and Sullivan, A. C. (1986) J. Pharmacol. Exp. Ther.

12. Arch, J. R. S., Ainsworth, A. T., Cawthome, M. A., Piercy, V., Sennitt, M. V., Thody, V. E., Wilson, C., and Wilson, S. (1984) Nature 309, 163-165

13. Wilson, C., Wilson, S., Piercy, V., Sennitt, M. V., and Arch, J. R. S. (1984) Eur. J. Pharmacol. 100,309-319

14. Bojanic, D., Jansen, J. D., Nahorski, S. R., and Zaagsma, J. (1985) Br. J. Pharmol . 84,131-137

110-122

167

J. Physiol. 228, E552-E563

Physiol. 248, E397-E402

236,681-688

15.

16.

17.

18.

19.

Bond, R. A., and Clarke, D. E. (1987) Br. J . Pharmacol. 91,683- 686

Bond, R. A., and Clarke, D. E. (1988) Br. J. Pharmacol. 96,723- 734

Challis, R. A. J., Leighton, B., Wilson, S., Thurlby, P. L., and Arch, J. R. S. (1988) Biochem. Phurmacol. 37,947-950

Arch, J. R. S., Piercy, V., Thurlby, P. L., Wilson, C., and Wilson, S. (1989) in Hormones, Thermogenesis and Obesity (Lardy, H., and Stratman, F., ed) pp. 465-476, Elsevier Science Publishers, B. V., Amsterdam

Emorine, L. J., Marullo, S., Briend-Sutren, M.-M., Patey, G.,

20. Okayama, H, KGaichi, M., Brownstein, M., Lee, F., Yokota, T., and Atai, K. (1987) Methods Enzymol. 164’3-28

21. Aviv, H., and Leder, P. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 1408-1412

22. Gocayne, J., Robinson, D. A., FitzGerald, M. G., Chung, F.-Z., Kerlavage, A. R., Lentes, K.-U., Lai, J., Wang, C.-D., Fraser, C. M., and Venter, J. C. (1987) Proc. Natl. Acad. Sci. U. S. A.

23. Arakawa, S., Gocayne, J. D., McCombie, W. R., Urquhart, D. A., Hall, L. M., Fraser, C. M., and Venter, J. C. (1990) Neuron 2, 343-354

24. Chung, F.-Z., Wang, C.-D., Potter, P. C., Venter, J. C., andFraser, C. M. (1988) J. Bwl. Chem. 263,4052-4055

25. Lehrach, H., Diamond, D., Wozney, J. M., and Boedtker, H. (1977) Biochemistry 16,4743-4751

26. Machida, C. A., Bunzow, J., Searles, R. P., VanTol, H., Tester, B., Neve, K. A., Teal, P., Nipper, V., and Civelli, 0. (1990) J. Biol. Chem. 266,12960-12965

27. Robinson, D. A. (1988) Molecular Characterization of Cardiac b- Adrenermk ReceDtors. Ph.D. thesis. State Universitv of New

~~ ~

84,8296-8300

York at Buffalo’ 28. Dixon. R. A. F.. Sisal. I. S.. and Strader. C. D. (1988) Cold S w i m

I L I . . Harbor Symp. Quant. Bibl. 63, 487-497

29. Venter, J. C., Fraser, C. M., Kerlavage, A. R., and Buck, M. A. (1989) Biochem. Pharmacol. 38, 1197-1208

30. Fraser, C. M. (1989) J. Bwl. Chem. 264,9266-9270 31. Strader, C. D., Sigal, I. S., and Dixon, R. A. F. (1989) FASEB J.

32. Blum-Kaelin, D., Isler, D., Moeglen, C., Strom, G., and Meier, M. K. (1991) in Adrenoceptors: Structure, Mechanisms, Func- tion, Advances in Pharmacological Science (Szabadi, E., and Bradshaw, C. M., eds) pp. 315-316, Brikhauser Verlag, Base1

33. Assimacopoulos-Jeannet, F., Giacobino, J.-P., Seydoux, J., Glr- ardier, L., and Jeanrenaud, B. (1982) Endocrinology 110,439-

”

3,1825-1832

34.

35.

36.

37.

443 Marette, A., Geloen, A., Collet, A., and Bukowiecki, J. (1990) Am.

J. Physiol. 268, E320-328 Muzzin, P., Revelli, J.-P., Ricquier, D., Meier, M. K., Assimaco-

poulos-Jeannet, F., and Giacobino, J.-P. (1989) Biochern. J.

Frielle, T., Collins, S., Daniel, K. W., Caron, M. G., Lefkowitz, R. J., and Kobilka, B. K. (1987) Proc. Natl. Acad. Sci. U. S. A.

Chung, F.-Z., Lentes, K.-U., Gocayne, J. D., FitzGerald, M. G., Robinson, D. A., Kerlavage, A. R., Fraser, C. M., and Venter,

261,721-724

84, 7920-7924

J. C. (1987) FEES Lett. 211,200-206