Proc. Natl. Acad. Sci. USA Vol. 90, pp. 10230-10234, November 1993 Neurobiology ,u opiate receptor: cDNA cloning and expression JIA BEI WANG*, YASUO IMAI*, C. MARK EPPLERt, PAUL GREGOR*, CHARLES E. SPIVAK*, AND GEORGE R. UHL** *Molecular Neurobiology Branch, Addiction Research Center, National Institute on Drug Abuse, National Institutes of Health, and Departments of Neurology and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21224; and tAgricultural Research Division, American Cyanamid Co., Princeton, NJ 08543 Communicated by Vincent P. Dole, July 23, 1993

ABSTRACT ,u opiate receptors recognize morphine with cDNA confers p. opioid receptor properties on COS cells; the high affinity. A 2.1-kb rat brain cDNA whose predicted translation product of this cDNA shares an amino acid translation product displays 63% identity with recently de- sequence with the purified ,u opioid receptor protein. These scribed 8 and K opiate receptor sequences was identified data thus document the biochemical nature of a principal through polymerase chain reaction and cDNA homology ap- brain receptor for analgesic and addicting ,u opiate ligands proaches. This cDNA recognizes a 10.5-kb mRNA that is and allow exploration of its expression. expressed in thalamic neurons. COS-cell expression confers naloxonazine-, Na+-, and GTP-sensitive binding of ,I but not 8 or K opioid ligands. Expressing cells bind morphine, MATERIALS AND METHODS [D-Ala2,N-methyl-Phe ,glyol5]enkephalin (DAMGO), and Cloning Candidate Rat Brain Opioid Receptor cDNAs. [D-Ala2,D-Leu5]enkephalin (DADLE) with nanomolar or sub- Candidate rat brain opioid receptor cDNAs were obtained nanomolar affinities, defining a ,u opiate receptor that avidly using several mRNA and cDNA sources and several oligo- recognizes analgesic and euphoric opiate drugs and opioid nucleotide primers for PCR amplification (17, 18). pPCR4A peptides. was a 700-bp subclone of a partial cDNA amplified from single-stranded whole rat brain cDNA using oligonucleotides Multiple receptor sites recognizing exogenous opiate drugs 5'-AGACCGCCACCAACATATAC-3' and 5'-GCTTG- and endogenous opioid peptides have been defined based on AAGTTCTCGTCCAGG-3' (17, 18). Sequence analysis re- radioligand binding and pharmacological experiments (1, 2). vealed an open reading frame predicting amino acids similar ,u, 8, and K opioid receptors, as well as several subclasses of to those ofthe murine 8 opioid receptor and identical to 23 aa these main types, have been described (e.g., refs. 1-5). 8 sequenced from two peptides purified from p. opiate receptor receptors display high affinities for the endogenous opioid protein preparations (26). The 700-bp insert was excised with enkephalin peptides, K receptors display high affinities for EcoRI, radiolabeled by random priming, and used to screen dynorphin peptides, and ,ureceptors display high affinities for 6 x 105 plaques from an oligo(dT)-primed rat cerebral cortex analgesic and addicting opiate drugs (3-7). AZAP cDNA library with cDNA inserts >1.5 kb (27). Hy- Opiate receptors may couple to guanine nucleotide binding bridization was performed at 30°C in buffer containing 29% (G) proteins. Opiates can alter GTP hydrolysis, GTP analogs (vol/vol) formamide and 6x standard saline citrate (SSC) and and pertussis toxin can change opiate receptor binding, and washed at 50°C in 0.4x SSC/0.1% SDS. Plasmids were opiates can influence G-protein-linked second messenger autoexcised from phage DNA from positive plaques as de- systems and ion channels (8-14). Recent elucidation of the scribed (27) and analyzed by restriction analyses and DNA sequences of 8 and K opioid receptors using ligand autora- sequencing. One 2.1-kb clone, termed RC8-1, and a second diographic receptor screening (15-17), cDNA homology, and overlapping 3-kb clone, RC18-1, contained the sequences expression approaches (17-19) also documents their resem- displayed by the 700-bp insert. RC8-1 was subjected to com- blance to other G-protein-coupled seven-transmembrane- plete sequencing of both strands, and RC18-1 provided sub- domain neuropeptide receptors. sequent confirmatory 5' sequence data, using automated and ,u opiate receptors are of especial interest for several manual methods and sequence analysis using Genetics Com- reasons. Their dispositions and pharmacologic properties puter Group software as described (27, 28). RC8-1 was ex- place them among the receptors most identified with the pressed as a pcDNAl (Invitrogen) subclone, pcDNAlRC8-1. analgesic and addicting properties of opiate drugs (20-24). Characterizing RC8-1 as a ,u Opiate Receptor cDNA. COS Pharmacologic experiments have suggested that ,u opiate cells were transfected with pcDNA1RC8-1 at 20 pg per 107 receptor subtypes may have therapeutic implications. Selec- cells and cultured for 3 days in Dulbecco's modified Eagle's tive agonists acting at naloxonazine- and [D-Ala2,D-Leu5]- medium containing 10% (vol/vol) fetal bovine serum as enkephalin (DADLE)-sensitive Mi receptors might confer described (27, 29). CHO cells were transfected by lipofection analgesia without prominent respiratory depression (for ex- with 2 pg of pcDNA1RC8-1 and pSV2neo at 200 ng per 106 ample, see ref. 25). Further, a 23-aa sequence derived from cells, and clones were selected after 5 weeks of growth in a protein with properties of a ,u opioid receptor became medium containing G418 at 200-500 Ag/ml. available during the course of these cloning studies; the Radioligand binding was performed on membranes pre- sequence homologies of this peptide also suggested that ,u pared from washed cells at 4°C by homogenization in 50 mM opiate receptors fell into the G-protein-linked neuropeptide Tris buffer, by discarding material pelleted by a 15-min 1000 receptor family (26). In the present study, PCR and cDNA homology ap- Abbreviations: DAMGO, [D-Ala2,N-methyl-Phe4,glyol5]enkephalin; proaches have identified rat brain cDNAs with sequences Pen, penicillamine; DPDPE, [D-Pen2,Phe4,D-Pen5]enkephalin; similar to the murine 8 opioid receptor.§ Expression of one DADLE, [D-Ala2,D-Leu5]enkephalin; G protein, guanine nucleotide binding protein. iTo whom reprint requests should be addressed at: Molecular The publication costs of this article were defrayed in part by page charge Neurobiology, Box 5180, Baltimore, MD 21224. payment. This article must therefore be hereby marked "advertisement" §The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. L20684). 10230 Downloaded by guest on September 23, 2021 Neurobiology: Wang et al. Proc. Natl. Acad. Sci. USA 90 (1993) 10231

1 # # 50 x g centrifugation and retaining material pelleted by a 30-min MUOR1 ...... MDS STGPGNTSDC SDPLAQASCS PAPGSWLNLS HVDGNQSDPC MELVPSARAE .....DAFPS DORI ...... LQSSPLVNLS 46,000 x g centrifugation. Membrane fractions correspond- MESP TCSPSACLLP NSSSWFPNWA KOR1 ...... IQIFRGDPGP were .. MFPNAPPP GGCGEGVCSR to 50 of SOMR1 ...... LPHSSPSSSP ing ug protein (Bradford; Bio-Rad) resuspended FPEP ...... 6..... in 0.5 ml of Tris buffer with various labeled and unlabeled OBPR ...... MASP AGNLS.AWPG WGWPP .. PAA LRNLTSSPAP NEUROKR MASVPRGENW TDGTVEVGTH TGNLSSALGV TEWLALQAGN FSSALGLPAT drugs, nucleotides, and salts including 3H-labeled [D-Ala2,N- B2AR ...... ,,...... MP methyl-Phe4,glyol5]enkephalin ([3H]DAMGO; 60 Ci/mmol, 1 51# $ 100 Ci = 37 3H-labeled MUOR1 GLNRTGLGGN DSLCPQTG.. ...SPSMVTA ITIMALYSIV XCGLFGNFL. GBq; Amersham), [D-Pen2,4'-Cl-Phe4,D- DOR1 AFPSAGANAS GSPGARSA. ...S.SLALA IAITALY SAV CAVdLLGNVL Pen5ienkephalin ([3H]DPDPEpCl; 51 Ci/mmol; NEN, where KOR1 ESDSNGSVGS EDQQLESA.. ...HISPAIP VIITAVYSVV FVVGLVGNSL SOMR1 GPGSGAADGM EEPGRNSSQN GTLSEGQGSA ILISFIYSVV CLVGLCGNSM Pen is penicillamine), [3H]DADLE (37 Ci/mmol; NEN), and FPEP .... .METNSS LPTNISGGTP AVSAGYLFLD IITYLVFAVT FVLGVLGNGL [3H]U-69,593 {(5a,7a,8f3)-(+)-N-methyl-N-(7-[1-pyrrolini- OBPR TASPSPAPSW TPSPRPGPAH PFLQPPWAVA LWSLA.YGAV VAVAVLGNLV NEUROKR TQAPSQV ...... RANLTN QFVQPSWRIA LWSLA.YGLV VAVAVFGNLI nyl]-l-oxaspirol[4,5]dec-8-yl)benzacetamide} (57 Ci/mmol; B2AR HGNDSDFLLA PNGSRAPGHD ITQERDEAWV VGMAILMSVI VLAIVFGNVL Amersham) (30-32). Incubations for 150 min at 22°C were 101 150 terminated by filtration through GFB filters (Whatman) and MUOR1 V.MIVRYTK MKTATNIYIF ..1.... W'5LP.FQSVN YLMGTWPFGT DOR1 VMFGIvRYTK LKTATNIYIF NLAL...ALA. t STLP. FQSA YL14ETWPFGE three 4-ml washes with Tris buffer at 4°C. Radioactivity was KOR1 VMFVIIRYTK MKTATNIYIF NLALADALVT TTMP.FQSAV YLMNSWPFGD measured scintillation and data was ana- SOMR1 VIYVILRYAK MKTATNIYIL NLAIADELLM LSVP.FLVTS TLLRHWPFGA by liquid counting FPEP VIWVAG.FRM THTVTTISYL NLAVADFCFT STLPFFMVRK AMGGHWPFGW lyzed by EBDA and LIGAND (33). OBPR VIWIVLAHKR MRTVTNSFLV NLAFADAAMA ALNALVNFIY ALHGEWYFGA NEUROKR VIWIILAHKR MRTVTNYFLV NLAFSDASVA AFNTLINFIY GLHSEWYFGA Forskolin (10 ,uM)-stimulated cAMP accumulation was B2AR VITAIAKFER LQTVTNYFIT SLACADLVMG LAVVPFGASH ILMKMWNFGN assessed in COS and CHO cell cultures; some cells were 151 200 preincubated for 15 min with 1 mM 3-isobutyl-1-methylxan- MUOR1 ILCKIVISID :X:YM.FT ::LC.TMVDRYI AVCHPVKALD FRTPRNAKNV DORI LLCKAVLSID YYNMFTSIFT LTEMSVDRYI AVCHPVKALD FRTPAKAKLI thine and then incubated with forskolin, saline, morphine, KORI VLCKIVISID YYNMFTSIFT LTM4SVDRYI AVCHPVKALD FRTPLKAKII and/or DAMGO (1-10 ,uM) as described (ref. 34; kit RPA SOMR1 LLCRLVLSVD AVNMFTSIYC LTVLSVDRYV AVEHPIKAAR YRRPTVAKVV FPEP FLCKFVFTIV DINLFGSVFL IALIALDRCV CVLHPVWTQN HRTVSLAKKV 542, Amersham; kit KAPH2, Diagnostic Products, Los An- OBPR NYCRFQNFFP ITAVFASIYS MTAIAVDRYM AIIDPLKPR. .LSATATRIV geles). Transfected COS cells and expressing Xenopus NEUROKR NYCRFQNFFP ITAVFASIYS MTAIAVDRYM AIIDPLKPR. . LSATATKIV B2AR FWCEFWTSID VLCVTASIET LCVIAVDRYV AITSPFKYQS LLTKNKARVV oocytes were also assessed for expression of pcDNA1RC8-1 201 250 under whole-cell voltage clamp conditions with morphine MUORI WILSSA TKYRQ. .GSI DCTL.TFS.. ..HPTW.YWE applied by microsuperfusion as described and oocytes coin- DORI NCIWVLASG VGVPIMVMAV TQPR. .GAV VCML.QFP.. ..SPSW.YWD KOR1 NICIWLLASS VGISAIVLGG TKVREDVDVI ECSL.QFP.. ..DDEYSWW jected with pcDNA1RC8-1 and pcDNA-1-p1 serving as pos- SOMRI NLGVWVLSLL VILPIVVFSR TAANSD.GTV ACNM.LMP.. ..EPAQRWLV itive controls FPEP IIGPWVMALL LTLPVIIRVT T.VPGKTGTV ACTF.NFSPW TNDPKERIKV (35-38). OBPR IGSIWILAFL LAFPQCLYSK IKVMP...... GRTL.CYVQW PEGSRQHFTY ji Opiate Receptor Expression. RNA (20 pg) prepared from NEUROKR IGSIWILAFL LAFPQCLYSK IKVMP ..... GRTL.CYVQW PEGPKQHFTY mem- B2AR ILMVWIVSGL TSFLPIQMHW YRATHKQAID CYAKETCCDF FTNQAYAIAS rat tissues was electrophoresed, transferred to nylon branes as described (39), hybridized with radiolabeled RC8-1 251 300 = MUORI NLLKICV . -FIF&IM VlIICGLC )IILRLKSVRM LSGS . .. KE in 5x SSPE (lx SSPE 0.18 M NaCl/10 mM sodium DORI TVTKICV... .. FLFAFVVP ILIITIVCYGL MLLRLRSVRL LSGS ... KE phosphate, pH 7.4/1 mM EDTA)/1% SDS/50% (vol/vol) KORI LFMKICV .. ..FVFAFVIP VLIIIVCYTL MILRLKSVRL LSGS .... RE SOMR1 GFV.LYT... ..FLMGFLLP VGAICLCYVL IIAKMRMVPS RPAG .... S formamide/2.5 x Denhardt's solution/herring sperm DNA FPEP AVAMLTVRGI IRFIIGFSAP MSIVAVSYGL IATKIHKQGL IKSS ...... in OBPR HMIVI...... VLVYCFP LLIMGITYTI VGITLWGGEI PGDTCDKYQE (200 pg/ml) at 42°C overnight, and washed twice 0.4x NEUROKR HIIVI ...... ILVYCFP LLIMGVTYTI VGITLWGGEI PGDTCDKYHE SSC/0.5% SDS for 30 min at 52°C, and radioactive patterns .LVVMVFVYS QKIDKSEGRF HAQNLSQVEQ B2AR SIVSFYVP.. RVFQVAKRQL were phosphor imaged (39). For RNase protection assays, 5 301 350 of RNA was incubated with 32P-labeled 400-bp comple- MUORI KDRN ...... LRR IT RMVYYVA YU.VIIKALIT ,ug LRR ITRMVLVVG DOR1 KDRS ...... APWCWAP,i IFVIVWTLVD mentary RNA transcribed from the T7 promoter of pPCR4A LRR ITKLVLVVVA VFIICWTPIH KOR1 KDRN ...... IFILVEALGS linearized with BamHI and were de- SOMR1 TQRS ...... ERK ITLMVMMVVM VFVICWMPFY VVQLVNVFAE protected fragments FPEP ...... R PLRVLSFVAA AFFLCWSPYQ VVALIATVRI tected by gel electrophoresis and phosphor imaging (kit 1410; OBPR QLKA .R...... KRK VVRKZMIIVVV TFAICWLPYH IYFILTAIYQ

NEUROKR QLKA .. KRK VVKMMIIVVV TFAICWLPYH VYFILTAIYQ Ambion, Austin, TX). B2AR DGRSGHGLRS SSKFCLKEHK ALKTLGIIMG TFTLCWLPFF IVNIVHVIRA In situ hybridization used 10-,um sections through dien- 351 400 cephalon of perfusion-fixed rats and 50-base 35S-labeled MUOR1 I PETTF... LY-T CPN:VLY-APLENF KRCF. REF.. QTVSWHFCIA: ...... DORI INRRDPL.. VVAALHLCIA LGYANSSLNP VLYAFLDENF KRCF. RQL.. oligonucleotides complementary to bases indicated in Fig. 1, KOR1 TSHSTA.... ALSSYYFCIA LGYTNSSLNP VLYAFLDENF KRCF.RDF.. as described (40). Hybridization at 37°C overnight in a SOMRI QDDATVS ... Q. LSVI LGYANSCANP ILYGFLSDNF KRSFQRIL.. FPEP RELLQGMYKE IGIAVDVTSA LAFFNSCLNP MLYVFMGQDF RERLIHAL.. complex buffer was followed by washing at 50°C and emul- OBPR QLNRWKYIQQ VYLASFW... LAMSSTMYNP IIYCCLNKRF RAGFKRAFRW NEUROKR QLNRWKYIQQ VYLASFW... LAMSSTMYNP IIYCCLNKRF RAGFKRAFRW sion autoradiography (40, 41). B2AR NLIPKEV ...... YILLNW LGYVNSAFNP LIYC.RSPDF RIAFQELLCL 401 450 RESULTS MUOR1 .CIPTSSTIE QQNSTRVRQN TREHPSTANT VDRTNHQLEN LEAETAPLP. DORI .CRTPCGRQE PGSLRRPRQA TTRERVTACT PSP.0. PGGAAA... KORI .CFPIKMRME RQSTNRVR.N TVQDPASMRD VGGMNKPV ...... Among products of PCR amplification of rat brain cDNA SOMR1 CL... SWMD NAAEEPVDYY ATALKSRAYS VEDFQPENLE SGGVFRNGTC using oligonucleotides complementary to S opioid receptor FPEP .PASLERALT EDSTQTSDTA TNSTLPSAEV ALQAK ...... OBPR CPFIHVSSYD ELELKATRLH PMRQ.SSLYT VTRMESMSVV FDSNDGDSAR sequences was the 700-bp cDNA clone pPCR4A, which NEUROKR CPFIQVSSYD ELELKTTRFH PTRQ.SSLYT VSRMESVTVL FDPNDGDPTK B2AR RRSSSKTYGN GYSSNSNGRT DYTGEQSAYQ LGQEKENELL CEEAPGMEGF displayed an open reading frame with 71% amino acid se- quence identity to the 8 opioid receptor (17) and above- FIG. 1. Amino acid sequence of the rat brain , opiate receptor chance homology with sequences of other G-protein-linked predicted from the sequence of cDNAs RC8-1 and RC18-1 and receptors. This open reading frame contained the 23-aa comparisons of sequence to other G-protein-linked receptor family sequence from a ,u opioid receptor protein preparation (26) members. RC8-1 displayed open reading frames aligned with the (Fig. 1). murine 8 opioid receptor (DOR1) (17, 18), murine K opioid receptor rat cerebral cortical cDNAs with ra- rat somatostatin human Among hybridizing (KOR1) (19), receptor (SOMR1) (42), was a 2.1-kb N-formyl-methionine receptor (FPEP) (43), human opioid binding diolabeled pPCR4A hybridization probes protein (OPBR) (44), rat neuromedin K receptor (NEUK) (45), and cDNA, RC8-1, and a 3-kb cDNA, RC18-1 (Fig. 1). RC8-1 rat 32-adrenergic receptors (B2AR) (46) by using the program encoded an open reading frame of334 aa with 77% amino acid PILEUP. Boldface type and shading, transmembrane domain candi- homology and 63% amino acid identity to sequences of the dates; *, putative site for phosphorylation by protein kinase A; #, opiate receptor, 75% amino acid homology and 60% amino putative sites for N-linked glycosylation; double underline, targets of in situ hybridization oligonucleotide hybridization probes; single underline, sequences corresponding to the ,u opioid receptor protein opiate receptor sequences extending beyond aa 450 have been (26); italics, sequence from RC18-1 not translated in RC8-1. Non- truncated. Downloaded by guest on September 23, 2021 10232 Neurobiology: Wang et al. Proc. Natl. Acad. Sci. USA 90 (1993)

acid identity to sequences of the K opiate receptor, good homology to other neuropeptide receptors, and more distant homology to an "opiate binding protein" receptor and a catecholamine receptor. Hydrophobicity analyses revealed 200 seven hydrophobic domains of 20-24 aa whose sequences were especially conserved with the putative membrane- spanning domains of other G-protein-linked receptors. Sev- eral serine and threonine residues in putative intracellular domains were found in contexts favorable for protein kinase A and C phosphorylation (47). Subsequent RC18-1 sequence data revealed a longer open reading frame with 66 additional 1l500 ;< N-terminal amino acids and five consensus sequences for N-linked glycosylation (Fig. 1) (48). COS-cell expression of RC8-1 in the expression vector '240 0 1 2 3~0 4 65 pcDNA1 yielded naloxone-blockable high-affinity specific binding of [3H]DAMGO and [3H]DADLE (Table 1) that was 100- [3H]DAMGO, aM absent from cells transfected with vector alone (Fig. 2 and U2~~~~~~~~~ Table 1). There was no appreciable specific recognition of 0~~~~~~~~~. [3H]DPDPE or [3H]U-69,593 (Table 1). Scatchard analysis of r150- [3H]DAMGO and [3H]DADLE binding saturation experi- ments were most consistent with a single population of high-affinity binding sites for each ligand, with Kd values of 0.4 and 0.5 nM, respectively (data from Fig. 2; mean values 0 3 6 3152 for three experiments, 0.37 and 0.4, respectively). [3H]- DAMGO binding could be reduced by addition of Na+ or GTP to incubations (Table 1), but not by adding ATP (data not shown). Mg2+ addition enhanced [3H]DADLE's affinity. binding could be displaced by a number of [3H]DAMGO 0 2 4 6 8 opioid compounds in stereo-selective fashion. Pharmacolog- - B ically active (-)-naloxone and levorphanol isomers displayed substantially greater potency than pharmacologically less- active (+)-naloxone and dextrorphan isomers. Morphine [3H]DADLE, nM displaced binding with high affinity. DADLE, (-)-naloxone, naloxonazine, ethylketocyclazocine, and bremazocine also FIG. 2. Saturation analyses of binding of [3H]DAMGO (A) and shared this high affinity. DPDPE and para-chloro-DPDPE, [3H]DADLE (B) to membranes prepared from COS cells transiently relative 8-selective, and U-50,488 {trans-(±)-3,4-dichloro-N- expressing pcDNA1RC8-1. Receptor binding was carried out with 100 methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzacetamide} and nM naloxone added to parallel incubations to estimate nonspecific U-69,593, relatively K-selective, displayed substantially less binding. Values, fit using EBDA and LIGAND, indicate a good fit with This resembles those de- single-site models with Kd values of 0.4 aM for DAMGO and 0.5 nMfor potency. pharmacological proffle DADLE. Bma determinations for these two experiments are similar; scribed for ja opioid receptors, but not for 8 or K opioid values for two other smaller experiments performed using the same receptors, since ethylketocyclazocine and bremazocine batches of transfected COS cells are within 10%o of each other (data not share high affinities for ,u receptors (e.g., ref. 49). shown). No specific binding was noted in COS cells transfected with Initial Northern blot analyses ofthe distribution of mRNA pcDNA1 vector alone (data not shown). B, bound; F, free. hybridizing with radiolabeled RC8-1 hybridization probes suggested that relatively high expression levels of a 10.5-kb RNase-protection assays were able to detect protected frag- mRNA could be found in the thalamus (data not shown). ments consistent with presence of significant receptor mRNA Table 1. Radioligand binding to membranes from COS cells expressing pcDNA1RC8-1 Potency in displacing [3H]DAMGO binding Radioligand dissociation constants (Kd), nM Receptor Ligand (Kj), nM Ligand Mg2+ - NaCI GTP DAMGO 1.26 [3H]DAMGO 0.37 0.4 1.55 0.75 DADLE 0.77 [3H]DADLE 0.4 1.55 Levorphanol 0.74 Naloxonazine 1.6 Morphine 2.7 (-)-Naloxone 3.7 8 DPDPE >2000 [3H]pCI-DPDPE >1000 K Bremazocine 0.1 [3H]U-69,593 >1000 EKC 0.18 U-50,488 510 Other (+)-Naloxone >1000 Dextrorphan >1000 Radioligand binding assays were performed in 50 mM Tris HCl (pH 7.4) with 5 mM MgSO4, 50 mM NaCI, and 50 .M GTP added as indicated. K; values {Ki = IC5o/(l + [3H]DAMGO)/(4 x 10-10)} were determined from two to four experiments with SEM values of30%6 ofreported Ki values. Kd values were determined in two or three separate experiments with SEM values 10% of reported Kd values. ,B-Endorphin displayed a 0.8 nM Ki value in preliminary experiments (data not shown). Both GTP (line 1) and guanosine 5'-[,v,.imido]triphosphate (preliminary data not shown) reduced the affinity of [3H]DAMGO binding by -2-fold. Downloaded by guest on September 23, 2021 Neurobiology: Wang et al. Proc. Natl. Acad. Sci. USA 90 (1993) 10233 levels in thalamus, cerebral cortex, striatum, hypothalamus, interactions (10, 26, 50, 51). mRNA localization to specific midbrain, hippocampus, brainstem, and spinal cord but not thalamic and habenular neurons fits with the results of ,u cerebellum or liver (Fig. 3A). In situ hybridization studies receptor ligand autoradiography and studies of morphine- identified grain densities over neurons in several thalamic induced changes in glucose utilization (21-24, 52). The pro- nuclei (Fig. 3B). Hybridization was also concentrated in the tein encoded by RC8-1 is a member of the G-protein-linked medial portion of the lateral habenula (Fig. 3C). Thalamic seven-transmembrane-domain neuropeptide receptor family neurons (e.g., Fig. 3B) displayed 14.5 grains per 10 ,m2 and and contains 23 aa sequenced from a purified t. opiate lateral habenula neurons had 20.5 grains per 10 ,Am2, whereas receptor preparation that also displays evidence for G-pro- background values were 0.25 grains per 10 pUm2. tein coupling (26). Neither morphine nor DAMGO consistently reduced for- pL opiate receptors have been defined by high affinities for skolin-stimulated adenylate cyclase activity in transiently morphine, DADLE, and naloxonazine, compounds 1-10% as expressing COS cells (data not shown). CHO cells stably potent at 12 receptors (3, 6, 53-56). RC8-1 expression of expressing RC8-1 revealed modest morphine effects, with subnanomolar Kd values for DADLE, when tested in the cAMP levels reduced to 87 ± 5% ofcontrol values (P < 0.05; presence of magnesium, provides properties similar to those t test from experiment with or without 3-isobutyl-1-methyl- of Al receptors in calfthalamus (3, 30, 32, 57). Further studies xanthine pretreatment). Neither transfected COS cells nor of cDNAs closely homologous to RC8-1 may help to deter- expressing Xenopus oocytes revealed morphine-induced mine whether the l and t2 receptor binding proffes are changes in conductance at several holding potentials under produced by products of two different genes or by other whole-cell voltage clamp conditions, despite significant mechanisms. -aminobutyric acid (GABA) responses in GABApl-cotrans- p-receptor-mediated effects on second messengers and ion formed cells and robust [3H]DAMGO binding to membranes channels have been described in several cultured cell and prepared from sister cultures (data not shown). brain region preparations (14, 58-62). COS cells expressing RC8-1 failed to demonstrate consistent opiate-induced alter- ations in conductance or forskolin-stimulated adenyl cyclase DISCUSSION activity, although small effects were noted in several exper- Several features displayed by the RC8-1 cDNA are consistent iments. Morphine suppression of cyclase activity in stably with its representing a IL opiate receptor. The binding site expressing CHO cells could relate to the high levels ofRC8-1 conferred on COS cells expressing this cDNA recognizes expression in these cells or to their distinctive complements radiolabeled drugs selective for u receptors, but not those of G proteins and other regulatory machinery. binding selectively to 8 or K receptors. [3H]DAMGO binding N-Ethylmaleimide reduces u opiate receptor binding, im- is displaced in a fashion that indicates a u opiate receptor plicating possible sulfhydryl contributions to receptor func- proffle, with nanomolar affinities for morphine and the en- tion and possibly correlating with the substantial conser- kephalin analog DADLE and clear recognition of naloxona- vation of several cysteines among members ofthe G-protein- zine (e.g., refs. 3 and 49). Sodium influences, GTP effects, linked receptor family (54, 63). Sites for N-linked glycosy- and high-affinity recognition of 3-endorphin by the purified lation in the longer protein predicted from combining RC8-1 receptor protein are consistent with the opiate receptor and RC18-1 sequences are consistent with the substantial 1 29 4 5 6 7 8 9 10

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FIG. 3. Expression of ,A opiate receptor mRNA. (A) RNase protection (Phospholmager autoradiogram) ofa 400-bp fragment protected from digestion by 5 pg of ,u opiate receptor mRNA extracted from the following tissues. Lanes: 1, cerebral cortex; 2, striatum; 3, thalamus; 4, hypothalamus; 5, midbrain; 6, hippocampus; 7, brainstem; 8, cerebellum; 9, cervical spinal cord; 10, liver. When hybridization to the 400-bp ,tOR1 fragment was compared to hybridization to a 90-bp y'actin cDNA fragment to control for loading and background, radiation density ratio values derived from the Phospholmager (A opiate receptor/ractin) were 0.9%o, 0.9%6, 7.6%, 4.8%, 4.9%o, 2.2%, 2.3%, <0.1%, and 3.8% for the brain regions in lanes 1-9, respectively (data not shown). (B and C) Photomicrographs of emulsion autoradiograms of in situ hybridization grain densities overlying thalamic (B) and lateral habenula, medial aspect (C), neurons expressing ,u opiate receptor mRNA in a representative section through rat diencephalon. Similar results were obtained in experiments using sections from three brains. (B, x80; C, x32.) Downloaded by guest on September 23, 2021 10234 Neurobiology: Wang et aL Proc. Natl. Acad. Sci. USA 90 (1993) glycosylation displayed by the purified ,u opiate receptor 27. Shimada, S., Kitayama, S., Lin, C.-L., Nanthankumar, E., protein (26). Gregor, P., Patel, A., Kuhar, M. & Uhl, G. (1991) Science 254, These results and other recent data now document that at 576-578. least one representative of each ofthe major opioid receptor 28. Devereux, J., Haeberli, P. & Smithies, 0. (1984) Nucleic Acids types defined in classical pharmacological studies represents Res. 12, 387-395. a 29. Kitayama, S., Shimada, S. & Uhl, G. R. (1992) Ann. 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