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U Opiate Receptor: Cdna Cloning and Expression JIA BEI WANG*, YASUO IMAI*, C

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U Opiate Receptor: Cdna Cloning and Expression JIA BEI WANG*, YASUO IMAI*, C 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
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