- Email: [email protected]
Cloning of a human κ opioid receptor from the brain
Life Sciences, Vol. 56, No. 9 pp. PL 201~207,1995 Copyright 0 1995 Usevier Science Ltd Printed in the USA. All rights reserved 0024-320.5/95 $9.50 + .cm
Pergamon 00243205(94)00507-9 PHARMACOLOGY LETTERS Accelerated Communication CLONING
OF
A HUMAN
K OPIOID
RECEPTOR
FROM
THE
BRAIN
Jinmin Zhu, Chongguang Chen, Ji-Chun Xue, Satya Kunapulil, J. Kim DeRie12 and Lee-Yuan Liu-Chen Departments of Pharmacology and 1Physiology and 2Fels Institute for Molecular Biology and Cancer Research, Temple University School of Medicine, Philadelphia, PA 19140 (Submitted
November 30, 1994; accepted December 13, 1994; received in final form Decemberl6, 1994)
Abstract By using a rat Kopioid receptor cDNA as a probe to screen a human brain cDNA library, we isolated a 4.0-kb clone (~115) which encompasses a major portion of a human Kopioid receptor (l&or), extending from the amino acid residue #6 to the 3’-untranslated region. The extreme Sregion 232-bp fragment of ~115 was used as a probe to screen a human genomic DNA library. A 1.6kb fragment (d2) of one positive clone was found to extend from S-untranslated region to beyond the exon/intron junction at residue ArgW The genomic DNA fragment d2 and the cDNA clone ~115 were assembled to generate a clone (d2-~115) containing the entire coding sequence of hkor. Clone d2-z115 has an open reading frame of 1140 bp, which encodes for a 380-amino acid protein. The deduced amino acid sequence has 93.9% and 93.2% identity to rat and mouse K receptors, respectively. It also displays _ 60% identity to both human p and 6 receptors. Northern blot analysis showed that in the human brain there was a single hkor mRNA transcript of 6.0 kb. Among brain regions examined, the amygdala, caudate nucleus, hypothalamus and subthalamic nucleus contained high levels of hkor mRNA. Hkor was cloned into the expression vector pBKCMV and transiently expressed in COS-1 cells. Hkor had high affinity for [3H] diprenorphine, a nonselective opioid antagonist, and displayed stereospecific binding to naloxone. Kselective ligands (U50,488H and nor-BNI) had high affinities, whereas ~1and 6 selective ligands bound with much lower affinities. Dynorphin A (1-17) and a-neoendorphin, both endogenous Kpeptides, bound with high affinities. These binding characteristics confirmed that hkor is a Kreceptor, most likely ~1 type. Cloning of the human rcreceptor allows investigation of interactions of compounds with the human receptor, instead of rodent receptors, for development of better therapeutic agents. Key Words: opioid receptors, cDNA library, genomic DNA library
Wuction Opiates and opioid compounds act on receptors on the surface of cell membranes to produce their effects. The presence of three major types of opioid receptors - CI_, K, 6 - in the peripheral and central nervous system has been established by pharmacological and binding studies (1). K opioid receptors have been shown to mediate many physiological and pharmacological effects, including analgesia, diuresis, hypothermia, dysphoria and changes in neuroendocrine functions (1). Following the cloning of the mouse 6 opioid receptor (2,3), we (4) as well as several groups (5-9) cloned K opioid receptors from the rat and mouse. Both rat and mouse K receptors bind K agonists and antagonists with high affinities, yet bind l.t and 6 ligands with low affinities. Cloned rodent K receptors are coupled through G proteins to inhibition of adenylate cyclase. Correspondence should be sent to: Dr. Lee-Yuan Liu-Chen, Department of Pharmacology, Temple University School of Medicine, 3420 N. Broad St., Philadelphia, PA 19140. phone: (215) 7074188; fax: (215) 707-7068; e-mail: [email protected].
PL.202
Human ICOpioid Receptor
Vol. 56, No. 9, 1995
Cloning of human lr and 6 opioid receptors has been reported (10,ll). Recently, Mansson et al. (12) reported cloning of a human K receptor from the placenta by reverse transcription of mRNA followed by polymerase chain reaction (PCR) on cDNA. In the present study, we report the cloning of a human Kopioid receptor from the brain by conventional cDNA and genomic DNA library screening and expression of the clone in COS-1 cells for functional characterization. Materials and Methods Screening of human cDNA and genomic DNA libraries: A human fetal brain hZAPI1 cDNA library (Stratagene, La Jolla, CA) was plated according to the manufacturer’s instructions . An 849-bp probe corresponding to nucleotides 388-1237 of the rat 1copioid receptor ((4) with accession number L22536) was produced by PCR and labeled with [a-32P] dC!TP (3000 Wmmol, DuPont-NEN, Boston, MA) by use of random primers DNA labeling kit (GIBCO-BRL, Gaithersburg, MD). Approximately 15x106 cDNA clones were screened. Prehybridization of replica on Duralon-WTM membranes (Stratagene) was carried out in 50% formatm‘de/SxSSC/Sx Denhardt’s / 1.0% SDS / 0.1% sodium pyrophosphate / 100 pg/ml salmon sperm DNA (hybridization buffer) at 42’ C for 4 h. Hybridization was conducted with labeled probe (2 x 106 cpm/ml) in the hybridization buffer at 42’ C for 2 days. A final post-hybridization wash was performed in 0.2 x SSC / 0.1% sodium pyrophosphate at 65’ C for 30 min. Secondary and tertiary screening was carried out to purify the clones and h phage DNA of positive clones was prepared. pBlueScript plasmids were excised from h phages by in vivo excision with the ExAssist helper phage and transformed into SOLR cells. The plasmids were analyzed by restriction mapping and DNA sequence determined with automated and manual methods (13). Nucleotide sequence analysis was carried out with GCG programs. One clone with 4.0-kb insert, termed ~115, exhibited > 90% identity to the rat Kreceptor and contained 1125 bp upstream from the termination codon. To obtain the 5’-region of the human Kreceptor, we screened a human lymphocyte genomic DNA library in ADASH. The probe, corresponding the extreme 5’-end 232 bp of 2115, was produced by PCR and labeled by [a-32P]dCTP. One of the positive clones, 28 kb in length, was digested with HindIII. A fragment of 1.6 kb, termed d2, was positive by southern blot hybridization. D2 was subcloned and DNA sequence determined (13). This fragment contained the 5’-region of the human K receptor. There is an overlap of 240 bp between clone ~115 and clone d2. A common ApaI site within the overlap sequence was used to assemble the entire coding sequence of the human K opioid receptor. Z115 in pBlueScript was digested with ApaI and PstI and d2 with Sac1 and ApaI. The fragments were isolated and ligated into the mammalian expression vector pBK-CMV (Stratagene) pretreated with Sac1 and PstI. The clone is termed pBK-CMV-hkor. Northern blot analysis: Human multiple tissue northern blot was purchased from CLONETECH (Palo Alto, CA). Two pg of poly(A)+ RNA derived from several regions of the human brain was run in each lane and transferred onto a nylon membrane. The human K opioid receptor cDNA probe was lab&d with [a-32P]dCTP. Hybridization of radiolabeled probe to RNA on nylon membrane was carried out in 42” C for 24 h with 2 x 106 cpm/ml labeled probe in 50% formamide / 5x SSPE / 10x Denhardt’s / loo&ml salmon sperm DNA / 2% SDS. The blot was washed with 0.1 x SSC / 0.1% SDS at 55°C for 30 min. The membrane was then exposed to X-ray film for 2-3 days. Transient expression of the human K opioid receptor in COS-1 cells: pBKCMV-hkor was transfected into COS-1 cells as described with DEAE-dextran-chloroquin method (14,15) (DEAE-dextran, Pharmacia, Piscataway, NJ; chloroquin, Sigma, St. Louis, MO) at 10 pg DNA per 100~mm dish. Cells were harvested for 48-60 h following transfection and membranes were prepared for binding assays (4). Receptor binding: Opioid receptor binding was conducted with [fH]diprenorphine (Amersham, Arlington Heights, IL) according to our published procedure (4). Binding was carried out at 25’C for 1 h in duplicate in a volume of 1 ml with 30-60 pg protein. Saturation experiments
Vol. 56, No. 9, 1995
Human
K
Opioid
Receptor
PI/203
were performed with 8 concentrations of [fI-IJdiprenorphine (ranging from 0.02 nM to 2 nM). ()Naloxone (10 @vI) (DuPont, Wilmington, DE) was used to define nonspecific binding. Competitive inhibition of [3HJdiprenorphine binding was performed with 0.4-0.5 nM [QIJdiprenorphine and 910 concentrations of a unlabeled drug. Drugs used were U50,488H (Upjohn, Kalamazoo, MI), norBNI, naltrindole (RBI, Natick, MA), DAMGO, DPDPE, dynorphin A (l-17), dynorphin A (2-17), a-neoendorphin, CTAP (Peninsula, Belmont, CA), and (+)Naloxone (DuPont, Wilmington, DE). Bound and free ligand were separated by rapid filtration under reduced pressure over GF/B filters pre-soaked with 0.5% polyethyleneimine. Binding data were analyzed with EBDA and LIGAND programs (16). Protein contents of membrane preparations were determined by the bicinchoninic acid method of Smith et al. (17) (BCA reagent, Pierce Co., Rockford, IL). Results and Discussion Sequence analysis showed that clone 2115 isolated from the human fetal brain cDNA library did not encompass the entire coding region. The nucleotide sequence of this clone, 4.0 kb in length, corresponds to amino acid residues 6-380 and 3’-untranslated region of the assembled hkor (Fig. 1). From clone d2, isolated from the human genomic DNA library, we obtained the sequence 413 bp upstream from the initiation codon and partial sequence of 1.2 kb downstream from the initiation codon. Within clone d2, there is an exon/intron junction after Arg86. There is an overlap of 240 bp between clone 115 and clone d2 up to the exon/intron junction. The nucleotide sequences within this stretch are 100% identical. Within this overlap, there is a common ApaI site, which was utilized to assemble the full-length coding region. This recombinant clone contains an open-reading frame of 1140 bp, which translates into a protein of 380 amino acids. The deduced amino acid sequence and the putative seven transmembmne helices (TMHs) are shown in Fig. 1. The identity of the amino acid sequence of hkor to other opioid receptors are as follows: 93.9% to the rat K receptor (4), 93.4% to the mouse K receptor (5), 60.2% to the human p receptor (lo), and 59.1% to the human 6 receptor (11). Alignment and comparison of these sequences are shown in Fig.1. Many important features are conserved: two consensus Asn-linked glycosylation sites in the N-terminal domain; a pair of cysteine residues, which may form a disulfide bond between the first and second extracellular loops; several potential phosphorylation sites within the third intracellular loop and the C terminal domain. The deduced amino acid sequence of hkor is identical to that of the human Kreceptor isolated from the human placenta (12), with the exception of Asp2 instead of Glu2. Non-conservative substitutions in the l&or, as compared to the rat and mouse K receptors, occur mostly in the N-terminal domain and the C-terminal domain. Most notably, two Pro (Pro23 and Pro36) residues in the N-terminal domains instead of Leu and Ser. Whether these substitutions have functional significance remains to be determined. From the clone d2, we have obtained the nucleotide sequence of 413 bp upstream from the translation initiation codon. Where the transcription start site is not known. There is no TATA box or CAAT box within this stretch of nucleotide sequence. There is a kappa B element at -403 bp upstream from the initiation codon. Northern blot analysis of poly (A)+ RNA derived from different regions of the human brain showed a single mRNA transcript of 6.0 kb (Fig.2). This is similar in size to rat and mouse K receptor mRNAs (4,5). High levels of hkor mRNA were detected in the amygdala, caudate nucleus, hypothalamus and subthalamic nucleus. Moderate levels were found in the hippocampus and thalamus. The substantia n&a and corpus callosum contained low levels of hkor mRNA (Fig.2). This distribution pattern is in accord with the finding that in the human brain high levels of the K receptor were found in the amygdala, caudate-putarnen and hypothalamus and moderate levels in the hippocampus and thalamus (18). It is noteworthy that the subthalamic nucleus contains a high level of KX’eCCptOr mRNA, Suggesting that K receptors may play an important role in motor control.
PL-204
Human K Opioid Receptor
.......... ..MDSPIQIF ............ -E-__--............ -E_____.................... MDSSAAPTNA SNCTDALAYS
hkor mkor rkor hdor hmor
1
hkor mkor rkor hdor hmor
35 EPDSNGSAGS EDAQLEPAHI SPAIpvIITA
hkor mkor rkor hdor hmor
85 DYTKMKTAT ----____-_ ___-__-___
Vol. 56, No. 9, 1995
RGEPGPTCAP SACLPP.... --D-----S- s-_-L---___---- _-_-L_:::: .MEiPAPSAGAELQ.PPLFAN SCSPAPSPGS WVNLSHLDGN
NSSAWFPGWA _--S---N----S---N_ASDAYPSAFP LSDPCGPNRT
34
TMHl
84
-S----_"--
--Q-_-S---
---____---
_____-_---
__------__
-S---m-“-v
--Q-------
_---_____-
----___---
-c____----
SAGANASGPP G.... PGSAS -L-LAIA--- L--A-~_-- L--"----GNLGGRDSLCP P....TGSP. -MITAIT-M- L--I-C---- F--F---Y-TMH2 PFGDVLCyIy 134
N-SW
______---_ -----____-
_____-____
__A----___
__-----___
----____--
--A____---
-_______--
v__----___
-----____-
---A-S-L_-
--~-_-ET-
-_-EL-_-A-
v____--___
____----__
---A-S-L-
-mm--_GT_
-_-TI--.-_
DRYIAVCHPV -____--_---___--------___------______
KALDFRTPLK -___------_-____-----_----A--------~
184 ,B, -___----_-______----L------V -----V-N-I
VDVIECSLQF __--______ -A-____--_____-____ _____--__-A-G--VPIM -MAV-RP-D. .GAWCM-----AI-LPVM FMAT--Y-Q. .GS-D-T-T-
PDDDYSWWDL ---Es----___E______ -SPSW.Y--T SHPTW.Y-EN
234 Fm -------V-m _______V7_ VT-----L-LV--------
KDRNLRRITR _--______K ---______K --_S____-___-------
UlIXVVAVFY 284 ___-----_I -________I M-_-_-GA__ M_____---I
A ----____----___--------____-----_~---
hkor mkor rkor hdor hmor
135
hkor mkor rkor hdor hmor
185 -T-D
hkor mkor rkor hdor hmor
235 A
hkor mkor rkor hdor hmor
285 -1
hkor mkor rkor hdor hmor
334 DENFKRCFRD FCFPLKMRME RQSTSRVR.N TVQDPAYLRD IDGMNKPV.. 380
L_-----___
_---_-__--
I___---__-
__---CT_-
-A____-_--
-__-_--_-__----___--V-I---T-TM--e--T-
____--____
_----_____ -----___---G--L---R --G---v--m
SVRLLSGSRE ----_-____ ----____---____-_K___M_---K-
7mH6
hkor mkor rkor hdor hmor
I_______-I___---___ ---A-----V ___----_yV
TMH7 LVFUGSTSH STA.ALSSYY ---____--- -__._______----_-__ ---*V_____ I-WT-VDIDR WPLWAALH *J-K_-VTI.p E-TFQTV-WH
__---_____ -_--I-____ ____N---.---_______ -y--I_____ ---_N--we-------e-Q L-mCGmD PS-FS-P-EA ------_-SE --I-TSSNIQ-NST-I-Q-
333 ---_---___ ____-----L_-----A-____-__---
_-----SM-______SM__ -ARERVTACT -RDH-STANT
-__-V--___ -___V--------V----v C___V___--
VG------.. VG______.* PS......DG V-RT-HQLEN
. . . . . . . . . . . . . . . . . . . . . . . . . . .
PGGGRAA.. LElAETAPLP
Fig. 1. (Legend al fop of naf page.)
Vol. 56, No. 9, 1995
Human K Opioid Receptor
PL-205
Figure 1 shows deduced amino acid sequence of the human Kopioid receptor (hkor), and its alignment with those of the mouse Kreceptor (mkor) (5), the rat 1creceptor (rkor) (4), the human S receptor (hdor) (1 l), and the human j.t receptor (hmor) (10). Amino acid residue numbers refer to those of hkor, mkor and rkor. Nucleotide sequence of hkor has been deposited in the GenBank (accession number L37362). - indicates the same amino acid as in the human Kreceptor. **represent gaps introduced for sequence alignment. Seven putative transmembrane helices (TMHs) are underlined.
kb 9.5 7.5 4.4 2.4 1.35 Fig.2. Figure 2 shows distribution of hkor mRNA in some regions of the human brain. Poly(A)+ RNA derived from various brain regions was analyzed by northern blot as described in Methods. Note that there is a single transcript of 6.0 kb. Two pg of Poly(A)+ RNA was loaded in each lane. The source of Poly(A)+ RNA in each lane is as follows: 1, amygdala; 2, caudate nucleus; 3, corpus callosum; 4, hippocampus; 5. hypothalamus; 6. substantia nigra; 7. subthalamic nucleus; 8, thalamus. Exposure time was 3 days.
Membranes of COS-1 cells transfected with pBK-CMV-hkor exhibited high specific [%Ijdiprenorphine binding. Membranes of untransfected cells or cells transfected with the vector had no detectable specific binding. Binding of [XIjdiprenorphine was saturable and & and Bmax were estimated to be 0.08 nM and 375 + 36 fmole / mg protein (n=4). While (-)naloxone inhibited 0.3-0.5 nM [WIkliprenorphine binding with Ki of 4 nM, (+)naloxone did not inhibit binding even at 1 uM (Table l), indicating binding stereospecificity of hkor. [3H]Diprenorphine binding was potently inhibited by nor-BNI, a selective K antagonist, but much less by naltrindole, a selective 6 antagonist, or CTAP, a selective p antagonist (Table I). U50,488H, a selective Kagonist, was much more potent than DAMGO, a selective p agonist, or DPDPE, a selective 6 agonist, in inhibiting [Xkliprenorphine binding (Table 1). In addition, dynorphin A (1-17) and a-neoendorphin had high affinity for hkor, whereas dynorphin A (2-17) did not bind (Table 1). The Kl values of these drugs, listed in Table 1, in general agree with those in the literature (19-22). These data confiied that hkor represents an opioid receptor with K characteristics, most likely ~1 type.
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Human K Opioid Receptor
Vol. 56, No. 9, 1995
TABLE I Ki Values of Binding of Gpioid Compounds to the Human K Receptor Transiently COS-1 Cells
Compounds
Ki value (nM)
i.t-Selective ligands DAMGG CTAP
>lOOO >lOOO
&Selective ligands DPDPE Naltrindole
>lOOO 4.8f0.6
&elective ligands U50,488H Nor-BNI Dynorphin A( 1-17) a-Neoendorphin
0.68 f 0.040& 0.01 If 0.058f
Expressed in
0.16 0.003 0.001 0.008
others (-)-Naloxone (+)-Naloxone Dynorphin A (2-17)
3.9*0.3 >lOOO >lOOO
Data were expressed as mean + s.e.m. for each compound. independent experiments.
Each Kl value was derived from three
In summary, in this report we described the cloning of a human K opioid receptor from the brain and its deduced amino acid sequence. Upon transient expression in COS-1 cells, the clone displays binding characteristics expected of a K opioid receptor. The K receptor mRNA, 6.0 kb in size, is unevenly distributed in regions of the human brain. Cloning of the human K receptor allows us to examine interaction of drugs directly with the human receptor, instead of the rodent receptor as traditionally done, for development of better and safer therapeutic agents. Acknowledgment This work was supported by a grant from National Institute on Drug Abuse (DA 04745). We thank Jinling Yin for technical assistance, Dr. Jeffrey Benovic for helpful discussions and Dr. Thomas Frielle for the human genomic hDASH library. References 1. G.W. PASTERNAK. The Opiate Receptors, The Humana Press, Clifton, NJ (1988). 2. C. EVANS, D.E. KEITH,JR., H. MORRISON, K. MAGENDZO and R.H. EDWARDS, Science 258 1952-1955 (1992). 3. B. KIEFFER, K. BEFORT, C. GAVERIAUX-RUFF and C.G. HIRTH, Proc. Natl. Acad. Sci. U. S. A. Se 12048-12052 (1992). 4. S. LI, J. ZHU, C. CHEN, Y.-W. CHEN, J.K. DERIEL, B. ASHBY and L.-Y. LIU-CHEN, Biochem. J. 295 629-633 (1993). 5. K. YASUDA, K. RAYNOR, H. KONG, C.D. BREDER, J. TAKEDA, T. REISINE and G.I. BELL, Proc. Natl. Acad. Sci. U. S. A. a 6736-6740 (1993).
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6. M. NISHI, H. TAKESHIMA, K. FUKUDA, S. KATO and K. MORI, FEBS Lett. 330 77-80 (1993). 7. M. MINAMI, T. TOYA, Y. KATAO, K. MAEKAWA, S. NAKAMURA, T. ONOGI, S. KANEKO and M. SATOH, FEBS Lett. m 291-295 (1993). 8. F. MENG, G.-X. XIE, R.C. THOMPSON, A. MANSOUR, A. GOLDSTEIN, S.J. WATSON and H. AKIL, Proc. Natl. Acad. Sci. U. S. A. m 9954-9958 (1993). 9. Y. CHEN, A. MESTEK, J. LIU and L. YU, B&hem. J. 295 625-628 (1993). 10. J.-B. WANG, P.S. JOHNSON, A.M. PERSICO, A.L. HAWKINS, C.A. GRIFFIN and G.R. UHL, FEBS Lett. 338 217-222 (1994). 11. R.J. KNAPP, E. MALATYNSKA, L. FANG, X. LI, E. BABIN, M. NGUYEN, G. SANTORO, E.V. VARGA, V.J. HRUBY, W.. ROESKE and H.I. YAMAMURA, Life Sci. % 463-469 (1994). 12. E. MANSSON, L. BARE and D. YANG, B&hem. Biophys. Res. Comm. 202 1431-1437 (1994). 13. F. SANGER, S. NICKLEN and A.R. COULSON, Proc. Natl. Acad. Sci. U. S. A. 74 54635467 (1977). 14. B. CULLEN, Methods Enzymol. 152.684-704 (1987). 15. J.-C. XUE, C. CHEN, J. ZHU, S. KUNAPULI, J.K. DERIEL, L. YU and L.-Y. LIUCHEN, J. Biol. Chemi 269 30195-30199 (1994). 16. G.A. MCPHERSON, Computer Prog. Biomed. 1z 107-l 14 (1983). 17. P.K. SMITH, RI. KROHN, G.T. HERMANSON, A.K. MALLIA, F.H. GARTNER, M.D. PROVENZANO, E.K. FUJIMOTO, N.M. GOEKE, B.J. OLSON and D.C. KLENK, Anal. B&hem. 150 76-85 (1985). 18. R. QUIRION and C. PILAPIL, Receptors in the human nervous sysfem, F.A.O. Mendelsohn and G. Paxinos (eds), 103-121, Academic Press, Inc., San Diego, CA (1991). 19. J.A. CLARK, L. LIU, M. PRICE, B. HERSH, M. EDELSON and G.W. PASTERNAK, J. Pharmacol. Exp. Ther. 251461-468 (1989). 20. P.S. PORTOGHESE, M. SULTANA and A.E. TAKEMORI, Eur. J. Pharmacol. 146 185-186 (1988). 21. C.S. KONKOY and S.R. CHILDERS, B&hem. Pharmacol. ti 207-216 (1993). 22. A.E. TAKEMORI, B.Y. HO, J.S. NAESETH and P.S. PORTOGHESE, J. Pharmacol. Exp. Ther. 246 255-258 (1988).
Pergamon 00243205(94)00507-9 PHARMACOLOGY LETTERS Accelerated Communication CLONING
OF
A HUMAN
K OPIOID
RECEPTOR
FROM
THE
BRAIN
Jinmin Zhu, Chongguang Chen, Ji-Chun Xue, Satya Kunapulil, J. Kim DeRie12 and Lee-Yuan Liu-Chen Departments of Pharmacology and 1Physiology and 2Fels Institute for Molecular Biology and Cancer Research, Temple University School of Medicine, Philadelphia, PA 19140 (Submitted
November 30, 1994; accepted December 13, 1994; received in final form Decemberl6, 1994)
Abstract By using a rat Kopioid receptor cDNA as a probe to screen a human brain cDNA library, we isolated a 4.0-kb clone (~115) which encompasses a major portion of a human Kopioid receptor (l&or), extending from the amino acid residue #6 to the 3’-untranslated region. The extreme Sregion 232-bp fragment of ~115 was used as a probe to screen a human genomic DNA library. A 1.6kb fragment (d2) of one positive clone was found to extend from S-untranslated region to beyond the exon/intron junction at residue ArgW The genomic DNA fragment d2 and the cDNA clone ~115 were assembled to generate a clone (d2-~115) containing the entire coding sequence of hkor. Clone d2-z115 has an open reading frame of 1140 bp, which encodes for a 380-amino acid protein. The deduced amino acid sequence has 93.9% and 93.2% identity to rat and mouse K receptors, respectively. It also displays _ 60% identity to both human p and 6 receptors. Northern blot analysis showed that in the human brain there was a single hkor mRNA transcript of 6.0 kb. Among brain regions examined, the amygdala, caudate nucleus, hypothalamus and subthalamic nucleus contained high levels of hkor mRNA. Hkor was cloned into the expression vector pBKCMV and transiently expressed in COS-1 cells. Hkor had high affinity for [3H] diprenorphine, a nonselective opioid antagonist, and displayed stereospecific binding to naloxone. Kselective ligands (U50,488H and nor-BNI) had high affinities, whereas ~1and 6 selective ligands bound with much lower affinities. Dynorphin A (1-17) and a-neoendorphin, both endogenous Kpeptides, bound with high affinities. These binding characteristics confirmed that hkor is a Kreceptor, most likely ~1 type. Cloning of the human rcreceptor allows investigation of interactions of compounds with the human receptor, instead of rodent receptors, for development of better therapeutic agents. Key Words: opioid receptors, cDNA library, genomic DNA library
Wuction Opiates and opioid compounds act on receptors on the surface of cell membranes to produce their effects. The presence of three major types of opioid receptors - CI_, K, 6 - in the peripheral and central nervous system has been established by pharmacological and binding studies (1). K opioid receptors have been shown to mediate many physiological and pharmacological effects, including analgesia, diuresis, hypothermia, dysphoria and changes in neuroendocrine functions (1). Following the cloning of the mouse 6 opioid receptor (2,3), we (4) as well as several groups (5-9) cloned K opioid receptors from the rat and mouse. Both rat and mouse K receptors bind K agonists and antagonists with high affinities, yet bind l.t and 6 ligands with low affinities. Cloned rodent K receptors are coupled through G proteins to inhibition of adenylate cyclase. Correspondence should be sent to: Dr. Lee-Yuan Liu-Chen, Department of Pharmacology, Temple University School of Medicine, 3420 N. Broad St., Philadelphia, PA 19140. phone: (215) 7074188; fax: (215) 707-7068; e-mail: [email protected].
PL.202
Human ICOpioid Receptor
Vol. 56, No. 9, 1995
Cloning of human lr and 6 opioid receptors has been reported (10,ll). Recently, Mansson et al. (12) reported cloning of a human K receptor from the placenta by reverse transcription of mRNA followed by polymerase chain reaction (PCR) on cDNA. In the present study, we report the cloning of a human Kopioid receptor from the brain by conventional cDNA and genomic DNA library screening and expression of the clone in COS-1 cells for functional characterization. Materials and Methods Screening of human cDNA and genomic DNA libraries: A human fetal brain hZAPI1 cDNA library (Stratagene, La Jolla, CA) was plated according to the manufacturer’s instructions . An 849-bp probe corresponding to nucleotides 388-1237 of the rat 1copioid receptor ((4) with accession number L22536) was produced by PCR and labeled with [a-32P] dC!TP (3000 Wmmol, DuPont-NEN, Boston, MA) by use of random primers DNA labeling kit (GIBCO-BRL, Gaithersburg, MD). Approximately 15x106 cDNA clones were screened. Prehybridization of replica on Duralon-WTM membranes (Stratagene) was carried out in 50% formatm‘de/SxSSC/Sx Denhardt’s / 1.0% SDS / 0.1% sodium pyrophosphate / 100 pg/ml salmon sperm DNA (hybridization buffer) at 42’ C for 4 h. Hybridization was conducted with labeled probe (2 x 106 cpm/ml) in the hybridization buffer at 42’ C for 2 days. A final post-hybridization wash was performed in 0.2 x SSC / 0.1% sodium pyrophosphate at 65’ C for 30 min. Secondary and tertiary screening was carried out to purify the clones and h phage DNA of positive clones was prepared. pBlueScript plasmids were excised from h phages by in vivo excision with the ExAssist helper phage and transformed into SOLR cells. The plasmids were analyzed by restriction mapping and DNA sequence determined with automated and manual methods (13). Nucleotide sequence analysis was carried out with GCG programs. One clone with 4.0-kb insert, termed ~115, exhibited > 90% identity to the rat Kreceptor and contained 1125 bp upstream from the termination codon. To obtain the 5’-region of the human Kreceptor, we screened a human lymphocyte genomic DNA library in ADASH. The probe, corresponding the extreme 5’-end 232 bp of 2115, was produced by PCR and labeled by [a-32P]dCTP. One of the positive clones, 28 kb in length, was digested with HindIII. A fragment of 1.6 kb, termed d2, was positive by southern blot hybridization. D2 was subcloned and DNA sequence determined (13). This fragment contained the 5’-region of the human K receptor. There is an overlap of 240 bp between clone ~115 and clone d2. A common ApaI site within the overlap sequence was used to assemble the entire coding sequence of the human K opioid receptor. Z115 in pBlueScript was digested with ApaI and PstI and d2 with Sac1 and ApaI. The fragments were isolated and ligated into the mammalian expression vector pBK-CMV (Stratagene) pretreated with Sac1 and PstI. The clone is termed pBK-CMV-hkor. Northern blot analysis: Human multiple tissue northern blot was purchased from CLONETECH (Palo Alto, CA). Two pg of poly(A)+ RNA derived from several regions of the human brain was run in each lane and transferred onto a nylon membrane. The human K opioid receptor cDNA probe was lab&d with [a-32P]dCTP. Hybridization of radiolabeled probe to RNA on nylon membrane was carried out in 42” C for 24 h with 2 x 106 cpm/ml labeled probe in 50% formamide / 5x SSPE / 10x Denhardt’s / loo&ml salmon sperm DNA / 2% SDS. The blot was washed with 0.1 x SSC / 0.1% SDS at 55°C for 30 min. The membrane was then exposed to X-ray film for 2-3 days. Transient expression of the human K opioid receptor in COS-1 cells: pBKCMV-hkor was transfected into COS-1 cells as described with DEAE-dextran-chloroquin method (14,15) (DEAE-dextran, Pharmacia, Piscataway, NJ; chloroquin, Sigma, St. Louis, MO) at 10 pg DNA per 100~mm dish. Cells were harvested for 48-60 h following transfection and membranes were prepared for binding assays (4). Receptor binding: Opioid receptor binding was conducted with [fH]diprenorphine (Amersham, Arlington Heights, IL) according to our published procedure (4). Binding was carried out at 25’C for 1 h in duplicate in a volume of 1 ml with 30-60 pg protein. Saturation experiments
Vol. 56, No. 9, 1995
Human
K
Opioid
Receptor
PI/203
were performed with 8 concentrations of [fI-IJdiprenorphine (ranging from 0.02 nM to 2 nM). ()Naloxone (10 @vI) (DuPont, Wilmington, DE) was used to define nonspecific binding. Competitive inhibition of [3HJdiprenorphine binding was performed with 0.4-0.5 nM [QIJdiprenorphine and 910 concentrations of a unlabeled drug. Drugs used were U50,488H (Upjohn, Kalamazoo, MI), norBNI, naltrindole (RBI, Natick, MA), DAMGO, DPDPE, dynorphin A (l-17), dynorphin A (2-17), a-neoendorphin, CTAP (Peninsula, Belmont, CA), and (+)Naloxone (DuPont, Wilmington, DE). Bound and free ligand were separated by rapid filtration under reduced pressure over GF/B filters pre-soaked with 0.5% polyethyleneimine. Binding data were analyzed with EBDA and LIGAND programs (16). Protein contents of membrane preparations were determined by the bicinchoninic acid method of Smith et al. (17) (BCA reagent, Pierce Co., Rockford, IL). Results and Discussion Sequence analysis showed that clone 2115 isolated from the human fetal brain cDNA library did not encompass the entire coding region. The nucleotide sequence of this clone, 4.0 kb in length, corresponds to amino acid residues 6-380 and 3’-untranslated region of the assembled hkor (Fig. 1). From clone d2, isolated from the human genomic DNA library, we obtained the sequence 413 bp upstream from the initiation codon and partial sequence of 1.2 kb downstream from the initiation codon. Within clone d2, there is an exon/intron junction after Arg86. There is an overlap of 240 bp between clone 115 and clone d2 up to the exon/intron junction. The nucleotide sequences within this stretch are 100% identical. Within this overlap, there is a common ApaI site, which was utilized to assemble the full-length coding region. This recombinant clone contains an open-reading frame of 1140 bp, which translates into a protein of 380 amino acids. The deduced amino acid sequence and the putative seven transmembmne helices (TMHs) are shown in Fig. 1. The identity of the amino acid sequence of hkor to other opioid receptors are as follows: 93.9% to the rat K receptor (4), 93.4% to the mouse K receptor (5), 60.2% to the human p receptor (lo), and 59.1% to the human 6 receptor (11). Alignment and comparison of these sequences are shown in Fig.1. Many important features are conserved: two consensus Asn-linked glycosylation sites in the N-terminal domain; a pair of cysteine residues, which may form a disulfide bond between the first and second extracellular loops; several potential phosphorylation sites within the third intracellular loop and the C terminal domain. The deduced amino acid sequence of hkor is identical to that of the human Kreceptor isolated from the human placenta (12), with the exception of Asp2 instead of Glu2. Non-conservative substitutions in the l&or, as compared to the rat and mouse K receptors, occur mostly in the N-terminal domain and the C-terminal domain. Most notably, two Pro (Pro23 and Pro36) residues in the N-terminal domains instead of Leu and Ser. Whether these substitutions have functional significance remains to be determined. From the clone d2, we have obtained the nucleotide sequence of 413 bp upstream from the translation initiation codon. Where the transcription start site is not known. There is no TATA box or CAAT box within this stretch of nucleotide sequence. There is a kappa B element at -403 bp upstream from the initiation codon. Northern blot analysis of poly (A)+ RNA derived from different regions of the human brain showed a single mRNA transcript of 6.0 kb (Fig.2). This is similar in size to rat and mouse K receptor mRNAs (4,5). High levels of hkor mRNA were detected in the amygdala, caudate nucleus, hypothalamus and subthalamic nucleus. Moderate levels were found in the hippocampus and thalamus. The substantia n&a and corpus callosum contained low levels of hkor mRNA (Fig.2). This distribution pattern is in accord with the finding that in the human brain high levels of the K receptor were found in the amygdala, caudate-putarnen and hypothalamus and moderate levels in the hippocampus and thalamus (18). It is noteworthy that the subthalamic nucleus contains a high level of KX’eCCptOr mRNA, Suggesting that K receptors may play an important role in motor control.
PL-204
Human K Opioid Receptor
.......... ..MDSPIQIF ............ -E-__--............ -E_____.................... MDSSAAPTNA SNCTDALAYS
hkor mkor rkor hdor hmor
1
hkor mkor rkor hdor hmor
35 EPDSNGSAGS EDAQLEPAHI SPAIpvIITA
hkor mkor rkor hdor hmor
85 DYTKMKTAT ----____-_ ___-__-___
Vol. 56, No. 9, 1995
RGEPGPTCAP SACLPP.... --D-----S- s-_-L---___---- _-_-L_:::: .MEiPAPSAGAELQ.PPLFAN SCSPAPSPGS WVNLSHLDGN
NSSAWFPGWA _--S---N----S---N_ASDAYPSAFP LSDPCGPNRT
34
TMHl
84
-S----_"--
--Q-_-S---
---____---
_____-_---
__------__
-S---m-“-v
--Q-------
_---_____-
----___---
-c____----
SAGANASGPP G.... PGSAS -L-LAIA--- L--A-~_-- L--"----GNLGGRDSLCP P....TGSP. -MITAIT-M- L--I-C---- F--F---Y-TMH2 PFGDVLCyIy 134
N-SW
______---_ -----____-
_____-____
__A----___
__-----___
----____--
--A____---
-_______--
v__----___
-----____-
---A-S-L_-
--~-_-ET-
-_-EL-_-A-
v____--___
____----__
---A-S-L-
-mm--_GT_
-_-TI--.-_
DRYIAVCHPV -____--_---___--------___------______
KALDFRTPLK -___------_-____-----_----A--------~
184 ,B, -___----_-______----L------V -----V-N-I
VDVIECSLQF __--______ -A-____--_____-____ _____--__-A-G--VPIM -MAV-RP-D. .GAWCM-----AI-LPVM FMAT--Y-Q. .GS-D-T-T-
PDDDYSWWDL ---Es----___E______ -SPSW.Y--T SHPTW.Y-EN
234 Fm -------V-m _______V7_ VT-----L-LV--------
KDRNLRRITR _--______K ---______K --_S____-___-------
UlIXVVAVFY 284 ___-----_I -________I M-_-_-GA__ M_____---I
A ----____----___--------____-----_~---
hkor mkor rkor hdor hmor
135
hkor mkor rkor hdor hmor
185 -T-D
hkor mkor rkor hdor hmor
235 A
hkor mkor rkor hdor hmor
285 -1
hkor mkor rkor hdor hmor
334 DENFKRCFRD FCFPLKMRME RQSTSRVR.N TVQDPAYLRD IDGMNKPV.. 380
L_-----___
_---_-__--
I___---__-
__---CT_-
-A____-_--
-__-_--_-__----___--V-I---T-TM--e--T-
____--____
_----_____ -----___---G--L---R --G---v--m
SVRLLSGSRE ----_-____ ----____---____-_K___M_---K-
7mH6
hkor mkor rkor hdor hmor
I_______-I___---___ ---A-----V ___----_yV
TMH7 LVFUGSTSH STA.ALSSYY ---____--- -__._______----_-__ ---*V_____ I-WT-VDIDR WPLWAALH *J-K_-VTI.p E-TFQTV-WH
__---_____ -_--I-____ ____N---.---_______ -y--I_____ ---_N--we-------e-Q L-mCGmD PS-FS-P-EA ------_-SE --I-TSSNIQ-NST-I-Q-
333 ---_---___ ____-----L_-----A-____-__---
_-----SM-______SM__ -ARERVTACT -RDH-STANT
-__-V--___ -___V--------V----v C___V___--
VG------.. VG______.* PS......DG V-RT-HQLEN
. . . . . . . . . . . . . . . . . . . . . . . . . . .
PGGGRAA.. LElAETAPLP
Fig. 1. (Legend al fop of naf page.)
Vol. 56, No. 9, 1995
Human K Opioid Receptor
PL-205
Figure 1 shows deduced amino acid sequence of the human Kopioid receptor (hkor), and its alignment with those of the mouse Kreceptor (mkor) (5), the rat 1creceptor (rkor) (4), the human S receptor (hdor) (1 l), and the human j.t receptor (hmor) (10). Amino acid residue numbers refer to those of hkor, mkor and rkor. Nucleotide sequence of hkor has been deposited in the GenBank (accession number L37362). - indicates the same amino acid as in the human Kreceptor. **represent gaps introduced for sequence alignment. Seven putative transmembrane helices (TMHs) are underlined.
kb 9.5 7.5 4.4 2.4 1.35 Fig.2. Figure 2 shows distribution of hkor mRNA in some regions of the human brain. Poly(A)+ RNA derived from various brain regions was analyzed by northern blot as described in Methods. Note that there is a single transcript of 6.0 kb. Two pg of Poly(A)+ RNA was loaded in each lane. The source of Poly(A)+ RNA in each lane is as follows: 1, amygdala; 2, caudate nucleus; 3, corpus callosum; 4, hippocampus; 5. hypothalamus; 6. substantia nigra; 7. subthalamic nucleus; 8, thalamus. Exposure time was 3 days.
Membranes of COS-1 cells transfected with pBK-CMV-hkor exhibited high specific [%Ijdiprenorphine binding. Membranes of untransfected cells or cells transfected with the vector had no detectable specific binding. Binding of [XIjdiprenorphine was saturable and & and Bmax were estimated to be 0.08 nM and 375 + 36 fmole / mg protein (n=4). While (-)naloxone inhibited 0.3-0.5 nM [WIkliprenorphine binding with Ki of 4 nM, (+)naloxone did not inhibit binding even at 1 uM (Table l), indicating binding stereospecificity of hkor. [3H]Diprenorphine binding was potently inhibited by nor-BNI, a selective K antagonist, but much less by naltrindole, a selective 6 antagonist, or CTAP, a selective p antagonist (Table I). U50,488H, a selective Kagonist, was much more potent than DAMGO, a selective p agonist, or DPDPE, a selective 6 agonist, in inhibiting [Xkliprenorphine binding (Table 1). In addition, dynorphin A (1-17) and a-neoendorphin had high affinity for hkor, whereas dynorphin A (2-17) did not bind (Table 1). The Kl values of these drugs, listed in Table 1, in general agree with those in the literature (19-22). These data confiied that hkor represents an opioid receptor with K characteristics, most likely ~1 type.
PL-206
Human K Opioid Receptor
Vol. 56, No. 9, 1995
TABLE I Ki Values of Binding of Gpioid Compounds to the Human K Receptor Transiently COS-1 Cells
Compounds
Ki value (nM)
i.t-Selective ligands DAMGG CTAP
>lOOO >lOOO
&Selective ligands DPDPE Naltrindole
>lOOO 4.8f0.6
&elective ligands U50,488H Nor-BNI Dynorphin A( 1-17) a-Neoendorphin
0.68 f 0.040& 0.01 If 0.058f
Expressed in
0.16 0.003 0.001 0.008
others (-)-Naloxone (+)-Naloxone Dynorphin A (2-17)
3.9*0.3 >lOOO >lOOO
Data were expressed as mean + s.e.m. for each compound. independent experiments.
Each Kl value was derived from three
In summary, in this report we described the cloning of a human K opioid receptor from the brain and its deduced amino acid sequence. Upon transient expression in COS-1 cells, the clone displays binding characteristics expected of a K opioid receptor. The K receptor mRNA, 6.0 kb in size, is unevenly distributed in regions of the human brain. Cloning of the human K receptor allows us to examine interaction of drugs directly with the human receptor, instead of the rodent receptor as traditionally done, for development of better and safer therapeutic agents. Acknowledgment This work was supported by a grant from National Institute on Drug Abuse (DA 04745). We thank Jinling Yin for technical assistance, Dr. Jeffrey Benovic for helpful discussions and Dr. Thomas Frielle for the human genomic hDASH library. References 1. G.W. PASTERNAK. The Opiate Receptors, The Humana Press, Clifton, NJ (1988). 2. C. EVANS, D.E. KEITH,JR., H. MORRISON, K. MAGENDZO and R.H. EDWARDS, Science 258 1952-1955 (1992). 3. B. KIEFFER, K. BEFORT, C. GAVERIAUX-RUFF and C.G. HIRTH, Proc. Natl. Acad. Sci. U. S. A. Se 12048-12052 (1992). 4. S. LI, J. ZHU, C. CHEN, Y.-W. CHEN, J.K. DERIEL, B. ASHBY and L.-Y. LIU-CHEN, Biochem. J. 295 629-633 (1993). 5. K. YASUDA, K. RAYNOR, H. KONG, C.D. BREDER, J. TAKEDA, T. REISINE and G.I. BELL, Proc. Natl. Acad. Sci. U. S. A. a 6736-6740 (1993).
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Opioid Receptor
PG207
6. M. NISHI, H. TAKESHIMA, K. FUKUDA, S. KATO and K. MORI, FEBS Lett. 330 77-80 (1993). 7. M. MINAMI, T. TOYA, Y. KATAO, K. MAEKAWA, S. NAKAMURA, T. ONOGI, S. KANEKO and M. SATOH, FEBS Lett. m 291-295 (1993). 8. F. MENG, G.-X. XIE, R.C. THOMPSON, A. MANSOUR, A. GOLDSTEIN, S.J. WATSON and H. AKIL, Proc. Natl. Acad. Sci. U. S. A. m 9954-9958 (1993). 9. Y. CHEN, A. MESTEK, J. LIU and L. YU, B&hem. J. 295 625-628 (1993). 10. J.-B. WANG, P.S. JOHNSON, A.M. PERSICO, A.L. HAWKINS, C.A. GRIFFIN and G.R. UHL, FEBS Lett. 338 217-222 (1994). 11. R.J. KNAPP, E. MALATYNSKA, L. FANG, X. LI, E. BABIN, M. NGUYEN, G. SANTORO, E.V. VARGA, V.J. HRUBY, W.. ROESKE and H.I. YAMAMURA, Life Sci. % 463-469 (1994). 12. E. MANSSON, L. BARE and D. YANG, B&hem. Biophys. Res. Comm. 202 1431-1437 (1994). 13. F. SANGER, S. NICKLEN and A.R. COULSON, Proc. Natl. Acad. Sci. U. S. A. 74 54635467 (1977). 14. B. CULLEN, Methods Enzymol. 152.684-704 (1987). 15. J.-C. XUE, C. CHEN, J. ZHU, S. KUNAPULI, J.K. DERIEL, L. YU and L.-Y. LIUCHEN, J. Biol. Chemi 269 30195-30199 (1994). 16. G.A. MCPHERSON, Computer Prog. Biomed. 1z 107-l 14 (1983). 17. P.K. SMITH, RI. KROHN, G.T. HERMANSON, A.K. MALLIA, F.H. GARTNER, M.D. PROVENZANO, E.K. FUJIMOTO, N.M. GOEKE, B.J. OLSON and D.C. KLENK, Anal. B&hem. 150 76-85 (1985). 18. R. QUIRION and C. PILAPIL, Receptors in the human nervous sysfem, F.A.O. Mendelsohn and G. Paxinos (eds), 103-121, Academic Press, Inc., San Diego, CA (1991). 19. J.A. CLARK, L. LIU, M. PRICE, B. HERSH, M. EDELSON and G.W. PASTERNAK, J. Pharmacol. Exp. Ther. 251461-468 (1989). 20. P.S. PORTOGHESE, M. SULTANA and A.E. TAKEMORI, Eur. J. Pharmacol. 146 185-186 (1988). 21. C.S. KONKOY and S.R. CHILDERS, B&hem. Pharmacol. ti 207-216 (1993). 22. A.E. TAKEMORI, B.Y. HO, J.S. NAESETH and P.S. PORTOGHESE, J. Pharmacol. Exp. Ther. 246 255-258 (1988).