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Department of Zoology, 3029 Cordley Hall, Oregon State University, Corvallis, Oregon 97331, USA
(Requests for offprints should be addressed to S Bradford; Email: bradforc{at}science.oregonstate.edu)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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) opioid receptor has been isolated from the brain of a urodele amphibian, the rough-skinned newt Taricha granulosa. The deduced protein contains 385 amino acids and possesses features commonly attributed to G protein-coupled receptors, such as seven putative transmembrane domains. The newt receptor has 75% sequence identity to opioid receptors cloned from mammals, and 66% sequence identity to the opioid receptor reported for the zebrafish, with the greatest divergence in the extracellular N-terminus, the second and third extracellular loops and the intracellular C-terminus. Membranes isolated from COS-7 cells expressing the newt receptor possessed a single, high-affinity (Kd =1.5 nM) binding site for the -selective agonist U69593
[GenBank]
. In competition binding assays, the expressed newt receptor displayed high affinity for the -selective agonists GR89696, dynorphin A(1–13), U69593
[GenBank]
, U50488
[GenBank]
and BRL52537 as well as the -selective antagonist nor-binaltorphimine and the non-selective antagonist naloxone. Rank order potencies and affinity constants were similar in competition binding studies that used either whole brain homogenates or membranes isolated from COS-7 cells expressing the newt receptor. The expressed receptor displayed essentially no affinity for the 
-selective agonist DPDPE ([d-penicillamine, d-penicillamine]enkephalin), but showed moderate affinity for the µ-selective agonist DAMGO ([d-Ala-MePhe, Gly-ol]enkephalin) and moderately high affinity for nociceptin (orphanin FQ), the endogenous ligand for the opioid receptor-like (ORL)1 receptor. These findings support the conclusions that a gene for the opioid receptor is expressed in amphibians and that the pharmacology of the newt receptor closely matches mammalian opioid receptors. However, the functional dichotomy between the classic opioid receptors (, , µ) and ORL1 appears less strict in amphibians than in mammals.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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), delta (), mu (µ) and ORL1 (opioid receptor-like) represent the four main classes of opioid receptors, which are defined and distinguished by primary structures, anatomical distributions, pharmacological profiles and physiological functions. Opioid receptors belong to the superfamily of G protein-coupled receptors possessing seven transmembrane domains (Janecka et al. 2004, Waldhoer et al. 2004), and when activated, typically produce an inhibition of adenylyl cyclase activity (Reisine & Bell 1993) and the modulation of calcium and potassium conductances (Darlison et al. 1997).
opioid receptors (named for the prototypic ligand ketocyclazocine) selectively bind endogenous peptides derived from pro-dynorphin (Chavkin et al. 1982), as well as synthetic benzomorphans (Goldstein & Naidu 1989) and arylacetamides such as U50488
[GenBank]
(Lahti et al. 1982, Piercey et al. 1982) and U69593
[GenBank]
(Lahti et al. 1985). receptors participate in a wide range of physiological functions, including pain perception, feeding and drinking behavior, water balance, gut motility, temperature regulation and cardiovascular control (Bloom 1983). Because activation of receptors produces analgesia in the absence of primary reinforcement (Meng et al. 1993), the receptor has been an attractive target for development of analgesic drugs with low abuse potential. Although early agonists evaluated in humans showed a predisposition for eliciting central nervous system disturbances (altered perception, dysphoria, psychotomimesis) (Pfeiffer et al. 1986), -related drugs developed more recently do not provoke such adverse side effects and have been demonstrated to be neuroprotective and potentially effective in the treatment of stroke and ischemic brain attacks (Tortella et al. 1997).
Many early studies describing the isolation, solubilization, purification and partial characterization of opioid receptors were conducted using membranes prepared from the brains of toads (Bufo marinus) (Ruegg et al. 1980, 1981, Cone & Goldstein 1982, Simon et al. 1982) or frogs (Rana pipiens) (Benyhe et al. 1994, Simon et al. 1984, 1987, 1990, Stevens et al. 1987), because receptors were found to be highly abundant in the amphibian central nervous system. Early experiments with frogs and toads demonstrated the presence of immunoreactive dynorphin in the brain (Cone & Goldstein 1982) and reported antinociceptive responses to exogenously administered dynorphin (Stevens et al. 1987). In contrast, most studies describing molecular cloning of opioid receptors have focused on mammalian species; nucleic acid/amino acid sequence data correlated with binding pharmacology for non-mammalian species are nonexistent.
To date, full-length cDNA sequences have been reported for mouse (Yasuda et al. 1993), rat (Chen et al. 1993, Meng et al. 1993, Minami et al. 1993, Yakovlev et al. 1995), guinea pig (Xie et al. 1994) and human (Mansson et al. 1994, Simonin et al. 1995); a partial sequence for a bovine receptor (Li et al. 1996) is also available. Partial sequences for opioid receptors have been reported for two teleost fishes, white sucker (Catostomus commersoni) (Li et al. 1996, Darlison et al. 1997) and bass (Morone saxatilis) (Li et al. 1996), and a full-length cDNA sequence for a zebrafish (Danio rerio) receptor (‘ZFOR3') has been deposited in the GenBank database (Accession AF285173
[GenBank]
) along with the predicted amino acid sequence (GenBank Accession NP_878306
[GenBank]
). Among avian species, a opioid receptor has been identified and partially characterized pharmacologically in a passerine songbird (Junco hyemalis) (Deviche et al. 1993). Among amphibians, a cDNA sequence for a -like opioid receptor from the northern grass frog (R. pipiens) has been deposited in the GenBank database (Accession AF530573
[GenBank]
), but this sequence includes a base insertion that leads to a frameshift in the potential coding region. For this reason the identification or status of any protein(s) encoded by the grass frog cDNA sequence remains unclear.
Among urodele amphibians, no cloning experiments directed at opioid receptors have been reported to date, despite compelling evidence for the existence and physiological relevance of receptors in these animals. In male rough-skinned newts (Taricha granulosa), ethylketocyclazocine and bremazocine (-selective receptor agonists) suppress sexual behavior (amplectic clasping of females); the effect of bremazocine is reversed in a dose-dependent fashion by treatment with naloxone (a non-selective opioid antagonist) (Deviche & Moore 1987). Bremazocine suppresses spontaneous (Deviche et al. 1989) but not corticotropin-releasing factor-induced (Lowry et al. 1990) locomotor activity in male newts, supporting the idea that opioids may be important regulators of amphibian behavior. Finally, some but not all -selective agonists are capable of displacing [3H]corticosterone from the membrane glucocorticoid receptor identified in the Taricha brain (Evans et al. 2000). This membrane steroid receptor mediates rapid behavioral responses, such as suppression of amplectic clasping (Orchinik et al. 1991, Moore & Orchinik 1994), through a mechanism that is modulated by Mg2+ and guanyl nucleotides (Orchinik et al. 1992). Together, these observations have prompted speculation that the high-affinity membrane binding site for corticosterone is, in fact, located on a -like opioid receptor (Evans et al. 2000).
The current study was undertaken in an effort to characterize the opioid receptor(s) present in the brain of a urodele amphibian, the rough-skinned newt T. granulosa. Here we report the molecular cloning and heterologous expression of a full-length cDNA sequence that encodes the newt brain opioid receptor (nKOR). The deduced amino acid sequence of nKOR was compared with the other full-length opioid receptors that have been cloned to date. Binding pharmacology was characterized for nKOR transiently expressed in COS-7 cells, and compared with the results of similar binding assays performed using synaptosome preparations isolated from whole newt brains. This is the first time a non-mammalian opioid receptor has been cloned, expressed in a heterologous system, and characterized.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Newts were collected from local (Benton County, OR, USA) freshwater ponds. All animals were housed, handled and used in accordance with federal and institutional guidelines.
Degenerate PCR
Full-length cDNA sequences available from the GenBank database for various species (human, mouse, rat, pig, guinea pig, zebrafish) were aligned using ClustalW, and degenerate oligonucleotide primers were designed using Primer3 software (Rozen & Skaletsky 2000) based on highly conserved regions in the N-terminus and first intracellular loop. The sequences of the sense and the antisense primers (purchased from The Great American Gene Company) were 5'-GCACAT CTCYCCRGCCATCC-3' and 5'-GGTCCACRCTCA TCATGGTCAA-3' respectively. Total RNA was prepared from the pooled brains of six adult male newts according to the protocol supplied with the ToTALLY RNA kit (Ambion, Austin, TX, USA). This procedure was performed several times, on different dates, in order to obtain multiple RNA pools prepared independently. cDNA was obtained by RT-PCR using a cDNA first-strand synthesis kit (Fermentas, Hanover, MD, USA). This procedure was performed several times, on different dates, using different RNA templates. For degenerate PCR, primers, dNTPs, Taq polymerase and newt brain cDNA template were used in PCR reactions with the following cycling conditions: 94 °C x 3 min; 4 cycles of 94 °C x 45 s, 55 °C x 45 s, 72 °C x 1 min 30 s; 34 cycles of 94 °C x 30 s, 55 °C x 30 s, 72 °C x 1 min; terminal extension at 72 °C x 7 min. Negative controls included autoclaved water in place of the cDNA template. PCR products were separated on a 1.4% agarose gel, stained with ethidium bromide, and visualized with UV light. PCR products of the appropriate size (~309 bp) were purified from gels using a GelSpin DNA Purification Kit (MO BIO, Solana Beach, CA, USA), ligated into the pCR4-TOPO vector (Invitrogen), and transformed into TOP10 Escherichia coli competent cells. Following overnight incubation, plasmid DNA was isolated from minipreps and purified by alkaline lysis using a Qiaprep kit (Qiagen). Purified DNA was sequenced by the Central Services Laboratory at Oregon State University. Sequences were analyzed with a BLAST-N 2.0 search tool provided by NCBI.
Rapid amplification of cDNA ends (RACE)-PCR
Clones identified by degenerate PCR possessing the highest sequence identity to known opioid receptors were used to design gene-specific primers for RACE-PCR. The sense primer (for 3'RACE) was 5'-GGGA ACGTGCTCTGTAAAATAGTC-3'; the antisense primer (for 5'RACE) was 5'-CATGACCAAGGAGT TTCCCACCAG-3'. Gene-specific primers were purchased from One Trick Pony (Ransom Hill Bioscience, Ramona, CA, USA). 3'RACE-ready cDNA was prepared from newt brain RNA according to the FirstChoice RLM-RACE kit protocol (Ambion). 3'RACE-ready cDNA was used in conjunction with the nKOR gene-specific sense primer and the Ambion 3'‘Inner’ antisense primer in a 3'RACE-PCR reaction according to the kit protocol and recommended cycling conditions. Following agarose gel electrophoresis, a PCR product (approximately 1.4 kb) was gel purified, sub-cloned, and sequenced as described above.
5'RACE-ready cDNA was synthesized from newt brain RNA according to Ambion’s RLM-RACE kit protocol. 5'RACE-PCR reactions were carried out using the Ambion kit reagents and protocol, the Ambion ‘Outer RACE’ sense primer and a 5'RACE gene-specific antisense primer with the following cycling conditions: 94 °C for 3 min; 4 cycles of 94 °C x 45s, 62 °C x 45s, 72 °C x 1 min; 34 cycles of 94 °C x 30s, 62 °C x 30s, 72 °C x 1 min; and 72 °C x 7 min.
Regions of overlapping and identical sequences among the original degenerate PCR product and the 5'RACE and 3'RACE products were joined using SeqMerge software (Accelyrs, San Diego, CA, USA) to produce a contiguous sequence representing the full-length nKOR cDNA. New gene-specific primers were designed at the extreme 5' and 3' ends of the open reading frame, and the complete coding region was subsequently PCR amplified using a high-fidelity Pfx proof-reading polymerase (Invitrogen) and sub-cloned three separate times to obtain a consensus sequence.
Phylogenetic tree construction
Available full-length opioid receptor protein sequences for species other than T. granulosa were obtained from GenBank. Amino acid sequences were aligned using ClustalX (v1.81) (Thompson et al. 1997). The alignment was analyzed using the bootstrap and protein sequence parsimony components of PHYLIP 3.6 (Felsenstein 1989, 2004) to generate a phylogenetic tree. The tree was rooted in Rattus norvegicus somatostatin receptor type 1, representing a family of G protein-coupled receptors closely related to the opioid receptors (Pfeiffer et al. 2002).
Transient expression of nKOR in COS-7 cells
The full-length nKOR cDNA was sub-cloned into the mammalian expression vector pcDNA3.1/V5-His-TOPO (Invitrogen) and individual clones were analyzed for correct orientation with respect to the CMV promoter. COS-7 (African green monkey kidney) cells (American Type Culture Collection, Manassas, VA, USA) were cultured in 10 cm tissue culture plates at 37 °C under 5% CO2 in Dulbecco’s Modified Eagle’s Medium (Mediatech Cellgro, Herndon, VA, USA) supplemented with 2 mM L-glutamine (Invitrogen) and 10% fetal bovine serum (Hyclone, Logan, UT, USA). Transient expression was achieved by transfecting plates (90–95% confluency) using Lipofectamine 2000 reagent (Invitrogen) diluted with Opti-MEM medium (Invitrogen). For each 10 cm culture plate, 24 µg DNA in 1.5 ml Opti-MEM and 60 µl Lifpofectamine 2000 in 1.5 ml Opti-MEM were added to a plate containing 12 ml growth medium. Six hours after transfection, the lipofection mixture was aspirated and replaced with 14 ml of fresh growth medium.
Preparation of COS-7 cell membranes
Forty-eight hours after transfection, cells were washed twice with room temperature PBS and harvested under homogenization buffer (2 ml/plate) using a cell scraper. Suspended cells were homogenized in chilled buffer (25 mM Hepes, 100 µM phenylmethylsulfonyl fluoride (PMSF), 100 µg/ml trypsin inhibitor, 0.7 µg/ml leupeptin, 100 µg/ml bacitracin) using a chilled glass-on-glass Dounce homogenizer. Homogenates were centrifuged for 10 min at 45 000 g at 4 °C. Supernatants were discarded and pellets were resuspended in homogenization buffer (1 ml/plate) and centrifuged a second time. Supernatants were discarded and cell membrane pellets were resuspended in binding buffer (250 µl/plate) containing MgCl2 (see Binding Assays described below), snap-frozen in liquid N2, and stored at –80 °C until use. In the course of this study, multiple transfections were performed; however, tissues from different transfections were not pooled, and in a given binding assay all tissues used were obtained from a single transfection. The concentration of total protein in membrane preparations was determined using a colorimetric protein assay (Pierce, Rockford, IL, USA).
Preparation of neuronal P2 membranes from whole newt brains
Neuronal membranes (P2 membrane pellets) were prepared according to the method of Whittaker (1969), as modified by Orchinik et al.(1991). Brains were removed from anesthetized adult male newts and placed in 0.3 ml of chilled homogenization buffer (0.32 M sucrose, 5 mM HEPES, pH 7.45, 100 µM PMSF) per mg wet tissue weight. Brains were homogenized in a Teflon-on-glass Dounce homogenizer. The homogenate was centrifuged at 1000 g for 10 min at 4 °C, after which the supernatant was transferred to a clean centrifuge tube. The pellet (P1) was resuspended in the original volume of homogenization buffer and centrifuged again, as above. The new supernatant was added to the first supernatant and the pellet discarded. Pooled supernatants were centrifuged at 30 000 g for 40 min at 4 °C. The pellet (P2) was snap-frozen in liquid N2, thawed on ice, and resuspended in 0.18 ml/mg (original tissue weight) of 25 mM HEPES, 10 mM EDTA, pH 7.45, 100 µM PMSF. The suspension of P2 was incubated at 4 °C for 2 h, to allow for dissociation of endogenous ligand(s), and centrifuged at 40 000 g for 15 min at 4 °C. Supernatant was discarded and the final pellet was snap-frozen in liquid N2 and stored at –80°C.
Binding assays
Saturation and competition binding assays were performed in buffer consisting of 25 mM Hepes (pH 7.45) containing 10 mM MgCl2, 100 µM PMSF, trypsin inhibitor (100 µg/ml), leupeptin (0.7 µg/ml) and bacitracin (100 µg/ml). Frozen membrane pellets (COS-7 or P2) were thawed on ice and diluted with binding buffer to achieve a final protein concentration of 50 µg per binding reaction. For saturation experiments, various concentrations of [3H]U69593 (41.4 Ci/mmol; Perkin Elmer Life Sciences, Boston, MA, USA) were used in the presence of 10 µM nor-binaltorphimine (nor-BNI) (TOCRIS, Ellisville, MO, USA). Competition binding experiments were performed using 1–2 nM [3H]U69593 and various doses (10–12 to 10–4 M) of non-radioactive competitors. GR89696, U50488 [GenBank] , BRL52537and [D-Ala-MePhe, Gly-ol]enkephalin (DAMGO) were purchased from TOCRIS; dynorphin A(1–13) amide was from Phoenix Pharmaceuticals (Belmont, CA, USA); U69593 [GenBank] was from Sigma-Aldrich; nociceptin and [D-penicillamine, D-penicillamine] enkephalin (DPDPE) were from Multiple Peptide Systems (San Diego, CA, USA). Non-specific binding was defined as radioactivity remaining bound in the presence of 10 µM non-radioactive U69593 [GenBank] . Triplicate samples were run at each dose for both total and non-specific binding. Assays were conducted at 4 °C for 4 h with gentle mixing (80 r.p.m. on an orbital shaker). At the end of the incubation period, bound and free ligand were separated by rapid filtration over GF/C filters (pre-soaked with 0.25% polyethyleneimine for 30 min in inhibition experiments with peptide ligands) under vacuum using a Brandel (Gaithersburg, MD, USA) cell harvester. Filters were washed twice with 4 ml of chilled (4 °C) Hepes/MgCl2 (pH 7.45) buffer. Radioactivity in the filters was determined by liquid-scintillation counting on a Beckman LS 6500 scintillation counter. Binding data were analyzed using GraphPad Prism software (GraphPad Software, San Diego, CA, USA).
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The degenerate PCR procedure yielded an amplified product of 269 bp, representing a cDNA sequence encoding a peptide of 81 amino acids. BLAST analysis of the deduced amino acid sequence predicted a hypothetical protein characterized by putative conserved domains consistent with membership in the family of proteins containing seven transmembrane domains (rhodopsin family), and showed highest sequence identity to mammalian opioid receptors. RACE-PCR yielded additional nucleotide sequences overlapping the original ‘core’ sequence at each end, and allowed construction of a single, contiguous, nucleotide sequence.
The contiguous cDNA amplified from degenerate and RACE-PCR performed using newt brain cDNA template is shown in Fig. 1
. This sequence contains an open reading frame spanning nucleotides 1–1158. The sequence of the open reading frame was independently verified at least three times, using different preparations of newt brain cDNA prepared independently of one another. No products were amplified in negative controls that included autoclaved water in place of the cDNA template. Immediately upstream of the putative start codon shown in Fig. 1 is the sequence GTTATGG, a variation on the Kozak consensus sequence (for efficient initiation of transcription) (G/A)NNATGG (Kozak 1989). The open reading frame encodes a protein that is 385 amino acids in length and has an estimated molecular mass of 43 833 Da (in the absence of post-translational modification). BLAST analysis of this amino acid sequence detected seven conserved transmembrane domains and indicated highest sequence identity to G protein-coupled receptors in general, and to mammalian opioid receptors specifically. Hydrophobicity analysis also predicted the presence of seven transmembrane domains. Assignment of putative membrane-spanning regions are indicated in Figs 1 and 2, based on results obtained using online protein topology servers (Moller et al. 2001), including TMHMM2.0 (Krogh et al. 2001) and HMMTOP2.0 (Tusnady & Simon 2001). The predicted protein contains several sites for post-translational modification that are conserved among G protein-coupled receptors, including consensus sites for N-linked glycosylation (asparagine residues in the extracellular N-terminal domain), and amino acid substrates for palmitoylation and phosphorylation within the putative intracellular C-terminal domain. Cysteine residues in the first two putative extracellular loops provide for the formation of a disulfide bond. The sequence Asp-Arg-Tyr (DRY) in the second intracellular loop (immediately following TMIII) is also a feature conserved among members of the G protein-coupled receptor superfamily. The sequence shown in Fig. 1 has been deposited in the GenBank database (Accession AY725197
[GenBank]
).
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compares the deduced amino acid sequence of the nKOR with zebrafish and mammalian receptors (sequences from GenBank). The newt receptor is a protein consisting of 385 amino acids, and is larger than that described for mammals (380 amino acids for all four species) or the zebrafish (377 amino acids). This size difference is accounted for mainly by the fact that the extracellular N-terminal domain of nKOR is larger than the corresponding region in the other receptors listed. nKOR shares approximately 75% (74.2–76.0%) amino acid sequence identity with the mammalian receptors, but only 66% identity with the opioid receptor cloned from zebrafish. In general, the transmembrane domains, the intracellular loops and the first extracellular loop tend to be conserved among all species listed; the greatest divergences between the newt protein and that of the non-amphibian species occur in the extracellular N-terminal domain, the intracellular C-terminal domain and the second and third extracellular loops (all of which are highly conserved among mammals).
A tree generated by the PHYLIP (v3.6) protein sequence parsimony method demonstrates that the newt amino acid sequence groups with the kappa opioid receptors, as opposed to the three other opioid receptor types (, µ and ORL) that have been cloned from other vertebrates (Fig. 3).
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Saturation binding experiments using the -selective agonist [3H]U69593 revealed the presence of a specific, saturable, high-affinity (estimated Kd=1.514 nM) binding site on membranes isolated from COS-7 cells transiently expressing nKOR, as shown in Fig. 4. At a dose equivalent to the estimated Kd for [3H]U69593, specific binding was 95.4% of total binding. Saturation binding data were best fit using a one-site model for receptor–ligand interactions. No specific [3H]U69593 binding was observed in parallel experiments conducted with membranes isolated from cells that were not transfected. A pharmacological profile of the cloned newt receptor was constructed, based on the ability of various opioid ligands to displace [3H]U69593 from membranes of COS-7 cells transiently expressing nKOR (Fig. 5 and Table 1). The cloned receptor bound -specific ligands with high affinity (Ki=0.3–6.2 nM); the observed rank order potency was GR89696>dynorphin A(1–13)>nor-BNI>U69593>U50488>BRL52537.
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-selective agonist DPDPE (Ki=979 nM). Binding assays using neuronal P2 membranes
Competition binding experiments using neuronal P2 membranes prepared from Taricha brains yielded results very similar to those obtained with the cloned receptor transiently expressed in COS-7 cells (Fig. 6 and Table 1). P2 membrane pellets bound -specific ligands with high affinity (Ki=0.3–5.3 nM); the observed rank order potency for displacing [3H]U69593 was GR89696>dynorphin A(1–13)>nor-BNI>U50488> U69593
[GenBank]
>BRL52537.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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opioid receptor that is present in the brain of a urodele amphibian, the rough-skinned newt T. granulosa. This represents an extension of previous studies characterizing ligand binding in tissue homogenates of whole brains from frogs or toads (Benyhe et al. 1994, Newman et al. 2002, Simon et al. 1982, Simon et al. 1984), and purification protocols (Borsodi et al. 1986, Simon et al. 1987, 1990) providing evidence for distinct or -like receptors in the amphibian central nervous system.
Starting with total RNA isolated from newt brains, degenerate PCR amplified a cDNA fragment that showed high sequence homology to the mammalian receptors. Subsequently, RACE-PCR (using Taricha-specific oligo primers) allowed us to extend the original cDNA sequence in both directions, and culminated in amplification of a contiguous sequence representing a full-length cDNA encoding a protein of 385 amino acids. BLAST analysis of the deduced amino sequence again demonstrated highest sequence identity with known mammalian opioid receptors, and maximum parsimony analysis showed that the amino acid sequence clustered with the other vertebrate receptors that have been cloned. Analysis of the primary structure of the predicted protein revealed the presence of physical attributes shared by other G protein-coupled receptors that have been identified as opioid receptors. Among these are the presence of seven hydrophobic transmembrane domains, potential sites for N-linked glycosylation in the extracellular N-terminal domain, a conserved DRY motif at the beginning of the second putative intracellular loop, conserved cysteine residues in the second and third extracellular loops, and potential sites for palmitoylation and phosphorylation in intracellular loops as well as the intracellular C-terminal domain (Minami & Satoh 1995). Based on its sequence, we tentatively identified the cDNA we cloned from the brain of T. granulosa as the nKOR.
Membranes isolated from COS-7 cells transiently expressing nKOR possessed a saturable, high-affinity (Kd=1.51 nM) binding site for the -selective agonist [3H]U69593; no such binding was detected in COS-7 cells that were not transfected. This measurement agrees closely with values reported previously for [3H]U69593 in studies describing pharmacological profiles of mammalian receptors expressed in COS cells, including rat (Kd=1.46 nM) (Meng et al. 1993), guinea pig (Kd=1.17 nM) (Xie et al. 1994) and human (Kd=1.49 nM) (Simonin et al. 1995) receptors. The Kd values reported so far for [3H]U69593 in saturation binding assays employing amphibian brain tissues are slightly higher than our estimate for nKOR: 6.81 nM for the northern grass frog (R. pipiens) (Newman et al. 2002) and 15.3 nM in the European water frog (R. esculenta) (Benyhe et al. 1992).
In competition binding experiments designed to assess the affinity of various opioid ligands for the cloned newt receptor, membranes isolated from COS-7 cells transiently expressing nKOR showed high affinity for the -selective agonists GR89696, dynorphin A(1–13), U69593
[GenBank]
, U50488
[GenBank]
and BRL52537 as well as the -selective antagonist nor-BNI and the non-selective antagonist naloxone. In mammals, high affinity for both U69593
[GenBank]
and U50488
[GenBank]
is considered one means of pharmacologically distinguishing receptors from the other opioid receptor classes, since these agonists bind potently and specifically to receptors (Zukin et al. 1988, Clark et al. 1989) but do not bind to either or µ receptors (Raynor et al. 1996).
Competition binding assays conducted with neuronal P2 membranes prepared from Taricha brains revealed rank order potencies and affinity constants for ligands that matched closely the results of experiments employing membranes isolated from COS-7 cells transiently expressing nKOR. All together, our results are consistent with the idea that nKOR is, in fact, a newt opioid receptor, and that nKOR and the binding site present in whole newt brains are one and the same.
Like its mammalian counterparts, the expressed nKOR demonstrated little, if any, affinity for the-selective agonist DPDPE (Ki=979 nM). In contrast, the expressed nKOR showed moderate affinity for DAMGO (Ki=55.4 nM), unlike the cloned/expressed mammalian receptors, none of which demonstrate measurable affinity for this µ-selective agonist (Meng et al. 1993, Yasuda et al. 1993, Mansson et al. 1994, Xie et al. 1994, Simonin et al. 1995). Interestingly, the expressed nKOR showed moderately high affinity (Ki=23.2 nM) for nociceptin (orphanin FQ), an ORL1-selective agonist and the endogenous ligand for the ORL1 receptor (Meunier et al. 1995, Reinscheid et al. 1995). In mammals, nociceptin displays essentially no affinity for the canonical , and/or µ opioid receptors (Meunier et al. 1995, Reinscheid et al. 1995, Meng et al. 1996, Pan et al. 2002) and, despite a high degree of sequence homology shared among all of the opioid receptor classes, the ORL1 receptor in mammals does not recognize the classic opioid drugs that bind with high affinity to , and µ opioid receptors (Owens & Akil 2002). Such discrimination is especially interesting when it is appreciated that nociceptin (FGGFTGARK SARKLANQ) bears a significant degree of sequence identity to dynorphin A (YGGFLRRIRPKLKWDNQ) and belongs to the family of opioid peptides (Danielson et al. 2001).
We recently cloned, expressed and characterized an ORL1 receptor from the brain of Taricha (nORL1), and found that the expressed nORL1 binds nociceptin and dynorphin A(1–13) with high affinity (Walthers et al. 2005). Research into the physiology of mammalian opioid ligand–receptor interactions has supported the idea of a strict functional dichotomy, in which the classic opioids interact with µ, and/or receptors to produce analgesia and nociceptin–ORL1 interactions act in an opposing fashion (Reinscheid et al. 1998, Danielson et al. 2001). Our results suggest that, in contrast to mammals, newts have ORL1 and opioid receptors that recognize both nociceptin and dynorphin. This apparent lower specificity might reflect a more basal characteristic of opioid receptors.
Prior research shows that, among G protein-coupled receptors, the N-terminal domain and extracellular loops specify receptor–ligand selectivity (Akil et al. 1996). Studies involving expression of chimeric opioid receptors, as well as modeling experiments simulating receptor–ligand interactions suggest that the second extracellular loop of the receptor may play a crucial role in recognition of endogenous ligands (Wang et al. 1994, Raynor et al. 1996, Paterlini et al. 1997). We found that the N-terminal domain and the second and third extracellular loops (and to a lesser degree the first extracellular loop) of nKOR diverge from the amino acid sequences reported for the mammalian receptors that have been cloned to date. These differences might confer different standards for ligand selectivity and account for the fact that nKOR recognizes DAMGO and nociceptin, whereas mammalian opioid receptors do not. Li et al.(1996) noted that a valine residue that is highly conserved in the first extracellular loop of all mammalian receptors is replaced by a negatively charged glutamate residue in a partial cDNA sequence isolated for a frog receptor, and speculated that the amphibian receptor might have the capacity to bind nociceptin. It turns out that nKOR also possesses glutamate instead of valine at the position in question, and does bind nociceptin, as predicted.
Multiple, distinct receptor subtypes (1, 1a/b, 2, 2a/b, 3) have been proposed based partly on binding studies revealing multiple sites possessing different ligand affinities, partly on the basis of pharmacological experiments demonstrating selective interactions with various ligands, and partly on the basis of varying anatomical distributions (Zukin et al. 1988, Clark et al. 1989, Benyhe et al. 1990, Rothman et al. 1990, Wollemann et al. 1993). One group has reported that two transcripts are derived from a single gene encoding the rat receptor, demonstrating the potential for multiple receptor splice variants (Yakovlev et al. 1995). In general, however, molecular cloning studies have failed to identify cDNA sequences that would account for the multiplicity of receptor subtypes. Our results do not rule out the possibility that multiple nKOR subtypes and/or splice variants exist in the newt brain; however, we found no evidence for the expression of more than one type of receptor. In multiple, redundant, PCR procedures, we amplified a product that appeared as a single band on ethidium bromide-stained gels, and the sequence of the amplified cDNA was always the same. Additionally, we found that the binding affinities and rank order potencies for -selective ligands were essentially the same, regardless of whether binding assays were conducted using membranes isolated from COS-7 cells transiently expressing the cloned nKOR or neuronal P2 membranes prepared from whole Taricha brains. Our observations are consistent with the notion that only one type of opioid receptor mRNA is expressed in newt brains.
Recent studies show that G protein-coupled receptors can function in homo- and hetero-oligomeric complexes (Dean et al. 2001, Angers et al. 2002, Lee et al. 2003); co-expression experiments demonstrate the formation of opioid receptor heterodimers that exhibit novel ligand-binding properties compared with either receptor expressed alone (Jordan & Devi 1999, George et al. 2000, Pan et al. 2002). For example, stable co-expression of and opioid receptors results in the formation of a functional – heterodimer exhibiting binding properties that match the 2, receptor subtype (Jordan & Devi 1999). Whether or not other functional combinations of opioid receptors might explain the apparent diversity of receptor subtypes remains unresolved. We have recently cloned full-length cDNA sequences for Taricha and µ opioid receptors, and it will be interesting to test the hypothesis that co-expression of two or more types of Taricha opioid receptors may generate novel binding profiles matching the pharmacology described for one or more of the proposed receptor subtypes.
Early studies with rough-skinned newts demonstrated rapid and potent suppression of courtship behavior (amplectic clasping of females) in males subjected to acute stress or treated with the steroid corticosterone (Moore & Miller 1984). Similarly, male newts treated with bremazocine and ethylketocyclazocine, both -selective agonists, showed rapid inhibition of amplectic clasping (Deviche & Moore 1987). Subsequently, it was discovered that there is a highly specific, saturable corticosterone-binding site in neuronal membranes isolated from Taricha brains (Orchinik et al. 1991) that appears to be a G protein-coupled receptor (Orchinik et al. 1992). In a series of ligand-binding competition experiments designed to screen various classes of ligands for ability to displace [3H]corticosterone from neuronal membranes, it was found that some, but not all, ligands were effective (Evans et al. 2000). Dynorphin A(1–13), U50488
[GenBank]
, bremazocine and ethylketocyclazocine were able to displace some (up to 70%) of [3H]corticosterone specific binding. U69593
[GenBank]
(a -selective agonist) and nor-BNI (a -selective antagonist) were found to be ineffective. These results, together with the behavioral data collected previously, were consistent with the hypothesis that the membrane corticosterone receptor in the Taricha brain is located on a opioid-like receptor (Evans et al. 2000). The Taricha brain receptor reported here binds all of the -selective ligands with high affinity, including dynorphin A(1–13), U50488
[GenBank]
, nor-BNI and U69593
[GenBank]
. In addition, membranes isolated from COS-7 cells transiently expressing nKOR showed low, or no, specific binding of [3H]corticosterone (data not shown). We conclude, therefore, that nKOR is not the membrane receptor responsible for binding corticosterone in the Taricha brain.
In summary, we have cloned a full-length cDNA encoding a opioid receptor from the brain of an amphibian, the rough-skinned newt T. granulosa. This receptor is similar to receptors cloned previously from other vertebrates, but shows interesting pharmacological differences with respect to ligand-binding profiles. The newt brain receptor does not appear to be the corticosterone-binding site described by our laboratory previously, but it remains possible that co-expression of two or more opioid receptor types (including the receptor) may result in the formation of an oligomeric complex possessing novel binding properties.
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Angers S, Salahpour A & Bouvier M 2002 Dimerization: an emerging concept for G protein-coupled receptor ontogeny and function. Annual Review of Pharmacology and Toxicology 42 409–435.[CrossRef][ISI][Medline]
Benyhe S, Varga E, Hepp J, Magyar A, Borsodi A & Wollemann M 1990 Characterization of kappa 1 and kappa 2 opioid binding sites in frog (Rana esculenta) brain membrane preparation. Neurochemistry Research 15 899–904.[CrossRef][ISI][Medline]
Benyhe S, Szucs M, Borsodi A & Wollemann M 1992 Species differences in the stereoselectivity of kappa opioid binding sites for [3H]U-69593 and [3H]ethylketocyclazocine. Life Sciences 51 1647–1655.[CrossRef][ISI][Medline]
Benyhe S, Simon J, Borsodi A, Wollemann M & Barnard EA 1994 [3H]dynorphin-8 binding sites in frog (Rana esculenta) brain membranes. Neuropeptides 26 359–364.[CrossRef][ISI][Medline]
Bloom FE 1983 The endorphins: a growing family of pharmacologically pertinent peptides. Annual Review of Pharmacology and Toxicology 23 151–170.[CrossRef][ISI][Medline]
Borsodi A, Khan A, Simon J, Benyhe S, Hepp J, Wollemann M & Medzihradszky K 1986 Purification of kappa-opioid receptor subtype to apparent homogeneity from frog brain. NIDA Research Monograph 75 1–4.
Chavkin C, James IF & Goldstein A 1982 Dynorphin is a specific endogenous ligand of the kappa opioid receptor. Science 215 413–415.
Chen Y, Mestek A, Liu J & Yu L 1993 Molecular cloning of a rat kappa opioid receptor reveals sequence similarities to the mu and delta opioid receptors. Biochemical Journal 295 625–628.
Clark JA, Liu L, Price M, Hersh B, Edelson M & Pasternak GW 1989 Kappa opiate receptor multiplicity: evidence for two U50,488-sensitive kappa 1 subtypes and a novel kappa 3 subtype. Journal of Pharmacology and Experimental Therapeutics 251 461–468.
Cone RI & Goldstein A 1982 A dynorphin-like opioid in the central nervous system of an amphibian. PNAS 79 3345–3349.
Danielson PB, Hoversten MT, Fitzpatrick M, Schreck C, Akil H & Dores RM 2001 Sturgeon orphanin, a molecular ‘fossil’ that bridges the gap between the opioids and orphanin FQ/nociceptin. Journal of Biological Chemistry 276 22114–22119.
Darlison MG, Greten FR, Harvey RJ, Kreienkamp HJ, Stuhmer T, Zwiers H, Lederis K & Richter D 1997 Opioid receptors from a lower vertebrate (Catostomus commersoni): sequence, pharmacology, coupling to a G-protein-gated inward-rectifying potassium channel (GIRK1), and evolution. PNAS 94 8214–8219.
Dean MK, Higgs C, Smith RE, Bywater RP, Snell CR, Scott PD, Upton GJ, Howe TJ & Reynolds CA 2001 Dimerization of G-protein-coupled receptors. Journal of Medical Chemistry 44 4595–4614.
Deviche P & Moore FL 1987 Opioid kappa-receptor agonists suppress sexual behaviors in male rough-skinned newts (Taricha granulosa). Hormones and Behavior 21 371–383.[CrossRef][Medline]
Deviche P, Lowry CA & Moore FL 1989 Opiate control of spontaneous locomotor activity in a urodele amphibian. Pharmacology, Biochemistry, and Behavior 34 753–757.[CrossRef][ISI][Medline]
Deviche P, Cotter P & Gulledge CC 1993 Identification, partial characterization, and hypothalamic distribution of kappa, mu, and delta opioid receptors in a passerine songbird (Junco hyemalis). Brain Research 614 220–226.[CrossRef][ISI][Medline]
Evans SJ, Searcy BT & Moore FL 2000 A subset of kappa opioid ligands bind to the membrane glucocorticoid receptor in an amphibian brain. Endocrinology 141 2294–2300.
Felsenstein J 1989 PHYLIP – Phylogeny Inference Package (Version 3.2). Cladistics 5 164–166.
Felsenstein J 2004 PHYLIP (Phylogeny Inference Package) version 3.6. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle, WA.
George SR, Fan T, Xie Z, Tse R, Tam V, Varghese G & O’Dowd BF 2000 Oligomerization of mu- and delta-opioid receptors. Generation of novel functional properties. Journal of Biological Chemistry 275 26128–26135.
Goldstein A & Naidu A 1989 Multiple opioid receptors: ligand selectivity profiles and binding site signatures. Molecular Pharmacology 36 265–272.[Abstract]
Janecka A, Fichna J & Janecki T 2004 Opioid receptors and their ligands. Current Topics in Medicinal Chemistry 4 1–17.[ISI][Medline]
Jordan BA & Devi LA 1999 G-protein-coupled receptor heterodimerization modulates receptor function. Nature 399 697–700.[CrossRef][Medline]
Kozak M 1989 The scanning model for translation: an update. Journal of Cell Biology 108 229–241.
Krogh A, Larsson B, von Heijne G & Sonnhammer EL 2001 Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. Journal of Molecular Biology 305 567–580.[CrossRef][ISI][Medline]
Lahti RA, VonVoigtlander PF & Barsuhn C 1982 Properties of a selective kappa agonist, U-50,488H. Life Sciences 31 2257–2260.[CrossRef][ISI][Medline]
Lahti RA, Mickelson MM, McCall JM & Von Voigtlander PF 1985 [3H]U-69593 a highly selective ligand for the opioid kappa receptor. European Journal of Pharmacology 109 281–284.[CrossRef][ISI][Medline]
Lee SP, O’Dowd BF & George SR 2003 Homo- and hetero-oligomerization of G protein-coupled receptors. Life Sciences 74 173–180.[CrossRef][ISI][Medline]
Li X, Keith DE Jr & Evans CJ 1996 Multiple opioid receptor-like genes are identified in diverse vertebrate phyla. FEBS Letters 397 25–29.[CrossRef][ISI][Medline]
Lowry CA, Deviche P & Moore FL 1990 Effects of corticotropin-releasing factor (CRF) and opiates on amphibian locomotion. Brain Research 513 94–100.[CrossRef][ISI][Medline]
Mansson E, Bare L & Yang D 1994 Isolation of a human kappa opioid receptor cDNA from placenta. Biochemical and Biophysical Research Communications 202 1431–1437.[CrossRef][ISI][Medline]
Meng F, Xie GX, Thompson RC, Mansour A, Goldstein A, Watson SJ & Akil H 1993 Cloning and pharmacological characterization of a rat kappa opioid receptor. PNAS 90 9954–9958.
Meng F, Taylor LP, Hoversten MT, Ueda Y, Ardati A, Reinscheid RK, Monsma FJ, Watson SJ, Civelli O & Akil H 1996 Moving from the orphanin FQ receptor to an opioid receptor using four point mutations. Journal of Biological Chemistry 271 32016–32020.
Meunier JC, Mollereau C, Toll L, Suaudeau C, Moisand C, Alvinerie P, Butour JL, Guillemot JC, Ferrara P, Monsarrat B et al. 1995 Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor. Nature 377 532–535.[CrossRef][Medline]
Minami M & Satoh M 1995 Molecular biology of the opioid receptors: structures, functions and distributions. Neuroscience Research 23 121–145.[CrossRef][ISI][Medline]
Minami M, Toya T, Katao Y, Maekawa K, Nakamura S, Onogi T, Kaneko S & Satoh M 1993 Cloning and expression of a cDNA for the rat kappa-opioid receptor. FEBS Letters 329 291–295.[CrossRef][ISI][Medline]
Moller S, Croning MD & Apweiler R 2001 Evaluation of methods for the prediction of membrane spanning regions. Bioinformatics 17 646–653.
Moore FL & Miller LJ 1984 Stress-induced inhibition of sexual behavior: corticosterone inhibits courtship behaviors of a male amphibian (Taricha granulosa). Hormones and Behavior 18 400–410.[CrossRef][Medline]
Moore FL & Orchinik M 1994 Membrane receptors for corticosterone: a mechanism for rapid behavioral responses in an amphibian. Hormones and Behavior 28 512–519.[CrossRef][Medline]
), post-translational palmitoylation (
) and phosphorylation (
).
