©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure and Expression of a Rat Opioid Receptor Gene (*)

(Received for publication, December 8, 1994; and in revised form, January 2, 1995 )

Alexander G. Yakovlev Karl E. Krueger (1) Alan I. Faden (§)

From the Departments of Neurology and Pharmacology and the Department of Cell Biology, Georgetown University Medical Center, Washington, D. C. 20007

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

A gene encoding the rat kappa opioid receptor (KOR) was cloned and characterized. Results of rat genomic library screening and genomic Southern blot analysis show the gene represented at one copy/haploid genome. Three introns are present within the gene; however, polymerase chain reaction using different sets of primers specifying neighboring exons indicates that alternative splicing does not occur. Using reverse transcriptionpolymerase chain reaction and primer extension techniques, we are able to demonstrate that two species of transcripts are differentially produced from the KOR gene in a tissue-specific manner. The first transcript that we designate as KOR1 is equivalent to the cDNA sequence reported by other groups and is believed to correspond to KOR subtype 1. KOR1 begins with exon 1 just downstream of two TATA boxes, whereas the second transcript, which we refer to as KORx, begins in intron 1 and thereby retains this intronic sequence in the mature mRNA. Within this intronic sequence there are two potential translation initiation codons that are in-frame with the proposed initiation codon of KOR. The potential open reading frame that starts further upstream in KORx may lead to the translation of a variant KOR protein having a novel peptide sequence at its amino terminus.


INTRODUCTION

It has long been recognized that there are multiple classes of opioid receptors. Martin et al.(1976) initially proposed the existence of three major types of opioid receptors, termed µ, , and kappa. These receptors exhibit distinct physiological actions and show different distributions within the central nervous system (Mack et al., 1984; Spain etal., 1985). Isoreceptors for these receptors have also been suggested (Cheng et al., 1992; Zukin et al., 1988). During the past 2 years, µ, , and kappa opioid receptors have been cloned and sequenced (Reisine and Bell, 1993). They belong to the superfamily bearing seven transmembrane-spanning regions. These receptors are negatively coupled to adenylyl cyclase, inhibiting the formation of cyclic AMP (Chen et al., 1993; Simon, 1991); these inhibitory actions are mediated by pertussis toxin-sensitive GTP-binding regulatory (G) proteins (Reisine and Bell, 1993).

kappa opioid receptors show selective affinity for the endogenous opioid dynorphin (Chavkin et al., 1982) and appear to serve a number of physiological roles including cardiovascular regulation, feeding behavior, fluid balance, modulation of anti-nociception, and temperature control, among others (Bloom, 1983). They have also been implicated in certain pathophysiological states including shock, stroke, and central nervous system trauma (Faden, 1993). Based upon selective interaction with various ligands these receptors have been divided into at least three subtypes: kappa(1), kappa(2), and kappa(3) (Cheng et al., 1992; Zukin et al., 1988).

Rat kappa opioid receptor cDNAs for the kappa(1) subtype have been cloned in various laboratories using different sources of tissue for RNA isolation (Chen et al., 1993; Li et al., 1993; Meng et al., 1993; Minami et al., 1993; Nishi et al., 1993). Alignment of cloned KOR (^1)sequences shows very few nucleotide substitutions that do not affect the amino acid sequence of the protein. However, the cDNA clone isolated by Minami et al.(1993) from a rat thalamus cDNA library differs from others by the break of homology at its 5`-end. Structural analysis of this 5`-portion of rat thalamus KOR cDNA clone revealed a possible extension of the open reading frame upstream from the ``first'' ATG codon. This observation suggests either the presence of alternative splicing in the KOR gene, recombination, or existence of different promoter regions. Our study reports the cloning of the KOR gene and reveals the molecular basis for the differences in these two types of KOR messages.


EXPERIMENTAL PROCEDURES

Probe Preparation

The probe for kappa(1) OR was obtained using the RT-PCR technique. Total cellular RNA was isolated from rat brain by acidic phenol extraction (Chomczynski and Sacchi, 1987). First strand cDNA was synthesized using 5 µg of total RNA in a mixture of 5 µM oligo(dT), 5 µM of random hexamer primers, and 200 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) according to the manufacturer's recommended reaction conditions in 20 µl of final reaction volume. Three µl of cDNA were added to the PCR mixture containing 0.5 unit of Hot Tub DNA polymerase and reaction buffer (1 times, Amersham Corp.), 250 µM each of dNTPs, and 1 µM of each of two amplification primers. Primers used in this PCR (sense primer: 5`-GCAACCAACATCTACAT-3`; antisense primer: 5`-AAACACCGCTTGAAGTT-3`) correspond to the most conservative OR mRNA regions. After 30 cycles (consisting of denaturing for 45 s at 94 °C; annealing for 45 s at 48 °C; and primer extension for 45 s at 72 °C), PCR products of 746 bp were cloned into pCRII vector (Invitrogen) and analyzed by restriction mapping with Sau3AI. Six clones corresponding to kappa(1) OR cDNA according to restriction mapping were sequenced by the dideoxy chain termination method using Sequenase II (U. S. Biochemical Corp.).

Screening of the Genomic Library

A Wistar rat genomic DNA library made in DASH vector (Stratagene) was screened using a 746-bp fragment of kappa(1) OR cDNA labeled with [alpha-P]dATP (3000 Ci/mmol, Amersham Corp.) by random priming (U. S. Biochemical Corp.). The first screening hybridization was performed at low stringency (30% formamide, 6 times SSPE at 42 °C). Individual clones were isolated after two additional rounds of plaque purification at higher stringency (50% formamide, 5 times SSPE at 42 °C). Isolated clones were then analyzed by restriction mapping with numerous restriction endonucleases followed by hybridization with the same probe used for library screening.


RESULTS AND DISCUSSION

Multiple subtypes of KOR have been described according to their abilities to bind different ligands (Cheng et al., 1992; Zukin et al., 1988). The molecular origin of such variability is not clearly understood, although data suggest the possibility of either alternative splicing of the KOR mRNA precursor (Yasuda et al., 1993) or the existence of multiple genes encoding different receptors. To characterize the structure of the KOR gene and begin to determine whether multiple KOR genes exist, a rat genomic library was screened with a cDNA probe of 746 bp derived from RT-PCR of rat brain mRNA. High stringency hybridization conditions led to the isolation of 15 separate clones from 10^6 plaques. None of these clones spanned the entire length of the primary transcript sequence. Two sets of overlapping clones, which together covered this entire region plus over 7 kilobase pairs of sequence flanking both ends, were partially sequenced.

Sequence determination of all exons and flanking regions revealed that three introns are present within this gene (Fig. 1). The exonic sequence is essentially identical to that described by other groups with the exception of several single base substitutions, most of which are in the 3`-noncoding sequence. As all KOR sequences previously reported show several substitutions when compared with one another, the genomic sequence we have determined matches that reported by Chen et al.(1993). The predicted open reading frame begins in exon 2 and is completed in exon 4.


Figure 1: Nucleotide sequence of rat KOR gene. Numbering is relative to the first tsp of exon 1 (as determined in Fig. 3) and continues only as exonic sequence. The intronic sequence is shown in lowercaseletters. The partial sequence into exon 4 is shown, but this exon was completely sequenced to the 3`-flanking region as deposited in the GenBank data base under accession number U17995. Transcription start points for KOR1 and KORx transcripts (described in Fig. 3) are indicated. The potential open reading frame of KORx that begins within the sequence corresponding to intron 1 of KOR1 is given in boldfacetype, whereas the beginning of the KOR1-encoded protein is shown in normaltype. Continuation of the open reading frame is not shown and is equivalent to that reported earlier (Meng et al., 1993). Additional upstream mRNA sequences derived by 5`-RACE for KOR1 and KORx are underlined. Possible TATA boxes for KOR1 and consensus sequences for a CAAT box and NF-kappaB transcription factor upstream of the tsps for KORx are enclosed in boxes. kb, kilobase pairs.




Figure 3: Primer extension analysis for KOR1 (left) and KORx (right). The locations of the transcription start sites in the KOR gene were determined by a modified primer extension technique. Extension primers (5`-TGCCCACGCTGCTTTCTGCT-3` (for KOR1) or 5`-TGAAATCCCCTTTCCCAC-3` (for KORx)) were chosen in order to obtain about 100-250 nt of extended cDNA fragments according to sequences determined after 5`-RACE experiments. Oligonucleotides were labeled by using [-P]ATP (6,000 Ci/mmol, Amersham Corp.) and T4 polynucleotide kinase (Life Technologies, Inc.). Twenty µg of yeast (control) or rat brain RNA, pretreated with RNase-free DNase I (Promega), were reverse-transcribed with 15 pmol of phosphorylated extension primer and 5 units of thermostable rTth DNA polymerase (Perkin-Elmer) in 30 µl of reaction mixture, as recommended by the manufacturer for reverse transcription. To amplify the signal, 30 cycles of 1 min at 95 °C for denaturation and 5 min at 70 °C for primer annealing and extension were performed. This process permits the thermostable rTth to reverse-transcribe the same RNA templates after subsequent denaturing of RNA/DNA hybrids and reannealing with oligonucleotide. The reaction products were analyzed by electrophoresis in denaturing 6% polyacrylamide gels beside a sequencing ladder from the corresponding region of rat KOR gene obtained with the same primer used in primer extension procedures. Locations of tsps mapped by this procedure are highlighted by arrows.



All other clones we isolated were found to overlap this same sequence, indicating no evidence of heterogeneity of KOR genes in this library screening. Southern blot analysis of rat genomic DNA was performed based on restriction mapping of the sequenced clones, which permitted us to choose specific restriction enzymes that would give a defined fragmentary pattern. With the exception of XhoI and BamHI, restriction sites for all of the enzymes were found within intron 3. Southern blots of digested rat genomic DNA hybridized with a probe spanning the coding sequence of the third and fourth exons revealed fragments of the expected sizes (Fig. 2). The absence of other hybridizing fragments fails to provide support for the existence of other genes highly homologous to KOR.


Figure 2: Southern analysis of the rat KOR gene. Rat genomic DNA was isolated as described elsewhere (Sambrook et al., 1989), digested with the specified restriction endonucleases, electrophoresed in a 0.8% agarose gel, and vacuum transferred (Zaitsev and Yakovlev, 1983) to nylon membrane (MSI). The blotted membrane was probed with a randomly primed 746-bp KOR cDNA fragment in 50% formamide, 6 times saline/sodium phosphate/EDTA, 0.2% polyvinylpyrrolidone (M(r) 360,000), 20 mM EDTA (pH 8.0), and 2% SDS at 42 °C for 16 h followed by three subsequent washings in 0.5 times SSC, 0.5% SDS at 68 °C for 15 min each.



We used RT-PCR to examine the possibility that different subtypes of KORs might be derived as a result of alternative splicing. Sets of primers specifying neighboring exons were used to amplify KOR sequence from total RNA of a number of rat tissues. In all cases only one KOR amplification product was observed (see Fig. 4). Recently, a murine µ opioid receptor gene was cloned and described as having three introns (Min et al., 1994). Alignments of the kappa and µ opioid receptor sequences indicate that introns 2 and 3 for KOR are at identical positions with respect to introns 1 and 2 of the µ opioid receptor gene. Although one could hypothesize that intron 2 (or intron 1 of the µ opioid receptor gene) may permit alternative splicing to insert additional amino acids into the first intracellular loop, as demonstrated for the D(2) dopamine receptor (Eidne et al., 1989), our results with RT-PCR did not reveal the existence of such variants for KOR.


Figure 4: Differential expression of KOR1 (A) and KORx (B) transcripts. The presence of KOR mRNAs in rat tissues was detected by RT-PCR. Briefly, total cellular RNA from tissues was isolated by acidic phenol extraction (Chomczynski and Sacchi, 1987) and treated for 30 min at 37 °C with RNase-free DNase I (Promega) at 1 unit/µg of RNA. Five µg of total RNA was reverse-transcribed with Moloney murine leukemia virus RT (Life Technologies, Inc.) in 20-µl reaction mixtures. The resulting cDNA (3 µl) was subjected to the first round of PCR amplification using 5`-AGCTAGATCACTAATGTGCCCTGCA-3` as the antisense primer and either 5`-ACCGGCAGAGCCTTCTTCCAGTCTT-3` as a sense primer for KOR1 or 5`-GGGGTAGGGGTAGAGGGTGGGAAAG-3` as the sense primer for KORx. After amplification for 30 cycles (94 °C, 45 s; 59 °C, 45 s; 72 °C, 90 s), the reaction products were diluted 10-fold with water, and 3 µl was then reamplified for 30 cycles using the same reaction conditions with a different set of nested primers. For this second round of PCR antisense primer 5`-TTCTCTCGAGAGCCCGAGAGGAGCC-3` was used in conjunction with KOR1 sense primer 5`-TGGACCCATCGAGGCTGAACAGCTA-3` or KORx sense primer 5`-TGCCTGCACAGGCAAAGTTTGTCTC-3`. PCR products were analyzed by electrophoresis in 2% agarose gel containing 0.5 µg/ml ethidium bromide.



Despite the apparent lack of gene heterogeneity or alternative splicing, we discovered a mechanism that may potentially give rise to different KOR species. The 5`-sequence provided by Minami et al.(1993) appeared to differ from that described by other investigators, and we noted that the difference in this 5`-sequence corresponds to intron 1. Based on the cDNA and genomic KOR sequences, we then hypothesized that different mechanisms may give rise to KOR mRNA that would either include or lack the intron 1 sequence. To better characterize the 5`-end of KOR mRNA we used 5`-RACE to identify additional upstream sequences. When amplification was performed using an antisense primer to exon 1, an additional 176 nt were observed to match the sequence determined from the genomic clone. When 5`-RACE was performed with an antisense primer to the 3`-end of intron 1, amplification products were obtained that extended upstream into the first intron. This result further supports the possibility that at least two KOR transcripts are produced.

Based on the informative sequence obtained by 5`-RACE, predictions on the approximate locations of the transcription start points can be made. Appropriate antisense primers for exon 1 and intron 1 were then designed for primer extension analysis. The primer to exon 1 gave rise to two pairs of extension products (Fig. 3) that corresponded to a sequence extending nearly 300 nt upstream of that reported previously for the cDNA (Meng et al., 1993). Within 26 base pairs upstream of both tsps are two potential TATA boxes, supporting the likelihood that the tsps have been accurately mapped. Furthermore, the spacing of these TATA boxes may help to explain why 2 pairs of tsps with equivalent spacing are observed.

The primer to intron 1 also yielded two major extension products that were found close to the 5`-end of intron 1 (Fig. 3). These results are consistent with the sequence obtained earlier by 5`-RACE. It therefore appears that at least two types of transcripts are produced from the KOR gene. The first starts just downstream from one of two TATA boxes ultimately requiring excision of intron 1. This transcript corresponds to a type 1 KOR cDNA sequence reported previously (Chen et al., 1993; Li et al., 1993; Meng et al., 1993; Nishi et al., 1993), referred to here as KOR1. The second transcript begins within intron 1 and thus retains this intronic sequence in the mature mRNA. We have designated this second transcript as KORx.

The sequence of intron 1 in the KORx transcript carries two additional ATG triplets followed by a peptide-encoding sequence, which is in-frame with the expected open reading frame for KOR that starts in exon 2 (Fig. 1). This implies that KORx mRNA may encode a variant of the receptor that contains additional amino acids at its NH(2)-terminal end. Both translation start sites contain purines located three nucleotides upstream that are known to be essential for efficient translation of eucaryotic mRNA (Kozak, 1989). However, the deduced peptide sequence of this putative amino-terminal extension shows no significant homology with any other known protein in the GenBank/EMBL data base at this date.

The finding of two distinct KOR mRNA species is indicative that alternative promoters are operational in the transcriptional regulation of this gene. To examine the possibility that different promoters are active in different tissues we used nested RT-PCR on total RNA from rat brain, lungs, spleen, spinal cord, kidney, testes, and heart. To identify the KOR1 mRNA sequence, sense primers within exon 1 were used, whereas to identify KORx mRNA, sense primers within intron 1 were used. Both sets of amplifications were performed with the same antisense primers used for exon 4, thus covering nearly the entire length of the open reading frame (for details, see Fig. 4). The KOR1 species was observed in all tissues examined except kidney and testes. In contrast, KORx was found only in kidney and brain.

The transcriptional mechanisms giving rise to alternate KOR mRNAs remain to be determined. The sequence upstream of exon 1 for KOR1 contains two potential TATA boxes at positions -38 and -12 nt. The mechanism for transcribing KORx likely involves the binding of an RNA polymerase II initiation complex near the junction between exon 1 and intron 1. This region is comprised primarily of exon 1 sequence, which for the KORx transcript may constitute a significant portion of its promoter. There is no TATA box within this region, but it is noteworthy that there is an NF-kappaB consensus recognition site and a CAAT box within 40 bp upstream of the proposed tsps for KORx.

Our results demonstrate that two transcripts are derived from a single gene encoding KOR. The existence of two KOR transcripts indicates that two different promoters may regulate the expression of the KOR gene in different tissues or under different conditions and/or that different translation products may be produced. Studies are in progress to determine the functional significance of these two KOR transcripts. The potential open reading frame initiating in intron 1 may lead to the translation of a variant KOR protein having an additional peptide sequence at its amino terminus. This possibility is supported by two important observations: 1) the reading frame present in intron 1 is in-frame with the normal KOR encoding sequence and 2) expression of this novel transcript shows a discrete pattern of expression, implying that different cell types may utilize this variant KOR protein.

Although up to three KOR subtypes have been hypothesized based on pharmacological data, the failure to detect other genes that encode a KOR protein and the apparent lack of alternatively spliced KOR messages do not help to clarify the molecular basis for these pharmacological KOR subtypes. Expression of KORx in brain and kidney, but not in the other tissues examined, does not correlate with the expected localization of any of the known KOR subtypes. This suggests that either KORx represents a previously undetected KOR subtype or that the pharmacological properties of KORx are not readily distinguishable from those of KOR1.


FOOTNOTES

*
This work was supported in part by Grant R49/CCR 306634 from the Centers for Disease Control (to A. I. F.). The principles enumerated in the ``Guide for the Care and Use of Laboratory Animals'' prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Resources, National Research Council (Department of Health and Human Services Publication 85-23) were adhered to. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U17993[GenBank], U17994[GenBank], and U17995[GenBank].

§
To whom correspondence should be addressed: Georgetown University Medical Center, 3900 Reservoir Rd. NW, Washington, D. C. 20007. Tel.: 202-687-7007; Fax: 202-687-2585; afaden{at}macpost.odr.georgetown.edu.

(^1)
The abbreviations used are: KOR, kappa opioid receptor; OR, opioid receptor(s); RT, reverse transcription; PCR, polymerase chain reaction; 5`-RACE, rapid amplification of cDNA 5`-ends; tsp, transcription start point; bp, base pair(s); nt, nucleotide(s).


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.