Post-Transcriptional Regulation of Mouse k-Opioid Receptor Expression

Three mRNA variants are generated from the mouse k-opioid receptor (KOR) gene. The expression patterns of these KOR mRNA variants in adult animal tissues and during developmental stages are examined. Furthermore, the biological significance of generating these variants is demonstrated with respect to two post-transcriptional mechanisms, i.e., mRNA stability and translation efficiency. Variants A and B are both transcribed from promoter 1 of the KOR gene and expressed from early developmental stages through adult life. Although their sequences differ only at a 30-nucleotide insertion for variant B, these two variants are distinct with regard to their expression patterns, mRNA stability, and translation efficiency. Variant A is expressed ubiquitously in all the tissues examined and has a longer t1/2 (12 h), whereas variant B is more specific to the central nervous system both preand postnatally and has a t1/2 of ;8 h. Variant C is transcribed from promoter 2 of the KOR gene and is most specifically expressed, being detected only in the brain stem, spinal cord, and thalamic/hypothalamic areas of postnatal animals. With regard to protein translation, variants B and C are significantly more efficient than variant A. This study provides the evidence for multiple levels of KOR regulation. The biological implication of the generation of KOR mRNA variants is discussed. Opiates exert a wide spectrum of pharmacologic effects, mediated by a family of G protein-coupled transmembrane receptors called opioid receptors. Three opioid receptor types, m, d, and k, are present (Goldstein and Naidu, 1989; Loh and Smith, 1990). The genes of the three opioid receptors have been cloned, and their expression patterns in animals have been examined with in situ hybridization, immunohistochemistry, and ligand binding assays (Rius et al., 1991; Elde et al., 1995; Kieffer, 1995; Knapp et al., 1995; Mansour et al., 1995; Zhu et al., 1998). The physiologic roles have only begun to be uncovered from studying genetically altered animal models (Mattes et al., 1996; Sora et al., 1997; Tian et al., 1997; Loh et al., 1998; Simonin et al., 1998; Schuller et al., 1999). Studies using antibodies, nucleic acid probes, and radioisotope-labeled ligands reveal different expression patterns of the three opioid receptors in the brain and spinal areas that are associated with pain sensation and behavior. Our recent studies in developing animals also show distinct patterns of expression of the three opioid receptor genes in embryonic stages, suggesting potential functions of opioid receptors in early animal development (Chen et al., 1999). Particularly, the k-opioid receptor (KOR) appears very early during animal development [embryonic day (E) 9.5]. Consistent with the observation that KOR is expressed in developing embryos before the formation of the nervous system, the KOR gene is found to be active in the stem cell populations of an embryonal carcinoma cell line P19 (Chen et al., 1999). The mouse KOR gene has been isolated in several laboratories, and its genomic structure has been determined (Liu et al., 1995). In the P19 cell line model, we have demonstrated the biologic activities of dual promoters of the mouse KOR gene (Lu et al., 1997). These two promoters can potentially generate transcript variants that either contain or lack exon 1 sequence (Lu et al., 1997). In addition, our own as well as other studies (Belkowske et al., 1995; Lu et al., 1997), have demonstrated the presence of another variant using an alternative, splicing acceptor site at intron 1. As a result of this alternative splicing, this mRNA variant encodes an additional 30 nucleotides between exons 1 and 2 (Lu et al., 1997). Despite the demonstration of these KOR mRNA variants in mouse brain and cell lines, it remains elusive as to whether these KOR mRNA variants represent biologically functional KOR mRNAs and whether they are expressed differentially in animal tissues. Furthermore, the biologic significance of generating KOR variants is unknown. To address these questions, we set up experiments, first to examine the expression of these KOR mRNA variants in This work was supported by Grants DA11190, DA11806, DA70554, and DA00564, and from the National Institute on Drug Abuse, National Institutes of Health. ABBREVIATIONS: KOR, k-opioid receptor; RT-PCR, reverse transcription polymerase chain reaction; bp, base pair; TNT, transcription/translation; Luc, luciferase; UTR, untranslated region; CNS, central nervous system. 0026-895X/00/020401-08$3.00/0 Copyright © The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 57:401–408 (2000). 401 at A PE T Jornals on N ovem er 7, 2017 m oharm .aspeurnals.org D ow nladed from adult animals as well as during development. Second, we addressed the biological significance of these variants in two post-transcriptional events, mRNA stability and translation efficiency. The results of this study provide the evidence, for the first time, of differential expression of KOR mRNA variants as well as post-transcriptional regulation of KOR expression. Materials and Methods Analyses of KOR mRNA Variant Expression. P19 embryonal carcinoma cells were maintained as described (Wei and Chang, 1996). RNA was isolated from normal adult (body weight 25–30 g) CD1 mouse tissues and P19 cells using the method of Charron and Drouin (1986). To differentiate the expression of KOR mRNA variants in small tissue samples such as dissected brain areas, conventional mRNA detection methods such as Northern blot and RNase protection methods are not appropriate because of the limit in sensitivity and sample size. Another method, in situ hybridization, was not chosen, because the sequence that allowed these variants to be detected differentially are too short (i.e., 30 nucleotides long). Therefore, reverse transcription polymerase chain reaction (RT-PCR) was used. RT was conducted using SuperScript II RT (Life Technologies, Grand Island, NY) and primed with oligo(dT) primers on 2 mg of RNA samples in a total volume of 20 ml. To differentiate between the two variants generated from the first promoter with alternative splicing that occurred at intron 1, the variant-specific 59 primers were designed according to specific exon 1/exon 2 junction sequences (Lu et al., 1997). These two KOR mRNA variants were called A and B. The 59 primer for variant A is 59-ATCAGCGATCTGGAGCT-39 and that for variant B (containing an insertion of 30 nucleotides) is 59TCAGCGATCTGGAGCCCC-39 (underlined residues showing the splicing junctions of each species of mRNA). The sequence of the 59 primer for the third variant (C), transcribed from the second promoter located in intron 1, is 59-ACAGGCAAAGTTTGTC-39. A common 39 primer, 59-GCAAGGAGCATTCAATGAC-39, was designed according to an exon 4 sequence that is present in all the three variants. Thus, KOR mRNA variants A, B, and C were amplified as 730 base pair (bp), 760-bp, and 800-bp fragments, respectively. To establish RT-PCR, the linear ranges of amplification cycles and the PCR inputs in relation to PCR products were first determined (see Results). Appropriate dilutions of RT products were each amplified in a 20 ml PCR using commercially provided buffer with a reaction cycle of 94°C for 45 s, 55°C for 45 s, and 72°C for 1 min, for a total of 30 cycles. A pair of actin-specific primers (59-TGGCCTTAGGGTGCAGGG-39; 59-GTGGGCCGCTCTAGGCACCA-39) (Rappollee et al., 1989) were included for 23 cycles in each reaction for internal controls. For all the RT-PCR experiments, a negative control without RT was included. After PCR, 5 ml of each sample was analyzed on Southern blots using [a-P]dCTP-labeled probes prepared from the mouse KOR cDNA spanning exon I to exon IV and from actin cDNA. Southern blot analyses were conducted according to the established protocols (Maniatis et al., 1990). To quantify PCR products, the hybridizing bands were quantified in a phosphoimager by using Image Quant software (Molecular Dynamics, Sunnyvale, CA). The KOR-specific signal was normalized to the actin-specific signal in each reaction to obtain a value representing the specific KOR variant expression. The expression of each variant in all the samples (see Figs. 3 and 4) was represented as the relative level of expression by arbitrarily setting the expressed level of one variant at a specific stage as the value of 1. The mean and S.D. values were obtained from at least three experiments. To determine the t1/2 of mRNA, experiments were conducted in P19 stem cells that expressed endogenous KOR variants A and B. Actinomycin D was added at a concentration of 2 mg/ml. At different time points, RNA was collected and subjected to RT-PCR analyses and phosphoimager quantification as described previously. In Vitro Transcription/Translation (TNT). The cDNA of each KOR mRNA variant was cloned into the PvuII site of pSP73 (Promega, Madison, WI) following the T7 promoter. A control vector was generated by fusing the 59-untranslated sequence of luciferase (Luc) reporter from pGL3 (Promega) to the upstream of the ATG codon of the KOR coding region, which also was cloned into pSP73 vector at the same PvuII site. TNT reactions were conducted using the TNTcoupled reticulocyte lysate reaction system (Promega) at 30°C for 60 min. In each reaction, 0.5 mg of specific KOR cDNA vector and 50 ng of an internal control vector (a cytochrome enzyme CYP26 expression vector also cloned in pSP73) (Haque and Anreola, 1998) were used as the starting material, and [S]methionine was added during translation. The condition was established to synthesize KOR and CYP26 proteins in a linear range. The transcription efficiency was monitored by examining RNA products in Northern blot analyses. The translated products from the same amount of KOR mRNA variants were analyzed on 12% SDS polyacrylamide gels and quantified using a phosphoimager as described previously. The signal of the KOR protein was normalized to the internal control CYP signal to obtain a value representing specific KOR protein expression in