Antisense Mapping the Mor-1 Opioid Receptor - Evidence for Alternative Splicing and A Novel Morphine-6-Beta-Glucuronide Receptor

Cornell University, Итак, New York, United States
FEBS Letters (Impact Factor: 3.17). 09/1995; 369(2-3):192-6. DOI: 10.1016/0014-5793(95)00757-Z
Source: PubMed


Although MOR-1 encodes a mu opioid receptor, its relationship to the pharmacologically defined mu receptor subtypes has been unclear. Antisense mapping now suggests that these subtypes result from alternative splicing of MOR-1. Three oligodeoxynucleotide probes targeting exon 1 and another oligodeoxynucleotide directed against the coding region of exon 4 block supraspinal morphine analgesia, a mu1 action, while five of six oligodeoxynucleotides directed against exons 2 and 3 are inactive. Inhibition of gastrointestinal transit and spinal morphine analgesia, two mu2 actions, are blocked only by the probe against exon 4 and not by those directed against exon 1. In contrast, the analgesic actions of the extraordinarily potent mu drug morphine-6 beta-glucuronide are blocked by six different antisense oligodeoxynucleotides targeting exons 2 and 3, but not by those acting on exons 1 or 4. These results suggest that the mu1 and mu2 receptor subtypes originally defined in binding and pharmacological studies result from alternative splicing of MOR-1 while morphine-6 beta-glucuronide acts through a novel, previously unidentified receptor which is yet another MOR-1 splice variant.

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Available from: Grace Rossi, Dec 20, 2013
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    • "Morphine and most clinical analgesic agents act through m-opioid receptors (MORs). Pharmacologic studies have defined several m receptor subtypes including m 1 , m 2 , and morphine-6b- glucuronide (M6G) receptors (Wolozin and Pasternak, 1981; Pasternak, 1993; Rossi et al., 1995, 1996; Reisine and Pasternak, 1996). However, a single m-opioid receptor gene (OPRM1) has been identified in all the species (Min et al., 1994; Giros et al., 1995; Liang et al., 1995), raising the possibility of alternative pre-mRNA splicing of the OPRM1 gene to generate multiple splice variants with diverse actions. "
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    ABSTRACT: The μ opioid receptor (MOR-1) gene, OPRM1, undergoes extensive alternative splicing, generating an array of splice variants. Of these variants, MOR-1A, an intron-retention carboxyl terminal splice variant identical to MOR-1 except for the terminal intracellular tail encoded by exon 3b, is quite abundant and conserved from rodent to humans. Increasing evidence indicates that miroRNAs (miRNAs) regulate MOR-1 expression and μ agonists such as morphine modulate miRNA expression. However, little is known about miRNA regulation of the OPRM1 splice variants. Using 3' rapid amplification cDNA end (RACE) and Northern blot analyses, the present study identified the complete 3' untranslated region (3'-UTR) for both mouse and human MOR-1A and their conserved polyadenylation site, and defined the role the 3'-UTR in mRNA stability using a luciferase reporter assay. Computer models predicted a conserved miR 103/107 targeting site in the 3'-UTR of both mouse and human MOR-1A. The functional relevance of miR-103/107 in regulating expression of MOR-1A protein through the consensus miR-103/107 binding sites in the 3'- UTR was established by using mutagenesis and a miR-107 inhibitor in transfected HEK293 cells and Be(2)C cells that endogenously express human MOR-1A. Chronic morphine treatment significantly upregulated miR-103 and miR-107 levels, leading to downregulation of polyribosome-associated MOR-1A in both Be(2)C cells and the striatum of a morphine tolerant mouse, providing a new perspective on understanding the roles of miRNAs and OPRM1 splice variants in modulating the complex actions of morphine in animals and humans.
    Preview · Article · Dec 2013 · Molecular pharmacology
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    • "The greater potency of M6G has been attributed to greater efficacy in activating the MOR (Osborne et al. 2000; Ulens et al. 2001) or to its actions at a unique MOR subtype. The existence of a MOR-1 subtype with greater affinity for M6G than for morphine was first proposed by Rossi et al. (1995a) and was later confirmed by others (Brown et al. 1997; Mantione et al. 2002). Experiments using antisense probes or knockout mice have demonstrated the existence of splice variants of MOR-1 with differential affinity for morphine versus heroin and M6G (Rossi et al. 1995b; Matthes et al. 1996; Sora et al. 1997; Loh et al. 1998; Schuller et al. 1999; Unterwald et al. 1999; Pan et al. 2009). "
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    ABSTRACT: Heroin is rapidly metabolized to morphine that in turn is transformed into morphine-3-glucuronide (M3G), an inactive metabolite at mu-opioid receptor (MOR), and morphine-6-glucuronide (M6G), a potent MOR agonist. We have found that rats that had received repeated intraperitoneal injections of heroin exhibit measurable levels of M6G (which is usually undetectable in this species). The goal of the present study was to investigate whether M6G synthesis can be induced by intravenous (i.v.) heroin self-administration (SA). Rats were trained to self-administer either heroin (50 μg/kg per infusion) or saline for 20 consecutive 6-h sessions and then challenged with an intraperitoneal challenge of 10 mg/kg of heroin. Plasma levels of heroin, morphine, 6-mono-acetyl morphine, M3G, and M6G were quantified 2 h after the challenge. In vitro morphine glucuronidation was studied in microsomal preparations obtained from the liver of the same rats. Heroin SA induced the synthesis of M6G, as indicated by detectable plasma levels of M6G (89.7 ± 37.0 ng/ml vs. 7.35 ± 7.35 ng/ml after saline SA). Most important, the in vitro V (max) for M6G synthesis was correlated with plasma levels of M6G (r (2) = 0.78). Microsomal preparations from saline SA rats produced negligible amounts of M6G. Both in vivo and in vitro data indicate that i.v. heroin SA induces the synthesis of M6G. These data are discussed in the light of previous studies conducted in heroin addicts indicating that in humans heroin enhances the synthesis of the active metabolite of heroin and morphine.
    Full-text · Article · Oct 2011 · Psychopharmacology
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    • "However, to date only a single mu opioid receptor gene has been identified, raising the questions of how a single OPRM1 gene could explain the complex pharmacology of mu opioids in animals and humans. Our early antisense mapping studies suggested different exon combinations for the analgesic actions of the two mu agonists morphine and M6G, raising the possibility of alternative splicing in the OPRM1 gene [17,46]. Since then, much effort has been devoted to identifying these OPRM1variants. "
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    ABSTRACT: The mouse mu opioid receptor (OPRM1) gene undergoes extensive alternative splicing at both the 3'- and 5'-ends of the gene. Previously, several C-terminal variants generated through 3' splicing have been identified in the rat OPRM1 gene. In both mice and humans 5' splicing generates a number of exon 11-containing variants. Studies in an exon 11 knockout mouse suggest the functional importance of these exon 11-associated variants in mediating the analgesic actions of a subset of mu opioids, including morphine-6β-glucuronide (M6G) and heroin, but not others such as morphine and methadone. We now have examined 5' splicing in the rat. The current studies identified in the rat a homologous exon 11 and seven exon 11-associated variants, suggesting conservation of exon 11 and its associated variants among mouse, rat and human. RT-PCR revealed marked differences in the expression of these variants across several brain regions, implying region-specific mRNA processing of the exon 11-associated variants. Of the seven rat exon 11-associated variants, four encoded the identical protein as found in rMOR-1, two predicted 6 TM variants, and one, rMOR-1H2, generated a novel N-terminal variant in which a stretch of an additional 50 amino acids was present at the N-terminus of the previously established rMOR-1 sequence. When expressed in CHO cells, the presence of the additional 50 amino acids in rMOR-1H2 significantly altered agonist-induced G protein activation with little effect on opioid binding. The identification of the rat exon 11 and its associated variants further demonstrated conservation of 5' splicing in OPRM1 genes among rodents and humans. The functional relevance of these exon 11 associated variants was suggested by the region-specific expression of their mRNAs and the influence of the N-terminal sequence on agonist-induced G protein coupling in the novel N-terminal variant, rMOR-1H2. The importance of the exon 11-associated variants in mice in M6G and heroin analgesia revealed in the exon 11 knockout mouse implies that these analogous rat variants may also play similar roles in rat. The complexity created by alternative splicing of the rat OPRM1 gene may provide important insights of understanding the diverse responses to the various μ opioids seen in rats.
    Full-text · Article · Jan 2011 · Molecular Pain
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