Identification and characterization of seven new exon 11-associated splice variants of the rat μ opioid receptor gene, OPRM1.

Department of Neurology and Program in Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.
Molecular Pain (Impact Factor: 3.65). 01/2011; 7(1):9. DOI: 10.1186/1744-8069-7-9
Source: PubMed


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.

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Available from: Grace Rossi, Dec 20, 2013
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    • "The final set of splice variants are associated with exon 11 and contain only six transmembrane (6TM) domains, lacking TM1 present in the traditional full-length receptors (Pan et al., 2001; Xu et al., 2011). Initially thought to be of little significance, they have proven very important in understanding m-opioid pharmacology, as revealed by a series of knockout This work was supported, in part, by the National Institutes of Health National Institute on Drug Abuse [Grants R01-DA002615, R01-DA07242, and R01-DA06241]; the Harrington Research Institute; Mr. William H. Goodwin and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research; the Experimental Therapeutics Center of Memorial Sloan-Kettering Cancer Center (to G.W.P.); a fellowship from the PhRMA Foundation (to S.G.G.); and a core grant from the National Institutes of Health National Cancer Institute [Grant CA08748] (to the Memorial Sloan-Kettering Cancer Center). "
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    ABSTRACT: 3'-Iodobenzoyl-6β-naltrexamide (IBNtxA) is a potent analgesic in mice lacking many traditional opioid side-effects. In mice, it displays no respiratory depression, does not produce physical dependence with chronic administration and shows no cross tolerance to morphine. It has limited effects on gastrointestinal transit and shows no reward behavior. Biochemical studies indicate its actions are mediated through a set of MOR-1 splice variants associated with exon 11 that lack exon 1 and contain only six transmembrane domains. Like the mouse and human, rats express exon 11-associated splice variants that also contain only six transmembrane domains, raising the question of whether or not IBNtxA would have a similar pharmacological profile in rats. Given systemically, IBNtxA is a potent analgesic in rats, with an ED50 value of 0.89 mg/kg, s.c., approximately 4-fold more potent than morphine. It shows no analgesic cross tolerance in morphine-pelleted rats. IBNtxA displays no respiratory depression as measured by blood oxygen saturation. In contrast, oximetry shows an equianalgesic dose of morphine lowers blood oxygen saturation values by 30%. IBNtxA binding is present in a number of brain regions, with the thalamus standing out with very high levels and the cerebellum with low levels. In conclusion, as in mice, IBNtxA is a potent analgesic in rats with a favorable pharmacological profile and reduced side-effects.
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    • "Hence, compared with morphine, M6G has a lower volume of distribution and clearance rate, a lower permeability across the blood brain barrier and a slower clearance from the brain. The most attractive hypothesis for M6G differential effects is based on the presence of specific MOR splicing variants having higher affinity or specificity than morphine [29] [30]. Interestingly, animal studies revealed that heroin and M6G act through receptors and mechanisms distinct from those of morphine. "
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    • "To reconcile these findings we propose that buprenorphine acts on a distinct opioid receptor type/subtype coupled with G q/11 , which is consistent with the observation that several splice variants of the μ opioid receptor having different affinities for agonists were identified in human and rodents (Bolan et al., 2004; Xu et al., 2011). "
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