Dataset Information

Generation of the mu opioid receptor (MOR-1) protein by three new splice variants of the Oprm gene.

ABSTRACT: Using 5' RACE, we have isolated four additional exons of the mu opioid receptor gene (Oprm), resulting in a gene spanning over 250 kb. The four new exons are contained within eight additional splice variants containing exon 11 at the 5' terminus. Exon 11, which is under the control of a previously unknown upstream promoter, and exon 12 are located approximately 10 kb and approximately 8 kb upstream from exon 1, respectively. Exon 13 and 14 are located between exons 1 and 2. The regional distributions of the variants, as determined by reverse transcription-PCR, varied among themselves and were distinct from that of MOR-1, implying region-specific RNA processing. Three variants (MOR-1H, MOR-1I, and MOR-1J) contained two potential translational start points, with the translational start point in exon 1 producing proteins identical to the original MOR-1 protein. When expressed, the receptor binding of these three variants was indistinguishable from that of MOR-1. The remaining eight proteins using the translation start point in exon 11 were all truncated, with three (MOR-1G, MOR-1M, and MOR-1N) predicting proteins of only six transmembrane domains and the rest giving proteins under 10 kDa. Western blots with an exon 11-specific antiserum revealed bands consistent with the six transmembrane domain proteins within the brain, but the shorter proteins were not detected. Thus, the MOR-1 protein can be generated by four different splice variants of the Oprm gene under the control of two physically distinct promoters. Although the truncated proteins are expressed in brain with a unique regional distribution, their functional significance remains unknown.

SUBMITTER: Pan YX

PROVIDER: S-EPMC61171 | biostudies-literature | 2001 Nov

REPOSITORIES: biostudies-literature

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Generation of the mu opioid receptor (MOR-1) protein by three new splice variants of the Oprm gene.

Pan Y X YX Xu J J Mahurter L L Bolan E E Xu M M Pasternak G W GW

Proceedings of the National Academy of Sciences of the United States of America 20011101 24

Using 5' RACE, we have isolated four additional exons of the mu opioid receptor gene (Oprm), resulting in a gene spanning over 250 kb. The four new exons are contained within eight additional splice variants containing exon 11 at the 5' terminus. Exon 11, which is under the control of a previously unknown upstream promoter, and exon 12 are located approximately 10 kb and approximately 8 kb upstream from exon 1, respectively. Exon 13 and 14 are located between exons 1 and 2. The regional distribu ...[more]

PMID: 11717463

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Project description:The acute and chronic effects of alcohol on the brain and behavior are linked to alterations in inhibitory synaptic transmission. Alcohol's most consistent effect at the synaptic level is probably a facilitation of γ-aminobutyric acid (GABA) release, as seen from several rodent studies. The impact of alcohol on GABAergic neurotransmission in human neurons is unknown, due to a lack of a suitable experimental model. Human neurons can also be used to model effects of genetic variants linked with alcohol use disorders (AUDs). The A118G single nucleotide polymorphism (SNP rs1799971) of the OPRM1 gene encoding the N40D (D40 minor allele) mu-opioid receptor (MOR) variant has been linked with individuals who have an AUD. However, while N40D is clearly associated with other drugs of abuse, involvement with AUDs is controversial. In this study, we employed Ascl1-and Dlx2-induced inhibitory neuronal cells (AD-iNs) generated from human iPS cell lines carrying N40D variants, and investigated the impact of ethanol acutely and chronically on GABAergic synaptic transmission. We found that N40 AD-iNs display a stronger facilitation (versus D40) of spontaneous and miniature inhibitory postsynaptic current frequency in response to acute ethanol application. Quantitative immunocytochemistry of Synapsin 1+ synaptic puncta revealed a similar synapse number between N40 and D40 iNs, suggesting an ethanol modulation of presynaptic GABA release without affecting synapse density. Interestingly, D40 iNs exposed to chronic intermittent ethanol application caused a significant increase in mIPSC frequency, with only a modest enhancement observed in N40 iNs. These data suggest that the MOR genotype may confer differential sensitivity to synaptic output, which depends on ethanol exposure time and concentration for AD-iNs and may help explain alcohol dependence in individuals who carry the MOR D40 SNPs. Furthermore, this study supports the use of human neuronal cells carrying risk-associated genetic variants linked to disease, as in vitro models to assay the synaptic actions of alcohol on human neuronal cells.

Project description:BackgroundThe 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.ResultsThe 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.ConclusionThe 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.

Dataset Information

Generation of the mu opioid receptor (MOR-1) protein by three new splice variants of the Oprm gene.

Publications

Generation of the mu opioid receptor (MOR-1) protein by three new splice variants of the Oprm gene.

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