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Stabilization of the ?-opioid receptor by truncated single transmembrane splice variants through a chaperone-like action.

ABSTRACT: The ?-opioid receptor gene, OPRM1, undergoes extensive alternative pre-mRNA splicing, as illustrated by the identification of an array of splice variants generated by both 5' and 3' alternative splicing. The current study reports the identification of another set of splice variants conserved across species that are generated through exon skipping or insertion that encodes proteins containing only a single transmembrane (TM) domain. Using a Tet-Off system, we demonstrated that the truncated single TM variants can dimerize with the full-length 7-TM ?-opioid receptor (MOR-1) in the endoplasmic reticulum, leading to increased expression of MOR-1 at the protein level by a chaperone-like function that minimizes endoplasmic reticulum-associated degradation. In vivo antisense studies suggested that the single TM variants play an important role in morphine analgesia, presumably through modulation of receptor expression levels. Our studies suggest the functional roles of truncated receptors in other G protein-coupled receptor families.

SUBMITTER: Xu J

PROVIDER: S-EPMC3774389 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Stabilization of the μ-opioid receptor by truncated single transmembrane splice variants through a chaperone-like action.

Xu Jin J Xu Ming M Brown Taylor T Rossi Grace C GC Hurd Yasmin L YL Inturrisi Charles E CE Pasternak Gavril W GW Pan Ying-Xian YX

The Journal of biological chemistry 20130611 29

The μ-opioid receptor gene, OPRM1, undergoes extensive alternative pre-mRNA splicing, as illustrated by the identification of an array of splice variants generated by both 5' and 3' alternative splicing. The current study reports the identification of another set of splice variants conserved across species that are generated through exon skipping or insertion that encodes proteins containing only a single transmembrane (TM) domain. Using a Tet-Off system, we demonstrated that the truncated singl ...[more]

PMID: 23760268

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Project description:BackgroundMost clinical opioids act through μ-opioid receptors. They effectively relieve pain but are limited by side effects, such as constipation, respiratory depression, dependence, and addiction. Many efforts have been made toward developing potent analgesics that lack side effects. Three-iodobenzoyl-6β-naltrexamide (IBNtxA) is a novel class of opioid active against thermal, inflammatory, and neuropathic pain, without respiratory depression, physical dependence, and reward behavior. The μ-opioid receptor (OPRM1) gene undergoes extensive alternative precursor messenger ribonucleic acid splicing, generating multiple splice variants that are conserved from rodents to humans. One type of variant is the exon 11 (E11)-associated truncated variant containing 6 transmembrane domains (6TM variant). There are 5 6TM variants in the mouse OPRM1 gene, including mMOR-1G, mMOR-1M, mMOR-1N, mMOR-1K, and mMOR-1L. Gene-targeting mouse models selectively removing 6TM variants in E11 knockout (KO) mice eliminated IBNtxA analgesia without affecting morphine analgesia. Conversely, morphine analgesia is lost in an exon 1 (E1) KO mouse that lacks all 7 transmembrane (7TM) variants but retains 6TM variant expression, while IBNtxA analgesia remains intact. Elimination of both E1 and E11 in an E1/E11 double KO mice abolishes both morphine and IBNtxA analgesia. Reconstituting expression of the 6TM variant mMOR-1G in E1/E11 KO mice through lentiviral expression rescued IBNtxA but not morphine analgesia. The aim of this study was to investigate the effect of lentiviral expression of the other 6TM variants in E1/E11 KO mice on IBNtxA analgesia.MethodsLentiviruses expressing 6TM variants were packaged in HEK293T cells, concentrated by ultracentrifugation, and intrathecally administered 3 times. Opioid analgesia was determined using a radiant-heat tail-flick assay. Expression of lentiviral 6TM variant messenger ribonucleic acids was examined by polymerase chain reaction (PCR) or quantitative PCR.ResultsAll the 6TM variants restored IBNtxA analgesia in the E1/E11 KO mouse, while morphine remained inactive. Expression of lentiviral 6TM variants was confirmed by PCR or quantitative PCR. IBNtxA median effective dose values determined from cumulative dose-response studies in the rescued mice were indistinguishable from wild-type animals. IBNtxA analgesia was maintained for up to 33 weeks in the rescue mice and was readily antagonized by the opioid antagonist levallorphan.ConclusionsOur study demonstrated the pharmacological relevance of mouse 6TM variants in IBNtxA analgesia and established that a common functional core of the receptors corresponding to the transmembrane domains encoded by exons 2 and 3 is sufficient for activity. Thus, 6TM variants offer potential therapeutic targets for a distinct class of analgesics that are effective against broad-spectrum pain models without many side effects associated with traditional opioids.

Dataset Information

Stabilization of the ?-opioid receptor by truncated single transmembrane splice variants through a chaperone-like action.

Publications

Stabilization of the μ-opioid receptor by truncated single transmembrane splice variants through a chaperone-like action.

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