Mingming Xu

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

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 ≈10 kb and ≈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.

Identification and characterization of six new alternatively spliced variants of the human μ opioid receptor gene, Oprm

The mu opioid receptor plays an important role in mediating the actions of morphine and morphine-like drugs. Receptor binding and a wide range of pharmacological studies have proposed several mu receptor subtypes, but only one mu opioid receptor (Oprm) gene has been isolated. Like the mouse and rat, the human Oprm gene undergoes alternative splicing. In the present studies, we have identified and characterized six new splice variants from the human Oprm gene using a reverse transcription-polymerase chain reaction strategy, yielding a total of 10 human splice variants of the mu opioid receptor MOR-1. All the variants identified contained exons 1, 2 and 3, but differed from MOR-1 itself and each other by splicing downstream from exon 3, resulting in different amino acid sequences. Northern blot analysis demonstrated expression of the variant mRNAs. Receptor binding assays established that these variants belonged to the mu opioid receptor family with limited differences in mu opioid ligand affinities and selectivity. However, adenylyl cyclase and [35S]GTPgammaS binding assays revealed major differences in both potency and efficacy among these variants. The dissociation between binding affinity, potency and efficacy for the opioids among these variants may provide insights into the wide range of opioid responses among these agents observed clinically and opens new avenues in designing selective drugs based upon their efficacy and potency rather simple binding affinity.

Identification and characterization of two new human mu opioid receptor splice variants, hMOR-1O and hMOR-1X

The mouse gene encoding the mu opioid receptor, Oprm, undergoes extensive alternatively splicing, with 14 variants having been identified. However, only one variant of human mu opioid receptor gene (Oprm), MOR-1A, has been described. We now report two novel splice variants of the human Oprm gene, hMOR-1O and hMOR-1X. The full-length cDNAs of hMOR-1O and hMO-1X contained the same exons 1, 2, and 3 as the original hMOR-1, but with exon O or exon X as the alternative fourth exon, respectively. Northern blots revealed several bands with the exon O probe in both human neuroblastoma BE(2)C cells and human brain and a single band (5.5kb) with the exon X probe in selected human brain regions. When transfected into CHO cells, both variants showed high selectivity for mu opioids in binding assays. These two new human mu opioid receptors are the first human MOR-1 variants containing new exons and suggest that the complex splicing present in mice may extend to humans.

Involvement of exon 11-associated variants of the mu opioid receptor MOR-1 in heroin, but not morphine, actions

Heroin remains a major drug of abuse and is preferred by addicts over morphine. Like morphine, heroin has high affinity and selectivity for μ-receptors, but its residual analgesia in exon 1 MOR-1 knockout mice that do not respond to morphine suggests a different mechanism of action. MOR-1 splice variants lacking exon 1 have been observed in mice, humans, and rats, raising the possibility that they might be responsible for the residual heroin and morphine-6β-glucuronide (M6G) analgesia in the exon 1 knockout mice. To test this possibility, we disrupted exon 11 of MOR-1, which eliminates all of the variants that do not contain exon 1. Morphine and methadone analgesia in the exon 11 knockout mouse was normal, but the analgesic actions of heroin, M6G, and fentanyl were markedly diminished in the radiant heat tail-flick and hot-plate assays. Similarly, the ability of M6G to inhibit gastrointestinal transit was greatly diminished in these exon 11 knockout mice, whereas the ability of morphine was unchanged. These findings identify receptors selectively involved with heroin and M6G actions and confirm the relevance of the exon 11-associated variants and raise important issues regarding the importance of atypical truncated G-protein-coupled receptors.

Identification of three new alternatively spliced variants of the rat mu opioid receptor gene: dissociation of affinity and efficacy.

Mu opioid receptors mediate the pharmacological actions of morphine and morphine‐like drugs, such as heroin. The mouse and human Oprm genes undergo splicing. In these present studies, we have identified and characterized three new MOR‐1 splice variants from the rat Oprm gene. Using an RT‐PCR approach, we isolated the new exons 7, 8 and 9 downstream of exon 3. The rat exons 7 and 9 were homologous to the mouse exons 7 and 9 while the rat exon 8 was not. Northern blot analysis with the new exon probes showed distinctive and abundant transcripts of the variants in the rat brain. Full‐length cDNA clones containing the new exons, rMOR‐1C1, rMOR‐1C2 and rMOR‐1D were obtained using an RT‐PCR approach. Each contained the same exons 1, 2 and 3 as the original rMOR‐1, followed by different combinations of the new exons in place of exon 4. In addition, we also isolated another new variant, rMOR‐1A, which contains only exons 1, 2 and 3, and is homologous to the human variant MOR‐1A previously identified. All the variants were highly mu‐selective in binding studies with little difference in affinities for the mu ligands among the variants. However, functional evaluation of assessments of the variants using agonist stimulated [35S]GTPγS binding assays revealed marked differences among the variants, both in terms of potency and efficacy of the drugs. The relative efficacy of a series of mu opioids to each other varied depending upon the variant studied. Efficacy in the [35S]GTPγS assay did not correlate with either receptor binding affinity or with potency. Thus, selectivity of opioid action might be achieved by designing compounds with varying efficacies at different MOR‐1 variants.

Isolation and characterization of new exon 11-associated N-terminal splice variants of the human mu opioid receptor gene.

Alternative splicing of the mu opioid receptor genes to create multiple mu receptor subtypes has been demonstrated in animals and humans. Previously, we identified a number of C‐terminal variants in mice, rats and human, followed by several N‐terminal variants associated with a new upstream exon in mice (exon 11). Behavioral studies in exon 11 knockout mice suggest an important role for the exon 11 variants in the analgesic actions of heroin and morphine‐6β‐glucuronide, but not morphine or methadone. We now have identified a homologous human exon 11 and three similar human exon 11‐associated variants, suggesting conservation of exon 11 and its associated variants across species. hMOR‐1i has an additional 93 amino acids at the tip of the N‐terminus but is otherwise identical to hMOR‐1. When expressed in Chinese hamster ovary cells, the additional 93 amino acids in hMOR‐1i had little effect on opioid binding, but significantly altered agonist‐induced G‐protein activation. hMOR‐1G1 and hMOR‐1G2 predicted six transmembrane domain variants, similar to those seen in mice. The regional expression of these exon 11‐associated variants, as determined by RT‐PCR, varied markedly, implying region‐specific alternative splicing. The presence of exon 11‐associated variants in humans raises questions regarding their potential role in heroin and morphine‐6β‐glucuronide actions in people as they do in mice.

Morphine regulates expression of μ-opioid receptor MOR-1A, an intron-retention carboxyl terminal splice variant of the μ-opioid receptor (OPRM1) gene via miR-103/miR-107.

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 that 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 and Northern blot analyses, we 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 human embryonic kidney 293 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.

A Heroin Addiction Severity-Associated Intronic Single Nucleotide Polymorphism Modulates Alternative Pre-mRNA Splicing of the μ Opioid Receptor Gene OPRM1 via hnRNPH Interactions

Single nucleotide polymorphisms (SNPs) in the OPRM1 gene have been associated with vulnerability to opioid dependence. The current study identifies an association of an intronic SNP (rs9479757) with the severity of heroin addiction among Han-Chinese male heroin addicts. Individual SNP analysis and haplotype-based analysis with additional SNPs in the OPRM1 locus showed that mild heroin addiction was associated with the AG genotype, whereas severe heroin addiction was associated with the GG genotype. In vitro studies such as electrophoretic mobility shift assay, minigene, siRNA, and antisense morpholino oligonucleotide studies have identified heterogeneous nuclear ribonucleoprotein H (hnRNPH) as the major binding partner for the G-containing SNP site. The G-to-A transition weakens hnRNPH binding and facilitates exon 2 skipping, leading to altered expressions of OPRM1 splice-variant mRNAs and hMOR-1 proteins. Similar changes in splicing and hMOR-1 proteins were observed in human postmortem prefrontal cortex with the AG genotype of this SNP when compared with the GG genotype. Interestingly, the altered splicing led to an increase in hMOR-1 protein levels despite decreased hMOR-1 mRNA levels, which is likely contributed by a concurrent increase in single transmembrane domain variants that have a chaperone-like function on MOR-1 protein stability. Our studies delineate the role of this SNP as a modifier of OPRM1 alternative splicing via hnRNPH interactions, and suggest a functional link between an SNP-containing splicing modifier and the severity of heroin addiction.

Stabilization of morphine tolerance with long-term dosing: Association with selective upregulation of mu-opioid receptor splice variant mRNAs

Significance Many assume that morphine tolerance is a continually progressive response, based mainly upon preclinical studies typically lasting a week or less. Yet, clinicians have long appreciated the ability to manage cancer pain in patients with stable opioid doses for months, implying that extended dosing may eventually stabilize the level of tolerance. To reconcile these differences, we examined tolerance over 6 wk and show that extended morphine dosing leads to progressive tolerance for 3 wk that then stabilizes for up to 6 wk and is associated with increases in the abundance of mu opioid receptor splice variant mRNA levels of as much as 300-fold. Chronic morphine administration is associated with the development of tolerance, both clinically and in animal models. Many assume that tolerance is a continually progressive response to chronic opioid dosing. However, clinicians have long appreciated the ability to manage cancer pain in patients for months on stable opioid doses, implying that extended dosing may eventually result in a steady state in which the degree of tolerance remains constant despite the continued administration of a fixed morphine dose. Preclinical animal studies have used short-term paradigms, typically a week or less, whereas the clinical experience is based upon months of treatment. Chronic administration of different fixed morphine doses produced a progressive increase in the ED50 that peaked at 3 wk in mice, consistent with prior results at shorter times. Continued morphine dosing beyond 3 wk revealed stabilization of the level of tolerance for up to 6 wk with no further increase in the ED50. The degree of tolerance at all time points was dependent upon the dose of morphine. The mRNA levels for the various mu opioid receptor splice variants were assessed to determine whether stabilization of morphine tolerance was associated with changes in their levels. After 6 wk of treatment, mRNA levels of the variants increased as much as 300-fold for selected variants in specific brain regions. These findings reconcile preclinical and clinical observations regarding the development of morphine tolerance.

Characterizing exons 11 and 1 promoters of the mu opioid receptor (Oprm) gene in transgenic mice

BackgroundThe complexity of the mouse mu opioid receptor (Oprm) gene was demonstrated by the identification of multiple alternatively spliced variants and promoters. Our previous studies have identified a novel promoter, exon 11 (E11) promoter, in the mouse Oprm gene. The E11 promoter is located ~10 kb upstream of the exon 1 (E1) promoter. The E11 promoter controls the expression of nine splice variants in the mouse Oprm gene. Distinguished from the TATA-less E1 promoter, the E11 promoter resembles a typical TATA-containing eukaryote class II promoter. The aim of this study is to further characterize the E11 and E1 promoters in vivo using a transgenic mouse model.ResultsWe constructed a ~20 kb transgenic construct in which a 3.7 kb E11 promoter region and an 8.9 kb E1 promoter region controlled expression of tau/LacZ and tau/GFP reporters, respectively. The construct was used to establish a transgenic mouse line. The expression of the reporter mRNAs, determined by a RT-PCR approach, in the transgenic mice during embryonic development displayed a temporal pattern similar to that of the endogenous promoters. X-gal staining for tau/LacZ reporter and GFP imaging for tau/GFP reporter showed that the transgenic E11 and E1 promoters were widely expressed in various regions of the central nervous system (CNS). The distribution of tau/GFP reporter in the CNS was similar to that of MOR-1-like immunoreactivity using an exon 4-specific antibody. However, differential expression of both promoters was observed in some CNS regions such as the hippocampus and substantia nigra, suggesting that the E11 and E1 promoters were regulated differently in these regions.ConclusionWe have generated a transgenic mouse line to study the E11 and E1 promoters in vivo using tau/LacZ and tau/GFP reporters. The reasonable relevance of the transgenic model was demonstrated by the temporal and spatial expression of the transgenes as compared to those of the endogenous transcripts. We believe that these transgenic mice will provide a useful model for further characterizing the E11 and E1 promoter in vivo under different physiological and pathological circumstances such as chronic opioid treatment and chronic pain models.