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.

Mu opioid receptors play an important role in mediating the actions of a class of opioids including morphine and heroin. Binding and pharmacological studies have proposed several mu opioid receptor subtypes: mu₁, mu₂, and morphine-6β-glucuronide (M6G). The cloning of a mu opioid receptor, MOR-1, has provided an invaluable tool to explore pharmacological and physiological functions of mu opioid receptors at the molecular level. However, only one mu opioid receptor (Oprm) gene has been isolated. Alternative pre-mRNA splicing has been proposed as a molecular explanation for the existence of pharmacologically identified subtypes. In recent years, we have extensively investigated alternative splicing of the Oprm gene, particularly of the mouse Oprm gene. So far we have identified 25 splice variants from the mouse Oprm gene, which are controlled by two diverse promoters, eight splice variants from the rat Oprm gene, and 11 splice variants from the human Oprm gene. Diversity and complexity of the Oprm gene was further demonstrated by functional differences in agonist-induced G protein activation, adenylyl cyclase activity, and receptor internalization among carboxyl terminal variants. This review summarizes these recent results and provides a new perspective on understanding and exploring complex opioid actions in animals and humans.

The rat μ-opioid receptor clone in which novel exon 5 was found in the place of exon 4 (MOR-1B) was one of the first MOR-1 variants described. We now have identified the mouse homolog of the rat MOR-1B as well as four additional variants derived from splicing from exon 3 into different sites within exon 5. The sequences of all of the variants were identical except for the intracellular tip of the C terminus encoded by exon 5, where each variant predicted a unique amino acid sequence ranging from 2 to 39 amino acids. All of the mMOR-1B variants were selective for μ-opioids in receptor-binding assays, as anticipated, because they all have identical binding pockets defined by the transmembrane domains. However, the relative potency and efficacy of μ-agonists to each other varied from variant to variant in guanosine 5′-O-(3-[³⁵S]thio)triphosphate-binding studies, as shown by morphine-6β-glucuronide, which was the most efficacious agent against mouse MOR-1B1 (mMOR-1B1) and the least efficacious agent against mMOR-1B2. mMOR-1B4 was quite unusual. Although mMOR-1B4 was μ-selective in receptor-binding studies and antagonists labeled mMOR-1B4 well, the binding affinities of most of the μ-agonists were far lower than those seen with mMOR-1, suggesting that the 39 amino acids at the C terminus of mMOR-1B4 influences the conformation of the receptor and its ligand recognition site itself either directly or through its interactions with other proteins. In conclusion, alterations in the amino acid sequence of the C terminus do not alter the μ-specificity of the receptor but they can influence the binding characteristics, efficacy, and potency of μ-opioids.

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.

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.