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

Abstract

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 [ ]enkephalin (DAMGO) and antagonists such as b-funaltrexamine also are highly selective for these receptors and have proven useful tools in exploring the pharmacology of these receptors. Evidence suggesting the existence of mu opioid receptor subtypes goes back several decades (Pasternak and Snyder 1975;Wolozin and Pasternak 1981), having initially been based upon detailed receptor binding approaches and the synthesis of antagonists highly selective for a subpopulation of mu opioid binding sites (Pasternak and Hahn 1980;Hahn et al. 1982). In vivo, these antagonists dissociated morphine analgesia from other actions, such as respiratory depression, the inhibition of gastrointestinal transit and even many of the components of dependence (Ling et al. 1984(Ling et al. , 1985(Ling et al. , 1986Heyman et al. 1988). In addition, differences among the mu opioids have also been noted. Clinicians have long appreciated the wide range of responses to different mu analgesics among their patients, leading to the recognition of the need to individualize therapy for patients. In addition to differences in the dose of drug needed to be effective among patients, the relative potencies of the drugs to each other also vary among patients. These observations can be observed in animal models as well. For

Morphine Regulates Expression ofμ-Opioid Receptor MOR-1A, an Intron-Retention Carboxyl Terminal Splice Variant of theμ-Opioid Receptor (OPRM1) Gene via miR-103/miR-107

“…Similar to other OPRM1 splice variants, mMOR-1A mRNA is differentially expressed in various brain regions . When expressed in Chinese hamster ovary cells, mMOR-1A and rMOR-1A display high m binding affinity and selectivity (Bolan et al, 2004;Pasternak et al, 2004;Xu et al, 2013). However, they revealed marked differences in agonistinduced total G protein stimulation determined by guanosine 59-O-(3-[ 35 S]thio)triphosphate binding as compared with other C-terminal splice variants (Bolan et al, 2004;Pasternak et al, 2004;Xu et al, 2013), suggesting the functional significance of the C-terminal tails in agonist-induced G protein coupling and signaling transduction.…”

“…All the full length C-terminal splice variants have identical transmembrane domains, which are encoded by exons 1, 2, and 3 and comprise the binding pocket, but contain a different intracellular C-terminal tail encoded by alternative 39 exons. Growing evidence suggests the functional importance of these C-terminal splice variants based upon region-specific expression (Abbadie et al, 2000a(Abbadie et al, ,b, 2001Pan et al, 1999Pan et al, , 2001), agonist-induced G protein coupling (Bolan et al, 2004;Pasternak et al, 2004;Pan et al, 2005a,b), receptor phosphorylation (Koch et al, 2001), internalization (Koch et al, 1998(Koch et al, , 2001, postendocytic sorting (Tanowitz et al, 2008), and morphine-induced itch .…”

“…The first reported C-terminal splice variant was the human MOR-1A (hMOR-1A) (Bare et al, 1994), followed soon afterward by the mouse and rat homologs, mMOR-1A and rMOR-1A (Bolan et al, 2004;Pasternak et al, 2004) (Fig. 1).…”

Alternatively spliced mu opioid receptor C termini impact the diverse actions of morphine

“…The truncations had little effect on ED 50 values, but significantly reduced DAMGO-and morphine-induced maxi- ]DAMGO), a mu agonist, with high affinity and with only subtle differences in selectivity for the endogenous opioid peptides dynorphin A and β endorphin (18,25,26). However, in [ 35 S]GTPγS-binding assays, these C-terminal splice variants displayed marked differences in mu opioid-induced G protein coupling in both potency (EC 50 ) and efficacy (% of maximal stimulation) (26,27), suggesting that the distal carboxyl terminal sequences influence mu agonist-induced receptor-G protein coupling and signal transduction. Multiple protein kinase phosphorylation sites are predicted from various alternative C-terminal sequences, and in vitro studies revealed differences in mu agonist-induced phosphorylation, internalization, and postendocytic sorting among several C-terminal splice variants (28,29).…”

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

“…This inability to reconcile the diverse pharmacology of -opioids with their binding selectivity led to the proposal of multiple -opioid-receptors (7), a concept initially proposed based on binding and pharmacological studies in animal models and subsequently confirmed with the identification of a large number of splice variants of the cloned -opioid-receptor MOR-1 in mice, rats, and humans ( Fig. 1A) (8)(9)(10)(11)(12)(13)(14)(15).…”