Elsevier

Neuroscience

Volume 133, Issue 1, 2005, Pages 209-220
Neuroscience

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

https://doi.org/10.1016/j.neuroscience.2004.12.033Get rights and content

Abstract

The μ 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 μ receptor subtypes, but only one μ 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 μ 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 μ opioid receptor family with limited differences in μ opioid ligand affinities and selectivity. However, adenylyl cyclase and [35S]GTPγS 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.

Section snippets

Primer design

Using Vector NTI (Informax, Bethesda, MD, USA), we identified potential mouse homologs of exon 5 in the human Oprm gene using a sequence alignment with an approximately 7.7 kb mouse genomic sequence containing exons 5a, 5b, 5c, 5d and 5e, an approximately 7.3 kb rat and a corresponding approximately 8 kb human corresponding genomic sequence obtained from the genome databases (NBCI and Ensembl). Three sets of antisense primers were designed based upon the alignment (see below). One of the

Cloning new splice variants from the human Oprm gene

To isolate potential human MOR-1 variants homologous to those identified in mice, we designed three sets of human antisense primers based upon the human genomic sequence alignment with the mouse and rat exon 5′s and then used these antisense primers together with two sense primers from 5′UTR of exon 1 in an RT-PCR strategy. We identified six new variants, hMOR-1B1, hMOR-1B2, hMOR-1B3, hMOR-1B4, hMOR-1B5 and hMOR-1Y, from Be(2)C, a human neuroblastoma cell line that expresses high level of μ

Discussion

Our previous studies revealed that the mouse Oprm gene undergoes extensive splicing to generate multiple MOR-1 variants (Bolan et al 2004, Pan et al 1999, Pan et al 2000, Pan et al 2001), with similar splicing patterns in the rat (Pasternak et al., 2004). A question arose as to whether the same complex splicing existed in the human Oprm gene. Recently, we isolated two splice variants, hMOR-1O and hMOR-1X, from the human Oprm gene (Pan et al., 2003), in addition to hMOR-1A which had been

Acknowledgments

This work was supported, in part, by a research grant to Y.-X.P. (DA13997), and a research grant and a Senior Scientist Award to G.W.P. (DA02615 and DA00220) from the National Institute on Drug Abuse and by a Core Grant to MSKCC from the National Cancer Institute (CA8748).

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