Splice variation of the mu-opioid receptor and its effect on the action of opioids

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British Journal of Pain
2014, Vol. 8(4) 133 –138
© The British Pain Society 2014
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DOI: 10.1177/2049463714547115
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Introduction
There is a growing appreciation of the complexity 
affecting an individual’s response to opioids, as well as 
a greater understanding of the differential response 
that individuals may have to different opioids. The 
complexity of an individual’s response to opioids is 
influenced not only by genetic and molecular factors 
but also by phenotypic determinants such as psychoso-
cial factors, gender and pain sensitivity. Although great 
efforts are being made to understand these factors, we 
have yet to identify predictable data that can contribute 
to a clinically meaningful treatment algorithm for per-
sonalising the prescribing of opioids. Such an algo-
rithm would help to guide the prescribing of opioids so 
that patients receive the opioid analgesics that are most 
suited to them.
Researching the factors that influence an individu-
al’s response to opioids involves investigating a wide 
range of parameters that include:
1. Genetic factors – deoxyribonucleic acid (DNA) 
sequence, genetic variation, gene–gene interac-
tion and splice variation.
Splice variation of the mu-opioid 
receptor and its effect on 
the action of opioids
Sophy K Gretton1 and Joanne Droney2
Summary points 
1. An individual’s response to opioids is influenced by a complex combination of genetic, molecular and 
phenotypic factors.
2. Intra- and inter-individual variations in response to mu opioids have led to the suggestion that mu-
opioid receptor subtypes exist.
3. Scientists have now proven that mu-opioid receptor subtypes exist and that they occur through a 
mechanism promoting protein diversity, called alternative splicing.
4. The ability of mu opioids to differentially activate splice variants may explain some of the clinical 
differences observed between mu opioids.
5. This article examines how differential activation of splice variants by mu opioids occurs through 
alternative mu-opioid receptor binding, through differential receptor activation, and as a result of the 
distinct distribution of variants located regionally and at the cellular level.
Keywords
Pain, opioid, OPRM1, splice variation, splice variants, mu-opioid receptor, mu opioids, genetic, 
pharmacogenetic
1St Peter’s Hospice, Bristol, UK
2The Royal Marsden NHS Foundation Trust, London, UK
Corresponding author:
Sophy K Gretton, St Peter’s Hospice, Charlton Road, Brentry, 
Bristol BS10 6NL, UK. 
Email: sgretton@gmail.com
547115 BJP0010.1177/2049463714547115British Journal of PainGretton and Droney
research-article2014
Original Article
134 British Journal of Pain 8(4)
2. Pharmacokinetic and pharmacodynamic factors 
– absorption, distribution, metabolism and 
excretion of opioids, drug activation of the 
receptor, influence of receptor dimerisation and 
incomplete cross tolerance.
3. Characterisation of pain phenotype, including 
type and chronicity of pain, pain sensitivity and 
brain connectivity.
4. Clinical factors – disease status, concomitant 
medication, biochemical and psychosocial fac-
tors, diet and so on.
5. Epigenic factors – the dynamic post-translational 
modifications that affect activation and activity 
of gene products.
A number of reviews have looked at the strength of 
evidence for many of these factors.1–5 In this review, we 
focus on the impact of splice variation of the mu-opioid 
receptor (MOR) on the clinical effect of opioids.
Mu opioids activate MORs
At the heart of an individual’s clinical response to opi-
oids is the binding of an opioid to, and activation of, an 
opioid receptor. The majority of opioids in clinical use 
are termed ‘mu opioids’ due to their selectivity for 
MOR in receptor binding assays. Morphine is the pro-
totypical mu opioid but other well-known mu opioids 
include codeine, diamorphine, morphine-6-glucuronide, 
oxycodone, fentanyl and methadone. Their interaction 
with the MOR is responsible for both analgesic effect 
and adverse events, such as respiratory depression, hal-
lucinations, itch and constipation.
Intra- and inter-individual variation in 
response to mu opioids
Clinicians have long been aware that a patient may 
respond differently to two mu opioids; a patient might 
achieve adequate pain relief and be spared side effects 
with one mu opioid, but they may continue to have 
pain and experience intolerable side effects with 
another. Furthermore, clinicians recognise that between 
individuals there may be a differential response to a 
given mu opioid, whereby one patient may benefit 
while another does not. These intra- and inter-individual 
variations in response to mu opioids have led to the 
suggestion that MOR subtypes exist. Without sub-
types, it is difficult to explain how supposedly similar 
drugs acting on the same receptor could give rise to 
such variable responses.
MOR subtypes
Mu, delta and kappa opioid receptors were discovered 
in the 1970s.6–8 They belong to the super-family of 
seven transmembrane G-protein coupled receptors; 
they were among the first neurotransmitter receptors 
to be identified and their discovery led to the detection 
of endogenous opioid peptides.9–12 Opioid receptor 
subtypes were first proposed in the 1980s when bind-
ing assays suggested the presence of multiple classes of 
binding site for opiates and enkephalins,13 and subse-
quent pharmacological studies supported this possibil-
ity.13–18 However, when the mu, delta and kappa opioid 
receptors were cloned in the early 1990s, they were 
found to originate from distinct opioid receptor genes, 
OPRM1,19–22 OPRD123,24 and OPRK1, respec-
tively.25–27 This discovery left scientists wondering how 
a single MOR gene, OPRM1, could generate a protein 
product that enabled such variable clinical response. 
Twenty years on, scientists have proven not only that 
MOR subtypes exist but also that they occur through a 
mechanism promoting protein diversity, called alterna-
tive splicing.28–30
Alternative splicing
‘Alternative splicing’ is the term given to the process in 
which a single gene gives rise to multiple protein prod-
ucts. This is made possible during the transcription of 
DNA into messenger ribonucleic acid (mRNA). DNA 
is comprised of genes, and genes are composed of 
exons, separated by introns; exons are the actual DNA 
sequences included in the translation of mRNA into 
the protein product, whereas introns are regions of 
DNA that are usually spliced out and removed during 
the process. ‘Splicing’ is the process of combining 
exons together, and ‘alternative splicing’ is the inclu-
sion of different combinations of exons within mRNA. 
Alternative splicing gives rise to different protein prod-
ucts contributing to protein diversity (Figure 1).
Alternative splicing explains how only a relatively 
small number of genes in the human genome (approxi-
mately 20,000–30,000) give rise to many more protein 
products; it is thought that approximately 60% of genes 
produce at least one alternative mRNA.31
MOR splice variants
The MOR was cloned in 1993 from a single gene, 
OPRM1.19–22 MOR1 is the name given to the primary 
MOR transcript, that is, the major form of OPRM1. 
MOR1 comprises four exons that encode architectur-
ally distinct regions of the receptor. Exon 1 codes for 
the extracellular amino terminus and first transmem-
brane domain, exons 2 and 3 code for the remaining 
six transmembrane domains and exon 4 codes for the 
final 12 amino acids of the intracellular carboxyl termi-
nus.32 (Figure 2)
Soon after its discovery, the importance of MOR1 
in morphine analgesia was confirmed in a series of 
Gretton and Droney 135
studies involving MOR1 gene knockout mice mod-
els.33More was learned later of its structure and func-
tion using the novel technique of ‘antisense mapping’, 
whereby individual exons could be targeted and 
degraded. These animal studies demonstrated that tar-
geting exon 1 resulted in a loss of morphine activity, 
despite preserving analgesia from diamorphine and 
morphine-6-glucuronide (M6G). Moreover, antisense 
probes targeting exons 2 and 3 had little effect on mor-
phine action, whereas they significantly diminished the 
activity of diamorphine and M6G.34,35 These findings 
have since been confirmed in exon-specific knockout 
mice models,36 strongly supporting the early assertion 
that diamorphine and M6G exert their pharmacologi-
cal actions through receptor mechanisms distinct from 
those of morphine.37 The possibility that these effects 
were being mediated via kappa- or delta-opioid recep-
tors was eliminated when diamorphine and M6G-
mediated analgesia persisted in a triple knockout 
mouse model.36
To date, nearly 20 human MOR splice variants have 
been characterised.28,38–40 Broadly speaking, these vari-
ants fall into two categories: variants associated with 
exon 1 and those associated with exon 11. Both exons 
are controlled by distinct promoters, which regulate 
the expression of their respective variants.
Exon 1–associated splice variants
The majority of exon 1–associated variants involve 
exon 4 being replaced by alternative exons. These vari-
ants give rise to full-length seven transmembrane 
G-protein coupled receptors. Because of this, the 
Figure 1. Alternative splicing occurs during transcription of DNA into mRNA, leading to an increased diversity of protein 
products arising from a single gene.
Figure 2. The MOR is a G-protein-coupled receptor. It 
has an extracellular amino terminus and an intracellular 
carboxyl tail. The receptor spans the cell membrane 
seven times resulting in three intracellular and three 
extracellular domains. The extracellular loops and the 
extracellular amino chain are important for ligand binding. 
The second and third intracellular loops as well as the 
carboxyl tail are important in the interaction between the 
receptors and G-proteins.
136 British Journal of Pain 8(4)
structure of the binding pocket (formed by folding of 
the seven transmembrane domains) is identical and the 
variants demonstrate similar affinity and selectivity for 
mu opioids in receptor binding assays. However, struc-
tural differences in the intracellular carboxyl terminus 
affect signal transduction following receptor activation. 
Binding assays in human41 and mice MOR splice vari-
ants42 reveal major differences in potency and efficacy 
among these variants. Moreover, there is poor correla-
tion between receptor activation and binding affinity, 
and the order in which mu opioids exert their maximal 
effect differs according to the variant.42 Thus, the abil-
ity of mu opioids to differentially activate splice vari-
ants may explain some of the clinical differences 
observed between the various mu opioids.
Exon 11–associated splice variants
Exon 11 is located upstream of exon 1, and novel exon 
11–associated splice variants have been identified in 
mice43, rats44 and humans.45 These variants account 
for approximately one-quarter of the total expression 
of MOR1 and its variants at the level of mRNA and 
protein in the whole brain of mice. Evidence from ani-
mal models suggest that exon 11–associated variants 
may be functionally and pharmacologically important, 
for example, disruption to exon 11 in mice has shown 
a significant reduction in the analgesic action of 
diamorphine, M6G and fentanyl, whereas morphine- 
and methadone-mediated analgesia were unaffected.40
MOR variants display regional specificity within the cen-
tral nervous system. Several of the exon 11–associated 
variants generate receptor proteins that are structur-
ally identical to MOR1. However, they differ from 
MOR1, and from each other, in their distribution and 
expression within specific regions of the central ner-
vous system (CNS).43,46 This level of regulation sug-
gests differential regional processing even among 
variants. Indeed, the regulation of MOR splice vari-
ants appears to be highly complex. There is evidence 
for cell-specific splicing, in which different cells 
express different splice variants,47 and for splice vari-
ants influencing the position of a receptor pre- and 
post-synaptically within a cell.48
Truncated variants form heterodimers to generate phar-
macologically active targets. A proportion of the exon 
11–associated variants predict proteins that lack exon 1. 
These variants encode proteins with six transmembrane 
domains rather than the full complement of seven. As 
described above, truncated exon 11–associated variants 
show regional mRNA distribution43 and they are impli-
cated in diamorphine and M6G-mediated analgesia.40 
However, they also appear to generate pharmacologi-
cally active targets through heterodimerisation. A study 
in mice demonstrated a novel naltrexone-derived agent 
binding to, and activating, the heterodimer formed 
between an exon 11–associated splice variant and a sec-
ond receptor protein, which may be the orphanin FQ 
receptor, ORL1. Not only did this study demonstrate 
the role of truncated variants in forming novel therapeu-
tic targets, but, by activation of the heterodimer, the 
novel agent was able to bring about analgesia without 
mediating any opioid-related side effects. These findings 
suggest that it may be possible to dissociate the analgesic 
properties of receptor activation from unwanted side 
effects.49
Splice variants encode single 
transmembrane protein products
Finally, a third class of variant has been identified com-
posed only of exons 1 and 4. This variant encodes a 
protein product with a single transmembrane domain. 
The functional importance of these variants has yet to 
be understood; however, there is evidence to show that 
single transmembrane variants modulate MOR1 
expression levels in the endoplasmic reticulum by 
dimerisation with the full-length seven-transmembrane 
MOR1.50
MOR splice variants in the clinical 
context
A small number of human studies have examined the 
impact of MOR subtypes in the context of clinical con-
ditions. In a study of CNS cell lines from the subjects 
infected with human immunodeficiency virus (HIV), 
researchers demonstrated that there are indeed specific 
differences in the MOR variant expression profile 
among CNS cell types (astroglia, microglia and neu-
rons), and that the expression levels of the these vari-
ants are differentially regulated by HIV type 1. 
Importantly, the data suggest that expression of MOR 
variants may be differentially regulated in the brains of 
HIV-infected subjects with varying levels of neurocog-
nitive impairment.51
The association between MOR splice variants and 
opioid-related side effects has also been examined; 
Chinese women undergoing gynaecological surgery 
were genotyped for single nucleotide polymorphisms 
(SNPs) in the OPRM1 gene. Results have shown an 
association between a SNP in the splice variant, 
MOR1X, and occurrence of fentanyl-induced emesis 
in the postoperative setting.52
Furthermore, in a model of mice with ‘morphine-
induced scratching’ (MIS), researchers propose that a 
MOR variant, MOR1D, is essential for MIS, whereas 
the major protein transcript of OPRM, MOR1, is nec-
essary for mediating analgesia and that these processes 
may be independent of each other.53
Gretton and Droney 137
Conclusion
The pharmacology of mu opioids is highly complex. So 
too is an individual’s clinical response to opioids. It is 
clear now that MOR subtypes exist and that they 
might, in part, explain why there is intra- and inter-
individual variation in response to opioids.
A growing body of evidence suggeststhat MOR 
splice variants are functionally and pharmacologically 
active, and that differential activation of variants by mu 
opioids occurs through alternative binding, through 
differential receptor activation and as a result of the 
distinct distribution of variants located regionally and 
at the cellular level.
We have learned that, in MOR splice variants, exon 
1 is necessary for morphine analgesia whereas exons 2 
and 3 are required for activity of diamorphine and 
M6G,34,35 and that novel exon 11 is important in medi-
ating activity of diamorphine, M6G and fentanyl but 
not morphine or methadone.40 Furthermore, heterodi-
mers of truncated variants have been shown to form 
novel therapeutic targets, and activation of novel tar-
gets has made it possible to dissociate analgesic effect 
from adverse effect.49 These data are exciting because 
they shed light on potential mechanisms underpinning 
differential pharmacological properties of the mu-
opioid system, and raise the possibility that novel ther-
apeutic agents might be engineered to activate analgesic 
targets, or be designed on the basis of their relative 
activation profile rather than simply on their binding 
affinity.
However, there remains an enormous challenge to 
turn this data into clinically meaningful algorithms to 
benefit patients. We will need to understand more 
about splice variants, realise their pharmacological and 
functional potential and establish to what extent this 
mechanism contributes to the overall clinical response 
of an individual to a given mu-opioid agonist such as 
morphine.
Conflict of interest
The authors declares that there is no conflict of interest.
Funding
This research received no specific grant from any funding 
agency in the public, commercial or not-for-profit sectors.
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