within the opioid
Amynah A Pradhan1,2, Monique L Smith1,2, Brigitte L Kieffer2,3and
Christopher J Evans1,2
1Semel Institute for Neuropsychiatry & Human Behavior, University of California Los Angeles,
Los Angeles, CA, USA,2Shirley and Stefan Hatos Center for Neuropharmacology, UCLA, Los
Angeles, CA, USA, and3Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre
National de la Recherche Scientifique/Institut National de la Santé et de la Recherche
Médicale/Université de Strasbourg, Illkirch, France
Amynah Pradhan, Semel Institute
for Neuropsychiatry & Human
Behavior, Hatos Center for
of California Los Angeles, 760
Westwood Plaza, Los Angeles, CA
90024-1759, USA. E-mail
GPCR; reward; pain; functional
selectivity; receptor biology
24 December 2011
24 May 2012
27 May 2012
The classic model of GPCR activation proposed that all agonists induce the same active receptor conformation. However,
research over the last decade has shown that GPCRs exist in multiple conformations, and that agonists can stabilize different
active states. The distinct receptor conformations induced by ligands result in distinct receptor–effector complexes, which
produce varying levels of activation or inhibition of subsequent signalling cascades. This concept, referred to as
ligand-directed signalling or biased agonism has important biological and therapeutic implications. Opioid receptors are Gi/o
GPCRs and regulate a number of important physiological functions, including pain, reward, mood, stress, gastrointestinal
transport and respiration. A number of in vitro studies have shown biased agonism at the three opioid receptors (m, d and k);
however, in vivo consequences of this phenomenon have only recently been demonstrated. For the m and d opioid receptors,
the majority of reported ligand selective behavioural effects are observed as differential adaptations to repeated drug
administration. In terms of the k opioid receptor, clear links between ligand-selective signalling events and specific in vivo
responses have been recently characterized. Drugs for all three receptors are either already used or are being developed for
clinical applications. There is clearly a need to better characterize the specific events that occur following agonist stimulation
and how these relate to in vivo responses. This understanding could eventually lead to the development of tailor-made
pharmacotherapies where advantageous drug effects can be selectively targeted over adverse effects.
ARM390, N,N-diethyl-4-(phenyl-piperidin-4-ylidenemethyl)-benzamide; BRET, bioluminescence resonance energy
transfer; DAMGO, [D-Ala2, N-MePhe4, Gly-ol]-enkephalin; DMR, dynamic mass redistribution; DPDPE,
[D-Pen2,5]enkephalin, [D-Pen2,D-Pen5]enkephalin; ESCRT, endosomal sorting complex required for transport; FRAP,
fluorescence recovery after photobleaching; GRKs, GPCR kinases; JDTic, (3R)-7-hydroxy-N-((1S)-1-([(3R,4R)-4-(3-
norBNI, norbinaltorphimine; SNC80, (+)-4-[(aR)-a-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-
diethyl benzamide; U50,488, trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide
GPCRs are the most abundant receptor class in the human
genome (Lagerstrom and Schioth, 2008), and as such, these
receptors regulate diverse biological functions. Given their
importance in physiological processes, they are the most
commonly targeted receptor class for pharmacological thera-
pies (Ma and Zemmel, 2002). Classical receptor theory had
postulated that GPCRs existed in equilibrium between an
inactive (R) and an active (R*) state, and that upon binding,
all agonists equally promoted the same subsequent receptor
regulation and signalling cascades (for review, see Kenakin,
2004). However, in the past 15 years, numerous studies have
challenged this idea, and the current view is that the receptor
British Journal of
960 British Journal of Pharmacology (2012) 167 960–969
© 2012 The Authors
British Journal of Pharmacology © 2012 The British Pharmacological Society
can exist in multiple states, and that agonists can initiate
selective receptor conformations, which in turn engage dis-
tinct signalling and receptor regulatory responses (Kenakin,
2011; Reiter et al., 2012). This concept has been referred to in
a number of ways including: ligand-directed signalling, func-
tional selectivity, biased agonism, ligand-biased efficacy, col-
lateral efficacy and stimulus trafficking (Galandrin et al.,
2007; Rajagopal et al., 2010; Vaidehi and Kenakin, 2010).
GPCRs can be modulated at a number of different levels.
Traditionally, it was thought that activation of G-proteins was
the primary way by which GPCRs signalled. This G-protein-
dependent signalling is mediated through Ga and Gbg
subunits, and includes regulation of adenylate cyclase,
phospholipases, multiple kinases and ion channels. More
recently, it has been shown that GPCRs can also mediate
G-protein-independent signalling, through proteins such as
b-arrestins and PDZ-containing proteins (Magalhaes et al.,
2012). b-Arrestin regulated responses are by far the best char-
acterized, and it appears that not only do b-arrestins mediate
receptor trafficking, but they also act as scaffolding molecules
on which a number of signalling cascades are initiated (for
review, see Sorkin and von Zastrow, 2009; Rajagopal et al.,
2010). Thus, functional selectivity may be observed by
ligands promoting G-protein-dependent or -independent sig-
nalling or both. The concept of ligand-directed signalling has
important implications from a therapeutic perspective and
holds the promise of designing drugs to selectively avoid
undesirable biological effects targeted by receptor activation.
This review will focus on ligand-directed signalling within
the family of opioid receptors as an example of GPCRs with
diverse structural and functional ligands and high therapeu-
Opium has been used for many centuries for its medicinal
and euphoric properties, and the use and abuse of this plant
ultimately led to the discovery of the endogenous opioid
system. One of the first breakthroughs in understanding the
unique pharmacology of opium occurred in 1806, when Frie-
drich Wilhelm Serturner isolated the primary active ingredi-
ent in opium and called it morphine, after Morpheus, the god
of dreams (Scott, 1969). The elucidation of the alkaloid struc-
ture of morphine led to the development of the synthetic
opioid heroin, which was found to be more potent than
morphine and even more problematic for triggering addictive
behaviours. Many other opioid agonists have since been char-
acterized, but to date, there are still no commercially avail-
able opioid therapeutics that are both effective analgesics and
free from abuse liability.
The opioid receptor family includes three members: the m,
d and k opioid receptors. The existence of opioid receptors
was discovered in 1973 by three separate groups, all using
opioid radioligand binding in brain homogenates (Pert and
Snyder, 1973; Simon et al., 1973; Terenius, 1973), and genes
encoding m, d and k receptors were subsequently cloned in
the early 1990s (Evans et al., 1992; Kieffer et al., 1992; Chen
et al., 1993; Minami et al., 1993). The m, d, and k opioid
receptors are encoded by Oprm1, Oprd1 and Oprk1 genes,
respectively. Opioid receptors are activated by a family of
naturally occuring endogenous peptides, the first of which
was discovered in 1975, and genes identified in the early
1980s. These neuropeptides, which include the enkephalins,
endorphins and dynorphins, are processed from larger pre-
cursor proteins encoded by Penk, Pdyn and Pomc genes.
Opioid receptors are located throughout the body, and regu-
late a number of important behaviours such as reward, pain,
stress, gastrointestinal transport and mood through receptors
in both the central and peripheral nervous systems (for recent
reviews, see Al Hasani and Bruchas, 2011; Sauriyal et al.,
The three opioid receptors show a high degree of
sequence homology, and a common opioid receptor binding
pocket within the helical transmembrane core has been pos-
tulated based upon modelling and structure activity studies
(Metzger and Ferguson, 1995; Paterlini, 2005). The greatest
divergence in sequence between the receptors occurs at extra-
cellular domains and in vitro studies using mutant receptors
have identified these regions as important for ligand selectiv-
ity (Kane et al., 2006). Likewise, these studies have identified
helical domain-mediated mechanisms for opioid receptor
activation within the membrane core receptor domain
(Decaillot et al., 2003), which is highly similar across the
three receptors. Very early on, in vitro receptor expression in
transfected cells identified the first example of opioid ligand-
directed trafficking (Arden et al., 1995; Keith et al., 1996), and
site-directed mutagenesis experiments also provided indirect
evidence for the existence of multiple active receptor confor-
mations (Befort et al., 1996), preparing the ground for biased
agonism at opioid receptors. As may be inferred from the
breadth of structural diversity of the peptide and alkaloid
agonists that bind to opioid receptors, not all ligands interact
with the same components of the receptor protein. Numer-
ous structure–activity studies have identified key amino acids
in the opioid receptors that selectively disrupt binding and
signalling of some but not all agonists (Kane et al., 2006). The
ligand diversity and therapeutic importance of opioid drugs
makes the opioid receptors excellent model GPCRs to under-
stand the basis of ligand-directed signalling.
Ligand-directed signalling at the m
Ligand directed signalling via the m opioid receptor has
important implications given the wide use of m opioid recep-
tor targeting drugs such as morphine, fentanyl, oxycodone
and heroin both as analgesics and drugs of abuse. In mice
null for the m opioid receptor, morphine loses both its anal-
gesic efficacy and rewarding properties (Matthes et al., 1996;
Contet et al., 2004), as well as many other well-described
biological activities (for review, see Gaveriaux-Ruff and
Kieffer, 2002), demonstrating that this receptor mediates
multiple effects of the prototypic opiate drug throughout
brain circuits. Given their therapeutic importance, agonists
that selectively induce discrete m opioid receptor signalling
complexes could be critical in developing pharmacotherapies
that dissociate m agonist-induced pain relief from reward and
opioids such as tolerance and hyperalgesia (Kieffer and Evans,
2002; Evans, 2004).
As with most GPCRs, ligand binding to the m opioid
receptor can induce receptor internalization, a complex regu-
Ligand-directed signalling at opioid receptors
British Journal of Pharmacology (2012) 167 960–969 961
latory process that can lead to diminished receptor activation
despite the continued presence of ligand [for review see
(Evans, 2004; Kelly et al., 2008)]. m Receptor internalization is
followed by receptor recycling back to the cell surface,
leading to restoration of receptor function (Koch et al., 2005).
Early evidence for agonist-selective trafficking was revealed
by the differential effects of morphine, DAMGO ([D-Ala2,
N-MePhe4, Gly-ol]-enkephalin), and fentanyl on receptor
trafficking in transfected cells, with morphine inducing poor
Further early work indicated differential phosphorylation
upon agonist activation, whereby morphine appeared to
induce little receptor phosphorylation compared to DAMGO
agonist-dependent signalling and desensitization, suggesting
biased responses involving GPCR kinases (GRKs), PKC and
b-arrestins (for review, see Evans, 2004; Kelly et al., 2008).
Recent technological developments have started to
further characterize ligand-directed signalling at the m opioid
receptor. A new approach, fluorescence recovery after pho-
tobleaching (FRAP), has revealed agonist-receptor-specific
biophysical events at the level of the plasma membrane
(Sauliere-Nzeh et al., 2010). In these studies, morphine trig-
gered diffusion of the fluorescent recombinant receptor in
neuroblastoma cells, which was pertussis toxin-sensitive. In
contrast, DAMGO induced a sucrose-dependent aggregation
to small isolated domains for half the receptors, and free
long-range receptor diffusion for the other half. Another
recent approach developed to investigate ligand-directed sig-
nalling at the m opioid receptor measured dynamic mass
redistribution (DMR) of the receptor upon agonist binding.
This promising approach provides real-time optical finger-
prints of GPCR signalling in living cells, and a first heat map
based on the numerical analysis of DMR parameters for
about 50 m ligands under 13 experimental conditions was
recently reported (Morse et al., 2011). This study revealed a
number of novel pharmacological properties for several com-
monly used opioid ligands; including differences in opioid
receptor affinity for specific G-proteins and activation of
divergent signalling cascades (Morse et al., 2011). Although
these studies may not definitively prove the existence of
ligand-directed signalling, they do provide the basis for
Agonist-directed signalling at the m opioid receptor is also
currently being characterized within native cell systems. In
the case of ERK phosphorylation, morphine was shown to
activate ERK pathways in the cytosol via PKCe leading to
ribosomal S6 kinase stimulation. In contrast, etorphine trig-
gered phospho-ERK translocation into the nucleus through a
b-arrestin pathway, modulating Elk-1 and gene expression.
First observed in HEK293 cells (Zheng et al., 2008), this dif-
ferential signalling event was later linked to spine stability
and morphology of hippocampal neurons, where morphine
decreased dendritic spine volume while etorphine, fentanyl
and DAMGO did not (Zheng et al., 2011). In addition, biased
agonism has been observed in primary cultures of dorsal root
ganglia cells where DAMGO but not morphine-activated P38
MAPK. Pharmacological blockade of P38 MAPK disrupted
desensitization of DAMGO but not morphine signalling to
calcium channels (Tan et al., 2009), and this supports previ-
ous data showing DAMGO-induced P38 activation regulates
endocytosis via the early endosomal antigen 1 and Rabeno-
syn5, both components of the early endosome (Mace et al.,
2005). Interestingly, in primary cultures from dorsal root
ganglia, DAMGO and clonidine, but not morphine, induced
cross-desensitization and co-internalization of both m recep-
tors and a2A-adrenoceptors. The cross-desensitization of
adrenoceptors and opioid receptors was disrupted both by the
absence of b-arrestin 2 and the blockade of P38 MAPK, sug-
gesting that biased agonism can also be important in regu-
lating signalling cascades initiated by other GPCRs (Tan et al.,
2009). In the case of morphine, desensitization of the m
opioid receptor in dorsal root ganglia was dependent on
b-arrestin 2, which was not observed for other higher efficacy
agonists (Mittal et al., 2012). Furthermore, whole cell patch
clamp experiments in locus coeruleus slices found that desen-
sitization of the m opioid receptor by morphine was mediated
by PKCa, while DAMGO used a GRK2-dependent mechanism
(Bailey et al., 2009).
A recent behavioural study demonstrated that JNK signal-
ling is selectively involved in morphine but not fentanyl
analgesic tolerance (Melief et al., 2010). Thus, inhibition of
JNK signalling either genetically or pharmacologically pre-
vented acute tolerance to morphine but not fentanyl. Con-
versely, GRK3 knockout mice maintained acute tolerance to
morphine but did not show acute tolerance to fentanyl
(Terman et al., 2004). Together, these data demonstrate the
existence of m agonist-specific signalling mechanisms in the
development of analgesic tolerance, with JNK and GRK3
kinases mediating distinct forms of tolerance. Effector
systems associated with m opioid receptors and the potential
distinct signalling complexes that probably operate in vivo are
schematized in Figure 1.
Finally, knock-in mice expressing a mutant m opioid
receptor that is able to internalize and recycle in response to
morphine showed increased analgesia and reward, and
reduced tolerance, dependence and addictive behaviour (Kim
et al., 2008; Berger and Whistler, 2011). Importantly, mor-
phine but not methadone produced enhanced analgesia and
low tolerance with methadone exhibiting similar effects in
knock-in and wild-type animals (Kim et al., 2008). These
observations show that facilitated receptor internalization
improved the drug profile
internalizing agonist (morphine) and suggest that the high-
internalizing properties of methadone contribute to optimal
analgesic efficacy and duration. m opioid receptor internaliz-
ing agonists and the associated signalling complexes there-
fore represent valuable molecular targets for more effective
analgesics (Berger and Whistler, 2010).
Together, these data demonstrate that agonist-biased sig-
nalling has behavioural consequences. These findings also
strongly suggest that morphine preferably recruits b-arrestin
2-mediated pathways in vivo, and recent evidence suggests
that other m agonists (methadone, fentanyl) may recruit
both b-arrestin 1 and 2 signalling (Groer et al., 2011). It will
be interesting to examine in vivo properties of novel com-
pounds such as herkinorin that, in contrast to all the
known m agonists including morphine, do not recruit
b-arrestin 2 (Tidgewell et al., 2008). Indeed, very recent work
suggests that herkinorin, as compared with morphine,
shows attenuated tolerance following chronic use (Lamb
et al., 2012).
AA Pradhan et al.
962 British Journal of Pharmacology (2012) 167 960–969
Ligand-directed signalling at the d
Compared with the more clinically relevant m opioid receptor
– at least at the time of writing – d opioid receptors have been
relatively understudied. However, recent advances in the
pharmacological and genetic tools used to study this receptor
have revealed its importance in a number of physiological
processes (Pradhan et al., 2011). Stimulation of d opioid recep-
tors does not result in many of the adverse effects associated
with m agonists, including addictive liability (Stevenson et al.,
2005; Codd et al., 2009), respiratory depression (Takita et al.,
1997; Codd et al., 2009) and constipation (Petrillo et al., 2003;
Codd et al., 2009). Although d agonists are poor analgesics in
acute pain (Gallantine and Meert, 2005), they are highly
effective in animal models of chronic inflammatory and neu-
ropathic pain (Fraser et al., 2000; Hurley and Hammond,
2000; Cahill et al., 2003; Nadal et al., 2006; Gaveriaux-Ruff
et al., 2008). Interestingly, d opioid receptors also modulate
emotional state. Genetic deletion of either the d opioid recep-
tor or its endogenous ligand, enkephalin, results in anxio-
genic and depressive-like behaviours (Konig et al., 1996; Filliol
et al., 2000), and d opioid receptor agonists produce anxiolytic
and anti-depressant effects (Broom et al., 2002a; Saitoh et al.,
2004; Perrine et al., 2006).
Agonist activation of the d opioid receptor can initiate
both G-protein-dependent and -independent signalling path-
ways. Agonist-induced activation of the d opioid receptor
leads to receptor desensitization, which for some agonists is
attributed to receptor internalization. d Opioid receptor
internalization has been observed following binding of
endogenous opioids (leu and met-enkephalin), peptides
([D-Pen2,5]enkephalin, [D-Pen2,D-Pen5]enkephalin (DPDPE),
deltorphin) and small molecules (SNC80) (Bradbury et al.,
2009; Pradhan et al., 2009). Unlike internalization of the m
opioid receptor, which results in rapid redistribution of most
receptors back to the cell membrane, d opioid receptors are
predominantly targeted for degradation through the endo-
somal sorting complex required for transport (ESCRT)
machinery (Henry et al., 2011).
Convergent evidence from a number of different in vitro
studies reveals ligand-directed signalling and trafficking of
the d opioid receptor. In bioluminescence resonance energy
transfer (BRET) studies, d opioid receptor ligands did not
equally engage b-arrestin 2. Most ligands were agonists for
inducing G-protein coupling to the receptor but showed vari-
able efficacy for arrestin-receptor interactions. In addition,
ligands that induced strong physical interactions with
G-proteins but weak or no b-arrestin 2 interaction acted as
competitive antagonists for arrestin binding (Molinari et al.,
2010). This subset of ligands could be explained as partial
Ligand-specific signalling complexes at the m opioid receptor. Treatment with morphine or DAMGO elicits differential signalling and trafficking of
the m opioid receptor. Activation of the m opioid receptor by the low-internalizing agonist morphine is thought to result in receptor desensitization
via a b-arrestin and PKC-dependent pathway. In contrast, the high-internalizing agonist DAMGO desensitizes the receptor in a GRK2-dependent
manner and recruits P38 MAPK, which appears to be critical for m opioid receptor desensitization and internalization. Such differences could
feasibly be explained by different receptor conformations that allow similar G-protein activation but different kinase recruitment and hence
desensitization. Further work is needed to explain how these ligand-specific complexes relate to tolerance.
Ligand-directed signalling at opioid receptors
British Journal of Pharmacology (2012) 167 960–969963
agonists for b-arrrestin 2 recruitment, and presumably
arrestin-mediated signalling; however, they did show full
agonist activity in recruiting G-protein subunits. In addition,
it was found that d opioid receptors were in constitutive
complexes with G-proteins, and that each d ligand induced a
specific conformation resulting in divergent activation of
second messenger transducers (Audet et al., 2008). Evidence
from BRET studies may more closely reveal true ligand
directed signalling, as this technique directly measures
protein–protein interactions that can reflect specific ligand-
induced receptor conformations.
Cellular studies examining ligand-specific desensitization
and receptor trafficking have also found biased agonism at
the d opioid receptor. Differential desensitization of the d
opioid receptor was observed following stimulation with
either peptide (DPDPE and deltorphin II) or alkaloid (etor-
phine) agonists (Allouche et al., 1999). Furthermore, each
class of ligand differentially activated kinases (Marie et al.,
2008) and distinctly mediated b-arrestin 1 recruitment
(Aguila et al., 2012) in order to initiate receptor desensitiza-
tion. In addition, agonists differentially regulated sorting of
the d opioid receptor following internalization. In SK-N-BE
cells expressing Flag-tagged human d opioid receptor, the
peptides DPDPE and deltorphin, and SNC80 appeared to
enkephalins and etorphine promoted receptor recycling
(Marie et al., 2003b; Lecoq et al., 2004). Taken together, these
in vitro studies indicate that the d opioid receptor exists in
multiple active conformation states.
Until recently, very little was known about the in vivo
consequences of agonist-induced d opioid receptor traffick-
ing. The development of a knock-in mouse model expressing
fluorescent d opioid receptor (DOR-eGFP) (Scherrer et al.,
2006; 2009) has opened the possibility to correlate ligand-
induced receptor trafficking with receptor function in vivo.
These animals express functional d opioid receptors at physi-
ological levels, which are directly visible in vivo. At behav-
iourally relevant doses, the prototypic agonist SNC80 (Bilsky
et al., 1995) produces internalization of DOR-eGFP through-
out the peripheral and central nervous systems of these
animals (Scherrer et al., 2006; Pradhan et al., 2009; Poole
et al., 2011). Acute administration of SNC80 also reversed
hyperalgesia in a model of inflammatory pain. However, this
initial treatment with SNC80 also produced robust receptor
internalization and G-protein uncoupling resulting in acute
behavioural desensitization (Pradhan et al., 2009). Subse-
quently, chronic treatment with SNC80 resulted in extensive
receptor degradation, as was predicted from in vitro studies,
leading to generalized behavioural tolerance to all agonist
effects (Pradhan et al., 2010) and Figure 2). Contrary to
SNC80, the d agonist ARM390 did not produce detectable
receptor phosphorylation or
agonist binding, although efficacy and potency for G-protein
activation (Marie et al., 2003a; Pradhan et al., 2009), and
analgesic properties (Pradhan et al., 2010) were similar. The
different receptor trafficking properties of ARM390 had
remarkable consequences in vivo. Acute treatment with
ARM390 did not produce behavioural desensitization, and d
opioid receptors remained located on the cell surface and
coupled to G proteins. However, chronic treatment ulti-
mately led to analgesic tolerance, despite unchanged d opioid
receptor number, G-protein coupling and cell membrane
localization. Unlike the generalized tolerance produced by
SNC80, ARM390 produced an analgesic tolerance, probably
due to changes in second messenger responses in pain-
specific pathways (Pradhan et al., 2010 and Figure 2). These
data indicate that ligand-specific trafficking of the d opioid
receptor can produce two distinct types of behavioural toler-
ance. From a therapeutic perspective, these findings imply
that non-internalizing d receptor agonists may be more effi-
cacious for the treatment of diseases unrelated to pain, such
as anxiety and depression.
In other studies examining tolerance following chronic
administration of systemically available d opioid receptor
agonists, analgesic tolerance was not observed in models of
chronic pain (Petrillo et al., 2003; Jutkiewicz et al., 2005;
Beaudry et al., 2009; Codd et al., 2009). To date, very few d
agonists have been characterized at the level of in vivo recep-
tor trafficking, and in order to truly determine the relation-
ship between d opioid receptor internalization and tolerance,
a thorough study examining a number of different ligands
under experimentally controlled conditions needs to be
A major caveat to the development of d agonists is that
some, but not all, d agonists also produce convulsions, an
effect that is specific to the activation of the d opioid receptor
(Broom et al., 2002b; Scherrer et al., 2006). Currently, it is
impossible to screen for this ligand-specific behavioural effect
in vitro. Understanding which signalling pathways mediate
this agonist-selective response would allow for the develop-
ment of in vitro screening tools for novel agonists, thus saving
time, money and the necessary behavioural studies. Impor-
tantly, this type of screen would further encourage the devel-
opment of d opioid receptor agonists for clinical use.
Ligand-directed signalling at the k
k Opioid receptors have been implicated in a number of
physiological responses, including nociception, stress, mood,
feeding, gut motility and diuresis. Therapeutically, k opioid
receptor agonists are being explored as alternatives to m anal-
gesics, as they have low abuse potential and produce minimal
effects on gastrointestinal transit and respiration. In addition,
k agonists may relieve or prevent hyperalgesia produced by
chronic use of m opioid receptor therapies (for review, see
Kivell and Prisinzano, 2010; Vanderah, 2010). However, the
clinical relevance of k agonists is limited as central activation
of k opioid receptors produces dose-dependent dysphoria and
some agonists such as salvinorin A produce hallucinations
(Gonzalez et al., 2006). The endogenous k ligand, dynorphin,
can be released during stress and produce behavioural corre-
lates of dysphoria, depression and anxiety, effects that have
been linked to pro-addictive behaviours and drug relapse (for
review, see Bruchas et al., 2010).
A recent study in mice examining dysphoria induced by k
opioid receptor activation has identified the signalling
pathway through which this behaviour is regulated. Condi-
tioned place aversion to the k agonist U50,488 and stress-
induced reinstatement of drug seeking (probably a result of
AA Pradhan et al.
964 British Journal of Pharmacology (2012) 167 960–969
dynorphin release) was mediated by the specific activation of
P38 MAPK by k opioid receptors in the dorsal raphe nucleus
(Land et al., 2009). In addition, k-opioid receptor activation
of the P38 MAPK pathway in glia also appeared to be impor-
tant for the development of hyperalgesia following peripheral
neuropathy (Xu et al., 2007). k Opioid receptors activate the
P38 MAPK pathway through G-protein-independent signal-
ling via GRK3 and b-arrestins (Bruchas et al., 2006). These
results suggest that the development of k ligands that only
activate G-protein-dependent events may produce analgesia
and circumvent the dysphoric effects. Although no such
ligands presently exist, this is a clear example of how the
characterization of the signalling pathways that mediate
specific behaviours may ultimately be used to tailor drug
A prime illustration of functional selectivity is observed at
the level of k antagonists. Unlike antagonists for the m and d
opioid receptors, certain k opioid receptor antagonists
(antagonist defined as ability to block k agonist activity both
in vitro and in vivo) have an extremely long duration of action.
For example, a single injection of the k selective antagonists,
JDTic, maintains continual blockade of k opioid receptors for
up to 3 weeks (Horan et al., 1992; Carroll et al., 2004; Bruchas
et al., 2007). This duration of action is in sharp contrast to the
pan-opioid receptor antagonist naloxone, which only lasts
for several hours. Recent work has shown that this long
duration of action is mediated by activation of JNK. Surpris-
ingly, norBNI and other long acting antagonists have been
found to activate JNK through the k opioid receptor, and
treatment with these antagonists was found to increase
phospho-JNK levels in the brain and spinal cord in wild-type
mice but not k opioid receptor knockout mice (Bruchas et al.,
2007; Melief et al., 2011). In addition, the administration of
the JNK inhibitor SP600125 blocked the long lasting antago-
nism induced by norBNI on the analgesic effects of the k
agonist U50, 488 (Bruchas et al., 2007). JNK1 in particular
appears to mediate long-term antagonism, as norBNI and
other long-lasting antagonists act as short duration competi-
tive antagonists in JNK1 KO mice (Melief et al., 2010; 2011).
Interestingly, this is a case where a functional antagonist is
not just a classical antagonist in blocking agonist binding but
a ‘collateral agonist’ for the JNK signalling cascade, the acti-
vation of which produces long duration inactivation of k
receptor signalling via a mechanism yet to be elucidated
(Bruchas et al., 2007). Therapeutically, k opioid receptor
antagonists are being developed for the treatment of stress,
anxiety and depression, and as an aid to curb drug relapse.
The behavioural consequences of functional selectivity at the d opioid receptor. SNC80 and ARM390 (ARM) have comparable selectivity and
potencies for the d opioid receptor, but highly distinct internalization properties. Systemic SNC80, but not ARM390, produces clear receptor
internalization in vivo as shown in slices from DOR-eGFP knock-in mice (representative images from the hippocampus and dorsal root ganglia)
(Pradhan et al., 2009). Chronic administration of either agonist produces two distinct forms of tolerance. Repeated administration of SNC80
produces widespread receptor down-regulation, thus resulting in a generalized tolerance where all d agonist-induced behaviours are affected. In
contrast, chronic administration of the low-internalizing agonist, ARM390, appears to affect d opioid receptors only at the level of the dorsal root
ganglia, thus producing tolerance at the level of pain processing (Pradhan et al., 2010).
Ligand-directed signalling at opioid receptors
British Journal of Pharmacology (2012) 167 960–969965
Understanding the mechanisms regulating the unique kinet-
ics of k opioid receptor antagonists has important implica-
tions for future drug design.
It should be recognized that beyond ligand-directed signal-
ling, there are multiple mechanisms that could readily
mediate differential in vivo activities of drugs targeting opioid
receptors. Factors such as intrinsic drug efficacy, pharmaco-
dynamics, drug selectivity and ligand accessibility to selective
receptor populations probably explain the differential effects
of many drugs. In addition, the ability of a ligand to differ-
entially activate splice variants of receptors or selectively acti-
vate homo- or heterodimeric receptor complexes are also
possibilities that may masquerade as ligand-directed signal-
ling (reviewed by Evans, 2004). Overall, biased agonism is
one of the many mechanisms by which opioid ligands could
produce diverse physiological effects.
The concept of biased agonism has profound implica-
tions, both in terms of understanding the complexity of
GPCR pharmacology and for facilitating drug development
(Bosier and Hermans, 2007; Galandrin et al., 2007). The very
recent crystal structures of all three opioid receptors with
ligands in place promises to visualize different receptor con-
formations as a result of different ligand interactions (Granier
et al., 2012; Manglik et al., 2012; Wu et al., 2012). The ability
of ligands to discretely activate particular signalling path-
ways, which may in turn regulate specific in vivo responses,
opens the possibility of separating desirable from adverse
drug effects. However, the evidence for this phenomenon is
primarily based on in vitro experiments using recombinant
cell systems, with only a few studies demonstrating clear
agonist-selective activity in vivo. A major challenge in GPCR
research will be to demonstrate the physiological relevance of
agonist-biased signalling and regulation. There is clearly a
need for further studies to bridge the gap between in vitro
findings showing differential agonist signalling cascades and
the in vivo behavioural consequence of signalling specificity.
In the opioid receptor field, the concept of biased agonism in
vivo is emerging, and the promise of biased m agonists that are
better analgesics with less abuse liability, or d and k agonists
with targeted and sustained efficacy in the treatment of pain
and mood disorders has now been validated in animal
models. Future drug development will be required to deter-
mine the clinical relevance of these findings.
All authors were supported by NIH-NIDA Grant DA05010 and
the Shirley and Stefan Hatos Research Foundation. AAP was
additionally supported by NIH-NIDA grant K99DA031243.
BK was also supported by the CNRS, INSERM, the Université
de Strasbourg and the French ANR grant IMOP. All drug and
molecular target (e.g. receptors, ion channels) nomenclature
conforms to the British Journal of Pharmacology Guide to Recep-
tors and Channels (Alexander et al., 2011).
Conflict of interest
The authors declare no conflict of interest in the preparation
of this manuscript.
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