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Evidence that Behavioral Phenotypes of Morphine in
b-arr2/Mice Are Due to the Unmasking of JNK Signaling
Nitish Mittal
1,2
, Miao Tan
2
, Onyemachi Egbuta
2
, Nina Desai
2
, Cynthia Crawford
1
, Cui-Wei Xie
2
,
Christopher Evans
2
and Wendy Walwyn*
,2
1
Department of Psychology, California State University, San Bernardino, CA, USA;
2
Department of Psychiatry and Biobehavioral Sciences, Stefan
Hatos Center for Neuropharmacology, Semel Institute, University of California, Los Angeles, CA, USA
The altered behavioral effects of morphine, but not most other mu agonists, in mice lacking b-arrestin 2, suggest that this scaffolding
protein regulates the signaling cascade of this commonly used analgesic. One of the cascades that could be regulated by b-arrestin 2 is
cJun-N-terminal kinase (JNK), which binds with b-arrestin 2 and modulates the analgesic effects of morphine. Using neurons lacking
b-arrestin 2 (b-arr2/) to examine this interaction, we found that b-arr2/neurons show altered intracellular distribution of JNK and
cJun, and that morphine, but not fentanyl, increased the nuclear localization of the phosphorylated, therefore activated, form of cJun, a
JNK target in dorsal root ganglia neurons. This suggests that deleting b-arrestin 2 affects the JNK cascade. We therefore examined
whether some of the behavioral phenotypes of mice lacking b-arrestin 2 could be a result of altered JNK signaling. Indeed, two different
JNK inhibitors reversed the enhanced analgesic effect of morphine, a known phenotype of b-arr2/mice, to + / + levels. Both the
reduced locomotor effect of morphine and the psychomotor sensitization to repeated morphine administration in b-arr2/mice were
also returned to + /+ levels by inhibiting JNK. In contrast, the behavioral effects of fentanyl were neither genotype-dependent nor
affected by JNK inhibition. Furthermore, a PKC inhibitor had a similar effect as inhibiting JNK in reducing the enhanced analgesic effect of
morphine in b-arr2/mice to + / + levels. In summary, removing b-arrestin 2 reveals mu receptor activation of the JNK cascade in a
ligand-specific manner explaining several behavioral phenotypes of b-arr2/mice.
Neuropsychopharmacology (2012) 37, 1953–1962; doi:10.1038/npp.2012.42; published online 11 April 2012
Keywords: mu opioid receptor; JNK; b-arrestin 2; morphine; analgesia; locomotion
INTRODUCTION
One of the many examples that the ubiquitously expressed
arrestins serve as more than terminators of receptor
signaling through initiating receptor internalization (Groer
et al, 2011; Shenoy and Lefkowitz, 2011; Shukla et al, 2011)
is the phenotype of mice lacking b-arrestin 2 (b-arr2/;
Bohn et al, 1999, 2002; Raehal and Bohn, 2011). As
morphine does not induce significant internalization of the
mu opioid receptor, the effects of morphine in b-arr2/
mice cannot be explained by this prototypical role of b-
arrestin 2. However, some progress has recently been made
in revealing several arrestin-dependent, yet internalization-
independent, signaling pathways of the mu opioid receptor
that explain some, but not all, effects of morphine in mice
lacking b-arrestin 2 (Arttamangkul et al, 2008; Dang et al,
2011; Quillinan et al, 2011; Walwyn et al, 2007).
Observations of ligand-specific signaling, or the ability of
different ligands to activate specific effectors, were first
made in the 1980s (Gee and Yamamura, 1983), but only
recently have become an accepted dimension of G-protein-
coupled receptor (GPCR) signaling (Reiter et al, 2012;
Urban et al, 2007). Such functional selectivity is believed to
result from the ability of different agonists of the same
GPCR to induce ligand-specific conformational changes of
the receptor. These distinct conformations recruit different
proteins to the agonist-bound receptor and activate down-
stream signaling cascades in an agonist-specific manner
(Galandrin et al, 2008). The opioid family of GPCRs are no
exception to such diversity; ligands of the mu, delta, and
kappa opioid receptors are known to activate functionally
distinct signaling cascades (Bruchas and Chavkin, 2010;
Melief et al, 2010; Peng et al, 2009; Pradhan et al, 2010).
The enhanced analgesic effect of morphine but not many
other mu agonists in mice lacking b-arrestin 2 suggests that
morphine, in activating a signaling cascade that is regulated
by b-arrestin 2, differs from other agonists (Bohn et al,
1999, 2002). As b-arrestin 2 is a multi-functional scaffolding
Received 13 September 2011; revised 29 February 2012; accepted 5
March 2012
*Correspondence: Dr W Walwyn, Department of Psychiatry and
Biobehavioral Sciences, Stefan Hatos Center for Neuropharmacology,
Semel Institute, University of California, Los Angeles, CA 90095, USA,
Tel: + 1 310 206 3231, Fax: + 1 310 825 7067,
E-mail: wwalwyn@ucla.edu
Neuropsychopharmacology (2012) 37, 1953 – 1962
&
2012 American College of Neuropsychopharmacology. All rights reserved 0893-133X/12
www.neuropsychopharmacology.org
and signal transduction protein (Xiao et al, 2007, 2010), the
signaling cascade responsible for such ligand specificity
remains unknown. However, the arrestins bind with, and
regulate, many components of the map kinase cascade, one
of which is the stress-activated protein kinase, cJun-N-
terminal kinase (JNK), a kinase that has recently been shown
to play an important role in ligand-directed signaling of the
mu and kappa opioid receptors (Melief et al, 2010).
As JNK binds with b-arrestin 2, this scaffolding protein
may modulate mu receptor activation of the JNK cascade
in a ligand-specific manner. Using mice lacking b-arrestin 2
to determine how this scaffolding protein regulates mu
receptor signaling, we have found that altered JNK activity
explains some of the behavioral phenotypes of mice lacking
b-arrestin 2.
MATERIALS AND METHODS
Animals
b-Arrestin 1 (also known as arrestin 2) or b-arrestin 2 (also
known as arrestin 3) mice, fully back-crossed into the C57Bl/
6 background, were kindly supplied by Dr Lefkowitz (HHMI,
University of North Carolina, Chapel Hill, NC). The
behavioral experiments used equal numbers of adult male
and female b-arrestin (b-arr) 1/2 + / + and /mice that
were 81±10 days old, 22.3±3.7g in weight and bred from
heterozygous matings. For the cellular experiments, early
postnatal b-arr2 + / + and /pups were obtained from
homozygous matings, one generation from heterozygous
breeders. All animal experiments were conducted in accor-
dance with the Guide for the Care and Use of Laboratory
Animals and followed institutionally approved animal care
and use protocols.
SDS–PAGE and Western Blot Analysis of JNK and
Phospho-JNK Proteins Levels
Dorsal root ganglia (DRGs) were collected from all spinal
levels of either b-arr2 + / + or /adult mice, weighed and
homogenized in the following lysis buffer; 20 mM Tris–HCl
pH 7.4, 0.32 M sucrose, 1 mM EDTA, 1 mM EGTA, 50 mM
NaF, 1 mM Na
4
P
2
O
7
,1Halt Protease and Phosphatase
Inhibitor Cocktail (Pierce, Thermo Scientific, Rockford, IL),
and protein content determined (BCA, Pierce). Protein
samples (50 mg/sample) were separated by SDS–PAGE (10%
NuPAGE gel; Invitrogen, Carlsbad, CA) and transferred
to an Immobilon PVDF membrane (0.45 mm; Millipore,
Billerica, MA). After several washes in TBS and a 120 min
blocking step (Casein/0.1% Tween-20, Thermo Scientific),
the membrane was incubated in anti-JNK or anti-phospho-
JNK at 1 : 100 (Thr183/Tyr185), from both Cell Signaling
(Danvers, MA) or glyceraldehyde 3-phosphate dehydrogen-
ase (GAPDH; 2.5 10
2
mg/ml, Thermo Scientific), o/n at
41C. The membrane was then washed in TBS with 0.1%
Tween-20, incubated in Stabilized Peroxidase-conjugated
secondary antibodies, anti-rabbit IgG for JNK (1 : 100) or
anti-mouse IgG for phospho-JNK (1 :100), or GAPDH
(1 : 10 000) for 120 min at RT (Pierce, Rockford, IL). The
labeled protein bands were visualized by chemilumines-
cence (Super signal Chemiluminescent Substrate, Pierce),
and signal intensity (pixels/mm
2
) quantified (ImageJ, NIH).
For each sample, the signal intensity of JNK or phospho-
JNK was normalized to that of GAPDH. Statistical analysis:
The mean of the three experiments for each of the six
samples are expressed as the mean±SEM and the differences
between groups determined by one-way ANOVA (Analyse-it
Software, Leeds, UK) with significance accepted at po0.05.
Cell Culture
DRGs from all spinal levels were harvested from early postnatal
(p0–1) b-arr2+/+ or /pups. After chemical dissociation
in trypsin (Invitrogen) and physical dissociation by trituration,
110
4
cells were plated on poly-D-lysine (Sigma, St Louis,
MO) and laminin (Invitrogen) coated coverslips of MatTek
dishes (MatTek, Ashland, MA) as previously described
(Walwyn et al, 2007). The cells were maintained for 24–48 h
in Neurobasal/B27 media containing NGFs (10 ng/ml) in vitro
after which the cells were treated and fixed for immunocy-
tochemistry or used to determine mu agonist inhibition of
voltage-dependent Ca
2+
channels (VDCCs).
Immunocytochemistry
b-arr2 + / + or /cells remained untreated or were
treated with morphine (NIDA) or fentanyl (Sigma) for
10 min, washed and fixed. After a series of washes, a 0.3%
Triton X100 permeabilization step, and a 10% serum
blocking step, anti-JNK3 (1 : 500, Santa Cruz Biotechnology,
Santa Cruz, CA) or anti-phosphorylated JNK (anti-phos-
pho-JNK: 1 : 100, Cell Signaling) were added. In a second,
matching, set of plates, primary antibodies against cJun
(1 : 500, Santa Cruz Biotechnology) and phosphorylated
cJun (anti-phospho-cJun, 1 : 250, Santa Cruz Biotechnology)
were added. After o/n incubation and further washes,
Alexa-555-conjugated donkey anti-goat/mouse (1 : 1000 for
phospho-cJun and phospho-JNK; Invitrogen) and Alexa-
488-conjugated donkey anti-rabbit/goat (1 : 1000 for cJun and
1 : 400 for JNK; Invitrogen) were added to each plate for
90 min at RT. After a final series of washes, the cells were
mounted in Prolong containing 40,6-diamidino-2-phenylin-
dole (DAPI; Invitrogen). Medium-sized DRGs (20–30 mM)
were visualized by a 60 oil immersion objective on a
Nikon TE2000 microscope and images were captured by a
Retiga 1300Exi CCD camera (QImaging, British Columbia,
Canada). All cells were imaged at the mid-nuclear level
using DAPI as a guide. Pixel intensity per unit area for each
of the three channels (RGB) was quantified by IPLab
(version 3.6.5, BioVision Technologies, Exton, PA). Statis-
tical analysis: Data from untreated cells were expressed as
mean±SEM of the nuclear/cytosolic signal intensity for
4100 cells/sample. The effect of different mu agonists on
cJun and phospho-cJun localization was determined by
comparing the percent change of the nuclear:cytosolic ratio
in the agonist treated vs untreated cells. Differences between
groups were determined using two-way ANOVA between
groups (Analyse-it Software) and significance accepted at
po0.05.
Analgesia
Thermal nociception was measured by the warm-water tail-
immersion assay in which the latency to remove the tail
Arrestin and JNK regulation of mu receptor function
N Mittal et al
1954
Neuropsychopharmacology
from a 49.5 1C water bath was measured. After a basal
response was obtained (average of three to four tests), one
of the JNK or PKC inhibitors, or matching vehicle, was
injected, another basal measurement was obtained 30 (PKC
inhibitor) or 60 (JNK inhibitor) min later and then
morphine or fentanyl was administered. The JNK inhibitors
used were: SP600125 (SP6; Anthra[1,9-cd]pyrazol-6(2H)-
one, 1,9-pyrazoloanthrone, 30 mg/kg i.p., EMD Chemicals,
Merck KGaA, Darmstadt, Germany) or BI78D3 (4-(2,3-
Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(5-nitrothiazol-2-ylthio)
-4H-1,2,4-triazol-3-ol) 25 mg/kg i.p., EMD Chemicals). Both
compounds inhibit the activation of all JNK isoforms by
binding with either the ATP docking site (SP6; Shin et al,
2002) or the D-domain consensus sequence in JNK to inhibit
the docking of upstream kinases (BI78D3; Stebbins et al,
2008). The PKC inhibitor used, Bisindolylmaleimide VIII
(Ro-31-7549, EMD Chemicals), inhibits PKCa,bI, bII, g,e
(Wilkinson et al,1993).Statistical analysis: Data are
expressed as the response in seconds (s), and as the mean±
SEM of each group. Differences between groups were deter-
mined by one- or two-way ANOVA with repeated measures
and factorial analysis at each time interval using Stat View
(v5). Significance was accepted at po0.05.
Locomotion
Locomotor activity was measured in 16.7 12.7 cm
2
boxes by
an infrared video-camera and Ethovision video tracking
software (v7.1; Noldus, Tacoma). The mice were habituated
for 15 min on the first day and, on each of the following 3
days, were given SP6 (30 mg/kg i.p.), or vehicle, 60 min before
morphine (10mg/kg s.c.) or fentanyl (0.2 mg/kg s.c.) and
placed in the locomotor boxes. Locomotion was assessed
over the following 30 min. Statistical analysis: Data are
expressed as mean±SEM and differences between groups
determined by one- or two-way ANOVA with repeated mea-
sures and factorial analysis at each time interval (Stat View).
Electrophysiology
Voltage-dependent Ca
2+
currents were recorded from
small–medium-sized DRG neurons 20–30 mmindiameter
under whole-cell voltage-clamp conditions as previously
described (Tan et al, 2003; Walwyn et al, 2007). Cells were
perfused with an external solution containing 10 mM
CaCl
2
, 130 mM tetraethylammonium chloride, 5 mM
HEPES, 25 mM d-glucose and 0.25 mMtetrodotoxinatpH
7.35. The patch electrode was filled with an internal
solution composed of 105 mM CsCl, 40 mM HEPES, 5 mM
d-glucose, 2.5 mM MgCl
2
, 10 mM EGTA, 2 mM Mg-ATP,
and 0.5 mM GTP at pH 7.2. Ca
2+
currents were evoked
every 10 s by 40-ms voltage steps from 80 to + 10 mV
using an Axopatch 200B patch-clamp amplifier. Capaci-
tance and series resistance were corrected with the
compensation circuitry on the amplifier. Series resistance
was compensated by 80–90%. Leak currents were sub-
tracted using a P/6 protocol. Recorded signals were
acquiredandanalyzedusingAxonpClampv8.0or9.0
software (Axon Instruments, Foster City, CA). Drug
application and desensitization protocols: The JNK in-
hibitor, SP6, was dissolved in DMSO as a 10-mM stock
solution and diluted with the culture medium to a final
concentration of 10 mM for application. The effect of SP6
on mu receptor–VDCC inhibition was assessed by a 4-h
pre-incubation with SP6 (10 mM). For the desensitization
experiments, b-arr2 + / + and /DRG neurons were
pre-incubated with 1 mM morphine, D-Ala
2
-MePhe
4
-Glyol
5
Enkephalin (DAMGO), or fentanyl, in the presence or
absence of 10 mM SP6 for 4 h, the media removed, and the
cells were washed by an B100 solution exchange before
being challenged by the full agonist, DAMGO (1 mM).
Statistical analysis: Mean Ca
2+
current amplitudes were
measured (pCLAMP 9.0; Axon Instruments, CA) between 5
and 10 ms after initiating the depolarizing step. Mean
current amplitudes were then plotted against time. Stable
recordings were fitted by a linear function to compare, by
extrapolation, control current amplitude to the current amplitude
recorded in the presence of opioid receptor agonists or SP6.
Data are expressed as mean±SEM and were compared using
ANOVA with a post hoc Tukey’s test with significance accepted
at po0.05. Recordings that exhibited marked rundown
(430%) were discarded as were significant outliers, five out
of the B300 + recordings (Prism v5.03).
RESULTS
DRG Neurons Lacking b-Arrestin 2 Show Altered JNK
and cJun Localization
JNK and phospho-JNK expression. As b-arrestin 2 facil-
itates GPCR activation of the JNK cascade (Willoughby
and Collins, 2005), genetically deleting b-arrestin 2 may
result in a compensatory adaptation in the expression
of JNK and phospho-JNK. This was examined by
SDS–PAGE and western blotting in b-arr2/and + / +
adult DRGs. No effect of genotype was found. The relative
intensities of the protein of interest normalized to that
of GAPDH were: JNK; 48 kDa: b-arr2 + / + ; 0.39±0.05,
b-arr2/: 0.33±0.05.54 kDa: b-arr2 + / + ; 0.23±0.06,
b-arr2/: 0.16±0.04. phospho-JNK; 54 kDa: b-arr2 + / + ;
0.61±0.13, b-arr2/: 0.64±0.04.
The basal intracellular location of JNK and cJun. As b-
arrestin 2 contains a nuclear export sequence resulting in its
constitutive exclusion from the nucleus (Song et al, 2006),
b-arr2/neurons may show altered nuclear or cytosolic
localization of proteins, such as JNK, that bind with b-
arrestin 2. We therefore examined the effect of deleting b-
arrestin 2 on the relative nuclear:cytosolic ratio of JNK,
phospho-JNK, and the major JNK target, cJun, and
phospho-cJun. b-arr2/DRG neurons showed a basal
increase in the nuclear localization of JNK, phospho-JNK,
and cJun, but not phospho-cJun (Figure 1b).
Ligand-induced changes in phospho-cJun location. We
next examined whether two mu agonists, morphine and
fentanyl, previously shown to activate the JNK cascade
(Melief et al, 2010) could alter the intracellular location of
phospho-cJun in b-arr2 + / + and /neurons. We focused
on phospho-cJun for a number of reasons; the activity of
this terminal kinase of the JNK pathway is finely regulated
by JNK (Hibi et al, 1993) and, importantly, the basal
location of phospho-cJun was unaffected by deleting
b-arrestin 2. We found that morphine increased the relative
Arrestin and JNK regulation of mu receptor function
N Mittal et al
1955
Neuropsychopharmacology
intensity of phospho-cJun in the nucleus of b-arr2/but
had no effect in + / + neurons. Fentanyl did not alter
phospho-cJun localization in + / + or /neurons
(Figure 1c). Neither ligand altered the nuclear:cytosolic
ratio of cJun (data not shown).
JNK Inhibition Reverses Behavioral Phenotypes of
Morphine in b-arr2/Mice
Using DRG neurons as a model, we have found altered basal
and mu agonist-induced changes in the intracellular
location of JNK and/or cJun, suggesting that deleting
b-arrestin 2 alters aspects of the JNK cascade. As this
cascade can modulate the behavioral effects of mu agonists
(Chen et al, 2008; Melief et al, 2010), we next examined
whether the behavioral phenotypes of b-arr2/mice
could be a result of altered JNK activity.
Analgesia. The role of JNK in thermal analgesia was
examined by administering one of two JNK inhibitors, SP6
or BI78D3, to b-arr2/and + / + mice, 60 min before
morphine (5 and 10 mg/kg s.c.) or fentanyl (0.2 mg/kg s.c.).
JNK
1.00
1.05
1.10
1.15
1.20 β-arr2+/+
β-arr2–/–
i cJun p-cJunp-JNK
a
ii iii iv
β-arr2+/+
β-arr2–/–
*
β-arr2+/+
β-arr2–/–
1.00
1.10
1.20
1.30
1.40
1.50
1.60 β-arr2+/+
β-arr2–/–
*
*
b
pcJun
cJun DAPI Merge
β-arr2–/–β-arr2+/+ β-arr2+/+ β-arr2–/– β-arr2–/–β-arr2+/+ β-arr2+/+ β-arr2–/–
Nuclear/Cytosolic
iii
cJun p-cJun
Cytosol 563 506
Nucleus 707 860
1.00
1.05
1.10
1.15
1.20
Nuclear/Cytosolic
Nuclear/Cytosolic
Nuclear/Cytosolic
1.00
1.05
1.10
1.15
1.20
1.25
***
85
90
95
100
105
110
β-Arr2–/–
110
β-Arr2+/+
85
90
95
100
105
Morphine
Fentanyl
i
c
ii
Morphine
Fentanyl
Nuclear/Cytosolic
(% untreated)
p-cJun
Nuclear/Cytosolic
(% untreated)
Figure 1 DRG neurons as a model to examine the JNK cascade. DRG neurons from early postnatal mice were used to examine the effect of deleting b-
arrestin 2 and different mu receptor agonists on the relative nuclear:cytosolic ratio of JNK and cJun. (a) The relative pixel intensity/unit area of the nuclear or
cytosolic labeling for JNK, phospho-JNK, cJun, or phospho-cJun was imaged and quantified in individual neurons. An example of the labeling and quantification
for one cell are shown in (ai, aii), respectively. (b) This method was used to quantify the nuclear:cytosolic ratio of JNK, phospho-JNK, cJun, and phospho-cJun,
in untreated b-arr2 + / + vs /neurons (n4100 neurons/sample). b-arr2/neurons showed a relative increase in the nuclear localization of JNK,
phospho-JNK, and cJun, (*po0.05 vs b-arr2 +/+), but not phospho-cJun (p¼0.75). (c) While morphine treatment (1 mM, 10 min) did not affect the
nuclear:cytosolic ratio of phospho-cJun in b-arr2 + / + neurons, this mu agonist did increase phospho-cJun in the nucleus vs cytosol of b-arr2/neurons.
.***po0.001 vs untreated. Scale bars ¼10 mm.
Arrestin and JNK regulation of mu receptor function
N Mittal et al
1956
Neuropsychopharmacology
Thermal analgesia was assessed by the warm-water tail-
immersion assay 20–30 min later and at regular intervals
thereafter. In b-arr2 + / + mice, neither of the JNK
inhibitors altered the analgesic profile of morphine, which
showed a comparable dose-response curve to previous
publications (Sora et al, 1997; Bohn et al, 2000). In contrast,
both SP6 and BI78D3, reversed the enhanced analgesic
effect of morphine in b-arr2/mice to wild-type levels
(Figure 2a and b). The analgesia induced by fentanyl was
neither genotype-dependent nor influenced by JNK. In
addition, the higher basal analgesia of b-arr2/
(2.19±0.05 s) vs + / + (2.03±0.05 s, po0.05) mice was
not affected by either SP6 or BI78D3. As PKC has been
shown to activate JNK (Lopez-Bergami et al, 2005; Melief
et al, 2010), we next examined whether the effect of
morphine in b-arr2/mice was also PKC-dependent. The
PKC inhibitor, Bisindolylmaleimide VIII, reversed the
enhanced effect of morphine in b-arr2/mice to wild-
type levels (Figure 2c). We also examined whether
b-arrestin 1 may be responsible for the effect of JNK in
b-arr2/mice but found no effect of deleting b-arrestin
1 or inhibiting JNK in the analgesic response to morphine
in these mice (Figure 2e).
Psychomotor effects of morphine
Acute locomotion:Another of the behavioral phenotypes
of b-arr2/mice is the reduced locomotor effect of
morphine (Bohn et al, 2003; Urs et al, 2011). We
hypothesized that, similar to the analgesic effect of
morphine, this may also be due to altered JNK activity.
The JNK inhibitor, SP6, or vehicle, was therefore adminis-
tered 60 min before assessing the locomotor effects of
morphine or fentanyl in b-arr2 + / + and /mice. SP6
had no effect in b-arr2 + / + mice, but SP6-treated /
mice showed an enhanced locomotor response to morphine
matching that of b-arr2 + / + mice (Figure 3a). By contrast,
the locomotor effect of fentanyl was neither genotype nor
SP6-dependent (Figure 3b).
Psychomotor sensitization:As the locomotor effect of a
single morphine injection was influenced by both genotype
and JNK inhibition, locomotor sensitization induced by
repeated morphine administration could similarly be
modulated by JNK in b-arr2/mice. We found that 3
days of repeated morphine administration resulted in less
locomotor sensitization in b-arr2/than + / + mice
(10 mg/kg; Figure 4a). This was reversed by SP6, adminis-
tered 60 min before morphine on each of the 3 test days
(Figure 4b). The locomotor sensitization induced by
repeated fentanyl administration was not influenced by
deleting b-arrestin 2 or inhibiting JNK (Figure 4c and d).
Possible cellular targets of the JNK cascade. Inhibiting JNK
reduced the analgesic efficacy of morphine 30 min after this
drug was administered. This suggests that JNK phosphor-
ylation of cytosolic targets, rather than the transcriptional
activity of cJun, modulates morphine analgesia. We there-
fore examined whether opioid coupling with VDCCs, a
Gbg-mediated second messenger cascade, may be influ-
enced by JNK in a ligand-specific manner. We examined
both the acute effect of inhibiting JNK on mu receptor–
VDCC coupling as well as desensitization of opioid
a
d
b
e
Morphine (10 mg/kg)
i
2
4
6
8
10
12
14
0
–60 0 30 60 90 120
Time (min)
Vehicle
SP6
BI78D3
ii
2
4
6
8
10
12
14
0
–60 0 30 60 90 120
Time (min)
Vehicle
SP6
BI78D3
i
0
2
4
Time (s)
Time (s)
Time (s)
Time (s)
Time (s)
Time (s)
Time (s)
Time (s)
Time (s)
Time (s)
6
8
10
12
–60 0 30 60 90 120
Time (min)
Morphine (5 mg/kg)
β-arrestin 2 +/+
β-arrestin 1+/+ β-arrestin 1–/–
β-arrestin 2–/ –
Vehicle
SP6
ii
0
2
4
6
8
10
12
–60 0 30 60 90 120
Time (min)
Vehicle
SP6
i
0
2
4
6
8
–60 0 20 40 60 80
Time (min)
Fentanyl (0.2 mg/kg)
Vehicle
SP6
ii
0
2
4
6
8
–60 0 20 40 60 80
Time (min)
Vehicle
SP6
i
0
2
4
6
8
10
12
–60 0 30 60 90 120
Time (min)
Morphine (10 mg/kg)
Vehicle
SP6
0
2
4
6
8
10
12
–60 0 30 60 90 120
Time (min)
ii
Vehicle
SP6
cii
2
4
6
8
10
12
14
0
–30 0 30 60 90 120
Time (min)
Vehicle
Bis-8
i
2
4
6
8
10
12
14
0
–30 0 30 60 90 120
Time (min)
Vehicle
Bis-8
Morphine (10 mg/kg)
Figure 2 The enhanced thermal analgesic effect of morphine in b-arr2/
mice is reduced to + / + levels by inhibiting JNK or PKC. The warm-water
(49.5 1C) tail-immersion assay was used to assess the analgesic effects of
different mu agonists in b-Arr2 + / + and /mice and the resultant data are
presented as the response (s; yaxis) over time (min; xaxis). (a, b) b-Arr2/
mice showed a dose-dependent increase in the thermal analgesic effects of
morphine ((a) 5 and (b) 10 mg/kg morphine s.c., 5 mg/kg: p¼0.12 vs b-arr2 + /
+, F
1,5
¼1.773; 10 mg/kg: po0.001 vs b-arr2+/+, F
1,5
13.227), which was
reversed by the JNK inhibitor, SP6 (aii) 5 mg/kg: po0.05 vs vehicle, F
1,5
¼2.422;
(bii) 10 mg/kg: po0.01 vs vehicle, F
1,5
¼4.501). BI78D3 (bii), similarly reversed
the enhanced analgesia seen in morphine-treated (10 mg/kg s.c.) b-arr2/
mice (po0.001 vs vehicle, F
1,5
¼6.142). Neither JNK inhibitor altered the b-arr2
+ / + response ((ai) 5 mg/kg: p¼0.06 vs vehicle, F
1,5
¼10.74; (aii) 10 mg/kg:
SP6:p¼0.94 vs vehicle, F
1,5
¼0.457; BI78D3: p¼0.71 vs vehicle, F
1,5
¼0.575)
(c) The PKC inhibitor Bisindolylmaleimide VIII (10 mg/kg i.p.) also reversed the
enhanced analgesia seen after morphine administration (10 mg/kg s.c.) in b-
arr2/mice (po0.001 vs vehicle, F
1,5
¼7.099), while having no effect in b-
arr2 + / + mice (p¼0.76 vs vehicle, F
1,5
¼0.937). (d) Mice treated with
fentanyl (0.2 mg/kg s.c.) showed no effect of the b-arrestin 2 deletion
(p¼0.84 vs b-arr2 + / + , F
1,5
¼0.403) or JNK inhibition (b-arr2 + / + :
p¼0.59 vs vehicle, F
1,5
¼0.748; b-arr2/:p¼0.96 vs vehicle, F
1,5
¼0.194).
(e) There was also no effect of deleting b-arrestin 1 (p¼0.10 vs b-arr2 + / + ,
F
1,5
¼1.883) or JNK inhibition on the morphine response of b-arr1/or
+/+ mice (SP6:b-arr1 + / + : p¼0.07 vs vehicle, F
1,5
¼2.089; b-arr1/:
p¼0.80 vs vehicle, F
1,5
¼0.224). Injection 1 ¼JNK or PKC inhibitor, or
matching vehicle. Injection 2 ¼morphine or fentanyl.
Arrestin and JNK regulation of mu receptor function
N Mittal et al
1957
Neuropsychopharmacology
receptor–VDCC inhibition following 4 h of opiate pre-
treatment. We found no effect of SP6 pre-treatment on the
acute VDCC inhibition induced by morphine, fentanyl or
DAMGO in either b-arr2 + / + or /neurons (Table 1).
However, desensitization induced by each of the ligands
revealed both genotype and JNK effects, as shown by a
1-mM test dose of DAMGO. In b-arr2 + / + neurons, the
desensitization induced by morphine (4 h, 1 mM) was
prevented by SP6 co-incubation. In b-arr2/neurons,
morphine did not induce significant desensitization, and
this was unaffected by SP6 co-incubation. Pre-incubation
with either DAMGO or fentanyl resulted in significant
desensitization in both b-arr2 + / + and /neurons that
was not altered by SP6.
DISCUSSION
When compared with other mu agonists, morphine induces
less internalization of the mu opioid receptor (Borgland
et al, 2003; McPherson et al, 2010; Zheng et al, 2011). This
suggests that the prototypical role of b-arrestin 2 in
initiating receptor internalization does not explain how
the effects of morphine, but not of other mu agonists, are
altered in mice lacking b-arrestin 2. Here, we demonstrate
that morphine activation of JNK in the absence of b-arrestin
2 explains ligand-specific behavioral phenotypes of b-
arr2/mice. These findings also demonstrate that the
JNK cascade may be activated in a ligand-specific manner,
providing further evidence of the role of this member of the
map kinase pathway in mediating ligand-directed signaling
of the mu receptor.
Compared with other mu agonists such as fentanyl, the
relative inability of morphine to cause significant receptor
phosphorylation, results in the recruitment of PKC to
morphine, but not fentanyl, bound mu receptors (Zheng
et al, 2011). As PKC is known to both scaffold with and
phosphorylate JNK (Lopez-Bergami et al, 2005; Ping, 2003;
Pontrelli et al, 2004), we propose that in wild-type mice,
morphine recruitment of PKC activates JNK but that b-
arrestin 2 recruitment limits further JNK activation. How-
ever, if b-arrestin 2 is absent and is coupled with the
inability of morphine to recruit b-arrestin 1 (Groer et al,
2011), sufficient PKC recruitment results in sustained
activation of the JNK cascade in b-arr2/neurons to
affect analgesia. In contrast, other mu agonists, such as
fentanyl differ from morphine as they are able to
phosphorylate the mu receptor and recruit either b-arrestin
1 or 2 (Groer et al, 2011; Zheng et al, 2011). In the absence
of b-arrestin 2, these agonists recruit b-arrestin 1, PKC
is not recruited and the JNK cascade is not significantly
activated.
Time (min)
Morphine 10 mg/kg
a
0
1000
2000
3000
4000
5000
6000
Total distance traveled (cm)
Vehicle
SP6
Fentanyl 0.2 mg/kg
b
0
500
1000
1500
2000
2500
5 1015202530
Distance traveled (cm)
Time (min)
0
2000
4000
6000
8000
10000
12000
14000
Total distance traveled (cm)
Vehicle
SP6
i
ii
β-arrestin 2 +/+
β-arrestin 2 −/−
0
200
400
600
800
1000
1200
1400
1600
5 1015202530
Distance traveled (cm)
Vehicle
SP6
ii
0
200
400
600
800
1000
1200
1400
1600
5 1015202530
Distance traveled (cm)
Vehicle
SP6
β-arrestin 2 −/−
βarr2+/+ βarr2−/−
0
500
1000
1500
2000
2500
5 1015202530
Distance traveled (cm)
iβ-arrestin 2 +/+
Vehicle
SP6
Vehicle
SP6
βarr2+/+ βarr2−/−
Time (min)
Time (min)
*
iii
iii
Figure 3 The reduced locomotor effect of morphine in b-arr2/mice is returned to + / + levels by inhibiting JNK. The locomotor effect of different
mu agonists in b-arr2 + / + and /mice is shown over the duration of the 30-min test in the line graphs with distance traveled (cm; yaxis) shown over
time (xaxis). The total distance traveled (cm; yaxis) is shown in the accompanying bar graph. (a) The locomotor effect of morphine was less in b-Arr2/
mice compared with their wild-type littermates (po0.01 vs b-arr2 + / + , F
1,11
¼14.251). SP6 (30 mg/kg i.p.) increased the effect of morphine in b-arr2/
mice (po0.001 vs vehicle, F
1,11
¼19.835) to + /+ levels (p¼0.52 vs b-arr2 + / + , F
1,14
¼0.429) but had no effect in b-arr2 + / + mice (p¼0.46 vs vehicle,
F
1,14
¼0.577). (b) Mice treated with fentanyl (0.2 mg/kg s.c.) showed no effect of genotype (p¼0.81 vs b-arr2 + / + , F
1,9
¼0.061) or JNK inhibition (SP6:b-
arr2 + / + : p¼0.68 vs vehicle, F
1,12
¼0.169; b-arr2/:p¼0.16 vs Vehicle, F
1,11
¼2.194).
Arrestin and JNK regulation of mu receptor function
N Mittal et al
1958
Neuropsychopharmacology
i
a
c
i
b
0
500
1000
1500
2000
2500
5 1015202530
Distance traveled (cm)
Time (min)
Day 1
Day 3
0
500
1000
1500
2000
2500
5 1015202530
Distance traveled (cm)
Time (min)
Day 1
Day 3
0
2000
4000
6000
8000
10000
Total distance traveled (cm)
Day 1
Day 3
0
2000
4000
6000
8000
10000
Total distance traveled (cm)
Day 1
Day 3
ii
0
2000
4000
6000
8000
10000
Total distance traveled (cm)
Day 1
Day 3
0
500
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1500
2000
2500
5 1015202530
Distance traveled (cm)
Time (min)
Day 1
Day 3
0
500
1000
1500
2000
2500
5 1015202530
Distance traveled (cm)
Time (min)
Day 1
Day 3
0
2000
4000
6000
8000
10000
Total distance traveled (cm)
Day 1
Day 3
0
1000
2000
3000
4000
5 1015202530
Distance traveled (cm)
Time (min)
0
5000
10000
15000
20000
Total distance traveled (cm)
Day 1
Day 3
0
1000
2000
3000
4000
5 1015202530
Distance traveled (cm)
Time (min)
Day 1
Day 3
0
5000
10000
15000
20000
Total distance traveled (cm)
Day 1
Day 3
0
5000
10000
15000
20000
Total distance traveled (cm)
Day 1
Day 3
d
ii
ii
ii
Vehicle - Morphine
β-arrestin 2 +/+ β-arrestin 2 −/−
SP6 - Morphine
Vehicle - Fentanyl
SP6 - Fentanyl
0
1000
2000
3000
4000
51015202530
Distance traveled (cm)
Time (min)
iDay 1
Day 3
Day 1
Day 3
1
3
Day
Day
0
5000
10000
15000
20000
Total distance traveled (cm)
0
1000
2000
3000
4000
5 1015202530
Distance traveled (cm)
Time (min)
i
Day 1
Day 3
**
**
***
*
*
Figure 4 The reduced psychomotor effect of morphine in b-arr2/mice is similarly returned to + / + levels by inhibiting JNK. These data are
presented in the same format as in Figure 2 showing both the distance traveled over time in the line graphs and total locomotion in the accompanying bar
graphs (ai) A 3-day protocol of successive morphine (10 mg/kg s.c.) treatment of b-arr2+ /+ mice resulted in an enhanced locomotor response to the same
dose of morphine by day 3 (po0.01 day 1 vs day 3, F
1,5
¼16.972). (aii) Conversely, similarly treated b-Arr2/mice showed no such increase (p¼0.41
day 1 vs day 3, F
1,6
¼0.765). (bi) Administration of SP6 (30 mg/kg i.p.), 60 min before each morphine injection had no effect on the locomotor sensitization in
b-arr2 + / + mice (po0.05 day 1 vs day 3, F
1,9
¼6.866) but reversed the lack of sensitization in the b-arr2/mice (po0.05 day 1 vs day 3, F
1,5
¼6.989) to
wild-type levels. (c, d) Mice treated with fentanyl (0.2 mg/kg s.c.) showed locomotor sensitization (po0.001 day 1 vs day 3, F
1,23
¼63.235), that was
unaffected by b-arrestin 2 deletion or SP6 administration (vehicle; p¼0.93 day genotype, F
1,23
¼0.007, SP6; p¼0.25 day genotype injection,
F
1,23
¼1.382). *po0.05, **po0.01.
Arrestin and JNK regulation of mu receptor function
N Mittal et al
1959
Neuropsychopharmacology
b-Arrestin 2 has been shown to bind with JNK3, but not
JNK1 or 2, to control GPCR activation of the JNK cascade
(McDonald et al, 2000; Miller et al, 2001; Song et al, 2006,
2009; Willoughby and Collins, 2005). Removing b-arrestin 2
may therefore alter both the location and control of JNK3 to
allow sustained JNK activation in b-arr2/mice. If JNK3
were indeed involved, this is a different isoform to the
JNK2-dependent regulation of receptor uncoupling follow-
ing repeated morphine administration (Melief et al, 2010).
In our experiments, acute mu receptor coupling with
VDCCs was unaffected by JNK inhibition, suggesting that
neither the mu receptor nor Gbg-coupled Ca
2+
channels are
downstream targets of the JNK activity unmasked by
deleting b-arrestin 2. In contrast, SP6 modulation of mu
receptor–VDCC desensitization in b-arr2 + / + neurons
shows a JNK dependency (Figure 5) that could be involved
in the JNK2 regulation of mu receptor–effector coupling
and morphine desensitization in vivo (Melief et al, 2010).
This raises an interesting hypothesis that the same isoform,
or possibly different JNK isoforms, may regulate different
signaling cascades of the mu receptor depending on
whether b-arrestin 2 is present or absent and the protocol
of morphine application.
The effects of morphine on locomotion and subsequent
sensitization in rodents are considered a model of the initial
phase of addiction in humans (Robinson and Berridge,
1993; Vanderschuren and Pierce, 2011). This response
requires D1 receptors, the phosphoprotein DARPP32
(Becker et al, 2001; Borgkvist et al, 2007; Urs et al, 2011)
and ERK (Borgkvist et al, 2008; Urs et al, 2011). We, and
others, have shown that the locomotor effect of morphine is
blunted in mice lacking b-arrestin 2 (Bohn et al, 2003; Urs
et al, 2011). As morphine induces the formation of a
b-arrestin 2/ERK complex in D1 neurons, it has been
suggested that this complex mediates the hyperlocomotor
effect of morphine (Urs et al, 2011). In using pharma-
cological tools to inhibit JNK in b-arrestin 2/mice, we
have shown that activation of the JNK cascade in the
absence of b-arrestin 2 explains the reduced locomotor
response of morphine in these mice. Taken together, these
data and those of Caron and colleagues (Urs et al, 2011)
suggest that b-arrestin 2 plays complementary roles in
this paradigm, one prevents JNK activation whereas the
other allows a b-arrestin/ERK complex to form in D1
neurons. We propose that both roles synergize, and
are necessary for the characteristic locomotor effect of
morphine to occur.
Although others have found that sensitization to morphine
is not b-arrestin 2-dependent (Bohn et al, 2003), we have
found that this context-specific response to multiple doses of
morphine is reduced in mice lacking b-arrestin 2. However,
our assay differed in several important ways; we examined
the first 30 min after morphine (30 vs 120 min), after 3 rather
than 9 days of repeated morphine, and our sensitization
protocol was context-associated. The initial loss of such
‘short-term’ sensitization in b-arr2/mice may therefore
not reflect a lack of, but rather an attenuation of the onset of
sensitization that may not be detectable in a context-
independent manner after an extended protocol of morphine
administration. Furthermore, as JNK inhibition recovered
the locomotor response at similar relative proportions for
each day of the test, this suggests that the JNK cascade does
not block morphine sensitization. However, inhibiting JNK
allows an initial locomotor response to morphine thus
permitting sensitization to develop.
Table 1 JNK Inhibition by SP6 has no Effect on Mu Receptor–VDCC Inhibition in b-arr2/or +/+ Neurons
Agonist Pre-treatment Morphine Fentanyl DAMGO
SP6 SP6 SP6
b-arr2+/+ 32.5±4.1 37.8±5.2 36.3±2.4 36.5±2.5 44.4±2.9 56.0±7.2
b-arr2/22.0±3.6* 23.6±2.8* 26.4±3.1* 24.4±4.4* 26.9±3.0** 23.1±4.4**
The effect of SP6 incubation (10 mM, 4 h) on Ca
2+
channel inhibition (%) by the mu opioid receptor agonists, morphine, fentantyl, and DAMGO (1 mM each) was
assessed in early postnatal dorsal root ganglia neurons from b-arr2+/+ and /mice.
*, **po0.05, 0.01 vs b-arr2+/+, same treatment.
−10
0
10
20
30
DAMGO (1μM) inhibition (%)
40
50
60
β-Arr2+/+
β-Arr2−/−
Baseline
DAMGO
Wash
β-Arr2+/+
β-Arr2−/−
Pre-treatment –M M + SP6 F F + SP6 D D + SP6
a
b
c
– M M + SP6 F F + SP6
Figure 5 Mu receptor-Ca
2+
channel inhibition as a possible cytosolic
JNK target. Mu receptor inhibition of voltage-dependent Ca
2+
channels
(VDCC) was examined in b-arr2 + / + and /DRG neurons. Exemplar
currents are shown above the bar graph, which summarizes the effect of
mu agonist pre-treatment coupled with JNK inhibition on mu receptor–
VDCC inhibition, n¼6–30 neurons/sample. Although JNK inhibition had
no effect on mu receptor–VDCC inhibition in b-arr2/or + / + neurons
(Table 1), the desensitization induced by a 4-h pre-treatment of b-arr2 + / +
neurons with morphine (1 mM) was prevented by SP6 co-incubation
(10 mM, 4 h). In contrast, b-arr2/neurons showed no desensitization to
morphine and no effect of SP6. Both fentanyl and DAMGO pre-treatment
desensitized mu receptor–VDCC inhibition in b-ar2 + / + and /neurons
but this was not affected by SP6 co-incubation. M ¼morphine; F ¼fentanyl;
D¼DAMGO. a: b-arr2 + / + neurons, po0.05 vs M, po0.001 vs F, F +
SP6, D, D + SP6. b: b-arr2/neurons; po0.05 vs D, D + SP6, po0.001
vs F, F + SP6, c: b-arr2 + / + neurons; po0.05 vs M + SP. Scale
bars ¼40 ms on the xaxis and 0.4 nA on the yaxis.
Arrestin and JNK regulation of mu receptor function
N Mittal et al
1960
Neuropsychopharmacology
Our findings, and those of several others, show that b-
arrestin 2 regulates mu receptor function through several
different pathways. For example, the enhanced basal
analgesia seen in b-arr2/mice results from an
enhanced level of constitutively active mu receptors
(Lam et al, 2011; Walwyn et al, 2007). Such constitutive
activity also explains the reduced VDCC inhibition by mu
agonists in b-arr2/neurons (Table 1; Figure 5; Lam
et al, 2011; Walwyn et al,2007).Thewell-known
attenuated morphine desensitization and tolerance of the
mu receptor in b-arr2/mice may result from b-arrestin
2 inhibition of mu receptor resensitization, independent of
receptor internalization (Dang et al, 2011; Quillinan et al,
2011). This could explain the lack of desensitization of
DAMGO-VDCC inhibition following chronic morphine
treatments in b-arr2/neurons seen in Figure 5. Our
data add yet another mechanism to b-arrestin 2 regulation
of mu receptor function. We show that the enhanced
morphine-induced analgesia and decreased locomotor
response in b-arr2/mice is JNK-mediated, demonstrat-
ing how b-arrestin 2 normally controls this pathway. It
would be of further interest to examine whether this role
of b-arrestin 2 in masking JNK activation may underlie
the enhanced reward, attenuated tolerance, or indeed
other pertinent phenotypes of morphine in mice lacking
b-arrestin 2.
ACKNOWLEDGEMENTS
This work was supported by NIH Grants DA05010 and
R24-DA025319. O Egbuta, N Desai, N Mittal, and WM
Walwyn are supported in part by Hatos Scholarships. N
Mittal was also supported by the Gates Millennium Scholars
program. Thanks to the laboratory of Robert Lefkowitz for
the b-arrestin-1 and 2 knockout mice.
DISCLOSURE
The authors declare no conflict of interest.
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