Journal of Neurochemistry, 2001, 76, 1887±1894
Direct binding of b-arrestins to two distinct intracellular domains
of the d opioid receptor
Bo Cen,* Qingming Yu,* Jun Guo,² Yalan Wu,* Kun Ling,* Zhijie Cheng,* Lan Ma² and
Gang Pei*
*Shanghai Institute of Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
²National Laboratory of Medical Neurobiology, Medical Center of Fudan University, Shanghai, China
Abstract
b-Arrestins regulate opioid receptor-mediated signal transduction and play an important role in opiate-induced analgesia
and tolerance/dependence. This study was carried out to
measure the direct interaction between b-arrestins and opioid
receptor. Immunoprecipitation experiments demonstrated that
b-arrestin 1 physically interacts with delta opioid receptor
(DOR) co-expressed in human embryonic kidney 293 cells in
an agonist-enhanced manner and truncation of the carboxyl
terminus of DOR partially impairs the interaction. In vitro data
from glutathione-S-transferase pull-down assay showed that
the carboxyl terminus (CT) and the third intracellular loop (I3L)
of DOR are both capable of and either domain is suf®cient for
binding to b-arrestin 1 and 2. Surface plasmon resonance
determination further revealed that binding of CT and I3L of
DOR to b-arrestin is additive, suggesting these two domains
bind at distinctly different sites on b-arrestin without considerable spatial hindrance. This study demonstrated for the ®rst
time the direct binding of b-arrestins to the two distinct
domains, the carboxyl terminus and the third intracellular loop,
of DOR.
Keywords: arrestin, interaction, opioid receptor, surface
plasmon resonance.
J. Neurochem. (2001) 76, 1887±1894.
Opioid receptor belongs to the G-protein-coupled receptor
(GPCR) family and is composed of three receptor types
known as d, k and m opioid receptors. Interaction of opioid
receptor with opioids produces strong analgesic effect.
However, chronic use of opioid drugs causes tolerance and
dependence and thus limits its clinical application and
results in opioid abuse. The molecular mechanisms underlying opioid tolerance and dependence are complex and not
well understood, but desensitization of opioid receptor has
been implicated as a possible mechanism (Nestler and
Aghajanian 1997). Studies indicate that b-arrestins are
important regulators of responsiveness mediated by opioid
receptors.
The arrestins comprise a family of intracellular proteins;
there are four members: visual arrestin, arrestin C (or
X-arrestin), b-arrestin 1 and b-arrestin 2. While visual
arrestin and arrestin C are primarily localized in the visual
system to regulate phototransduction (Kuhn 1978;
Murakami et al. 1993; Craft et al. 1994), b-arrestin 1 and
b-arrestin 2 are widely expressed, especially with high
levels in nervous and lymphatic tissues (Parruti et al. 1993)
and have been known to regulate the functions of many
important GPCRs (Pippig et al. 1993; Ferguson et al. 1996;
Aramori et al. 1997; Schlador and Nathanson 1997; Aragay
et al. 1998). Many studies have shown that signal
transduction mediated by opioid receptors is regulated by
b-arrestins through promoting receptor desensitization and
internalization (Kovoor et al. 1997; Zhang et al. 1998;
Appleyard et al. 1999; Li et al. 1999; Zhang et al. 1999).
Truncation of the carboxyl termini of d/k opioid receptors
reduced the rate of b-arrestin-dependent desensitization
Received August 30, 2000; revised manuscript received November 28,
2000; accepted December 1, 2000.
Address correspondence and reprint requests to Dr Gang Pei,
Shanghai Institute of Cell Biology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences, 320 Yue Yang Road,
Shanghai 200031, China. E-mail: [email protected]
Abbreviations used: CaMK, calmodulin-dependent protien kinase;
CT, carboxyl terminus; DOR, delta opioid receptor; DPDPE, [D-Pen5]encephalin; DSS, disuccinimidyl suberate; FBS, fetal bovine serum;
GPCR, G protein-coupled receptor; GST, glutathione-S-transferase;
HA, hemagglutinin; HEK, human embryonic kidney; I3L, third intracellular loop; MAPK, mitogen-activated protein kinase; MEM, modi®ed
Eagle's medium; PMSF, phenylmethylsulfonyl ¯uoride; SDS±PAGE,
sodium dodecyl sulfate±polyacrylamide gel electrophoresis; SPR,
surface plasmon resonance.
q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1887±1894
1887
1888 B. Cen et al.
(Kovoor et al. 1997; Appleyard et al. 1999). Our earlier
study showed that b-arrestin 1 interferes opioid receptor/G
protein coupling and differentially regulates the responsiveness of d, k and m opioid receptors (Cheng et al. 1998).
Terwilliger et al. (1994) reported that chronic morphine
administration increases b-arrestin levels in the rat locus
coeruleus. A recent study of Bohn and colleagues demonstrated that b-arrestin 2 knockout mice exhibit enhanced
morphine analgesia, indicating impairment of opioid
receptor desensitization in the b-arrestin de®cient mice
(Bohn et al. 1999). These studies provide strong evidence
for the physiologic importance of b-arrestins in modulating
the function of opioid receptors.
Much attention has been paid to the functional consequences of binding of b-arrestins to opioid receptors.
However, the characteristics and mechanisms of direct
interactions between b-arrestins and opioid receptors, the
receptor intracellular domains directly involved in b-arrestin
binding, are not well understood. The current study was
undertaken to analyze the direct physical interaction of
b-arrestins with the carboxyl terminus (CT) and the third
intracellular loop (I3L) of the d opioid receptor (DOR).
Materials and methods
Construction of expression vectors
Plasmids encoding hemagglutinin (HA) epitope-tagged mouse
DOR and a truncation mutant lacking carboxyl terminal 15
amino acids were constructed in pcDNA3 (Strategene, La Jolla,
CA, USA) as described previously (Pei et al. 1995; Guo et al.
2000). Glutathione S-transferase (GST) fusion protein constructs
for the carboxyl terminus of DOR (Gln331-Ala372) and the third
intracellular loop (Leu235-Ile259) were generated from the human
DOR cDNA clone (generously provided by Dr Jia-Bei Wang) by
ampli®cation using the polymerase chain reaction (PCR). The PCR
products were subcloned into pGEX-4T1 (Amersham/Pharmacia,
Piscataway, NJ, USA) with BamHI/XhoI sites. The GST fusion
protein construct for the third intracellular loop of human a2Aadrenergic receptor (a2-AR-I3L) was generously provided by Dr
Lin Li. Recombinant human b-arrestin 1 (Cheng et al. 1998) and
b-arrestin 2 (Ling et al. 1999) were subcloned into pET30a
(Novagen, Madison, WI, USA). All constructs were con®rmed by
DNA sequencing.
Expression and puri®cation of recombinant proteins in
Escherichia coli
Proteins were expressed in Escherichia coli BL21 (DE3) cells.
GST fusion proteins were induced with 100 mm isopropyl-b-dthiogalactoside (IPTG) for 2 h at 378C. Cell lysate was applied to
glutathione±Sepharose 4B beads (Amersham Pharmacia) and
fusion proteins were puri®ed according to the manufacturer's
instructions. Puri®ed GST fusion protein of a2-AR-I3L was
dialyzed and incubated with thrombin (Amersham Pharmacia) in
the ratio of 1 : 1000 (wt/wt, thrombin/fusion protein) at 228C for
24 h. GST tag was removed using glutathione±Sepharose 4B beads
and then thrombin was removed through centricon-30 (Amicon,
Beverly, MA, USA). Recombinant b-arrestin 1 and b-arrestin 2
were induced with 1 mm IPTG for 7 h at 378C. Cell lysate was
sequentially applied to 50% saturated (NH4)2SO4 solution, heparinand Q-Sepharose. Each batch of protein was analyzed by 10%
sodium dodecyl sulfate±polyacrylamide gel electrophoresis
(SDS±PAGE) and Coomassie blue staining, showing a purity of
more than 90%.
Cell cultures and transient transfection
Cells were obtained from American Type Culture Collection.
Human embryonic kidney (HEK) 293 cells were cultured in
modi®ed Eagle's medium (MEM; GIBCO/BRL, Gaithersburg,
MD, USA) supplemented with 10% fetal bovine serum (FBS) and
transfected using the calcium phosphate±DNA coprecipitation
method and used 48 h post-transfection as described previously
(Cheng et al. 1998). Neuroblastoma glioma NG108±15 and
THP-1 cells were cultured as described previously (Zhao et al.
1997; Zhao et al. 1999), lyzed by sonication in buffer A (20 mm
Tris-HCl pH 7.5, 1 mm EDTA, 100 mm NaCl, 0.1% Triton X-100),
and centrifuged for 10 min at 12 000 g at 48C to obtain cytosol
fraction.
Western blotting and immunoprecipitation
For the analysis of association of b-arrestin 1 and DOR, HEK293
cells transiently expressing HA-tagged DOR and b-arrestin 1 were
stimulated with or without 1 mm [d-Pen2,d-Pen5]encephalin
(DPDPE; Sigma, St Louis, MO, USA) at 378C for 10 min 48 h
following transfection and a ®nal concentration of 2 mm disuccinimidyl suberate (DSS; Pierce, Rockford, IL, USA) was added for
incubation of 60 min at room temperature. The cells were lyzed in
50 mm Tris-HCl pH 8.0, 5 mm EDTA, 150 mm NaCl, 1% NP-40,
0.1% SDS, 10 mm pyrophosphate, 0.5% deoxycholic acid, 10 mm
NaF, 10 mg/mL aprotinin, 10 mg/mL benzamidine, 2 mm phenylmethylsulfonyl ¯uoride (PMSF) and the lysate was centrifuged at
12 000 g for 30 min. The supernatant was incubated with
0.5 mg mouse monoclonal antibody 12CA5 (Roche Molecular
Biochemicals, Mannheim, Germany) against the HA epitope and
protein A-Sepharose (Amersham Pharmacia) at 48C for 4 h. The
immunocomplexes absorbed onto protein A-Sepharose were
resolved by 10% SDS±PAGE and subjected to western blotting
analysis with anti-b-arrestin antibodies (Cheng et al. 2000). The
direct western blotting analysis of cell lysate was carried out using
anti-b-arrestin antibodies and 12CA5 as described previously
(Zhao et al. 1999; Cheng et al. 2000). Phosphospeci®c polyclonal
antibodies against Tyr204-phosphorylated extracellular signalregulated kinase (ERK) 1 (p44) and ERK2 (p42) isoforms and
polyclonal antibodies against total ERK1/2 (phosphorylation-state
independent) were purchased from New England Biolabs, Boston,
MA, USA.
GST pull-down assay
Equal molar of GST fusion proteins (0.15 nmol, equal to 5 mg of
CT) bound to glutathione±Sepharose 4B beads were incubated on a
rotator with puri®ed b-arrestin 1 (0.2 mg), b-arrestin 2 (0.2 mg), or
cell cytosol fraction (150 mg of total protein) in 200 mL of buffer A
at 48C for 2 h. In competition experiments, 30 mm competitors
(a2-AR-I3L) were added. The beads were subsequently washed
with 600 mL of buffer A and eluted with 10 mm reduced
glutathione (GSH). Binding was quanti®ed by immunoblotting of
each fraction with anti-b-arrestin antibodies as compared with a
q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1887±1894
Interaction of d opioid receptor with b-arrestins
1889
10-fold range of known amounts of puri®ed b-arrestin 1 resolved
along with it.
Surface plasmon resonance (SPR) analysis
Real-time analysis of interaction between b-arrestin 1 and GST
fusion proteins was performed with a BIAcore-1000 instrument
(Pharmacia Biosensor AB, Uppsala, Sweden). b-Arrestin 1
(approximately 2000 response units) was immobilized to a CM5
biosensor chip by amine coupling according to the manufacturer's
instructions. A blank surface was prepared to examine non-speci®c
protein interactions and differences in the bulk refractive index due
to changes in buffers by applying the same treatment as for
immobilization of b-arrestin 1, but without injection of any protein.
The running buffer contained 20 mm Tris-HCl pH 7.5, 1 mm
EDTA, 100 mm NaCl, 0.005% Tween 20 and the ¯ow rate was
30 mL/min. The sensor surface was regenerated between assays by
treatment with 10 mm glycine, pH 2.0. The kinetic analysis of the
resulting binding curves was performed with BIAevaluation
software version 2.1 (Pharmacia Biosensor AB).
To determine the dissociation equilibrium constant (KD), ®ve
different concentrations of GST fusion proteins were injected over
immobilized b-arrestin 1 surface. Averages of the KD values were
obtained. The binding data from each curve were ®tted with either
one-site model or two-site model using the BIAevaluation software
version 2.1 (Pharmacia Biosensor AB). All models were veri®ed by
residual plots, which calculate the difference between the observed,
and the ®tted curves for each data point. Both association and
dissociation rate constants were determined from sensorgrams
producing a chi-squared (x2) value less than one.
Experiments using consecutive and simultaneous injection of
saturating level of CT and I3L were also attempted to test whether
they shared the same binding sites or occupied different binding
sites, respectively, on b-arrestin 1. Increasing concentrations of CT
or I3L were injected over the surface of immobilized b-arrestin 1 to
determine levels required for ligand saturation.
Results
Fig. 1 Association of DOR with b-arrestin 1. HEK293 cells were
cotransfected with plasmids of b-arrestin 1 and wild type DOR (WT),
DOR (D15), or GFP and the expression levels of b-arrestin 1 and
DOR were analyzed using antibodies against b-arrestins (a) and
12CA5, respectively (b), as described in Materials and methods. The
cells were incubated with or without 1 mM DPDPE at 378C for 10 min
followed by treatment of DSS. Cell lysate was immunoprecipitated
with 12CA5 and the presence of b-arrestin in the immunoprecipitates
was detected with b-arrestin antibodies following SDS±PAGE and
immunoblotting (c). (d) Scanning densitometry analysis of western
blot data in (c). *p , 0.05, compared with unstimulated control. (e)
DOR-mediated ERK phosphorylation. Cells cotransfected with
b-arrestin 1 and DOR (WT), DOR (D15) or GFP were incubated with
or without 1 mM DPDPE at 378C for 10 min. A fraction of the cell
lysate was subjected to SDS±PAGE and the phosphorylation of
ERK was determined by western blot analysis using phosphospeci®c p42/44 ERK antibodies. The results were quantitated by
scanning densitometry. *p , 0.05, compared with control (unstimulated cells cotransfected with b-arrestin 1 and GFP). Data are
means ^ SE of at least two independent experiments.
Association of b-arrestin 1 with DOR in HEK293 cells
Our earlier research showed that exogenous expression of
b-arrestin 1 in HEK293 cells strongly attenuates DORmediated signal transduction (Cheng et al. 1998), indicating
functional interaction of b-arrestin 1 with DOR in vivo. In
the present study, the potential physical interaction
between b-arrestin 1 and DOR in HEK293 cells was
assessed. The wild-type DOR (WT) or truncation mutant of
DOR lacking carboxyl terminal 15 amino acids DOR (D15)
was co-expressed with b-arrestin 1 in HEK293 cells and
immunoprecipitated with 12CA5 (Figs 1a and b). As shown
in Fig. 1(c), association of b-arrestin 1 with DOR (WT) was
detected and their interaction apparently was enhanced by
activation of the receptor by DOR agonist DPDPE. The
C-terminal truncated receptor DOR (D15) lacking GRK sites
required for agonist-dependent receptor phosphorylation
(Guo et al. 2000) could also be coprecipitated with
b-arrestin 1 but at an attenuated level (Figs 1c and d).
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Fig. 2 Interaction of the carboxyl terminus (CT) and the third intracellular loop (I3L) of DOR with b-arrestins. (a) The amino acid
sequences of CT and I3L of DOR fused with GST. (b) The puri®ed
GST and GST fusion proteins were detected by Coomassie blue
staining following SDS±PAGE (i). Cytosol fraction of THP-1 (ii) and
NG108±15 (iii) cells and puri®ed b-arrestin 1 (iv) and b-arrestin 2 (v)
were incubated with GST, I3L, or CT immobilized to glutathione±
Sepharose 4B. The proteins bound were eluted with 10 mM GSH
and analyzed with anti-b-arrestin antibodies following SDS±PAGE.
(c) Inhibition of b-arrestin 1 binding to CT and I3L by 30 mM puri®ed
a2-AR-I3L peptide. Results shown are representatives of at least
four independent experiments.
Thus, our data indicate that b-arrestin 1 could effectively
interact with DOR in vivo in an agonist-enhanced manner,
and this interaction is partially dependent on the carboxyl
terminus of DOR. These results implicate the possible
involvement of the intracellular domain other than the
cytoplasmic carboxyl tail of DOR in the interaction with
b-arrestins. To obtain additional pharmacological evidence
of an agonist-driven association of b-arrestin 1 with DOR
(WT) or DOR (D15), the function of such association
(Figs 1c and d) was investigated by measuring ERK
activation, which leads to the subsequent phosphorylation
and activation of targets such as protein kinases, transcription factors, and membrane proteins and regulates cell
growth and differentiation (Davis 1993). Phospho-speci®c
ERK polyclonal antibodies recognize only the phosphorylated p42 and p44 ERK isoforms and therefore have been
used widely to measure ERK activation. b-Arrestins play an
important role in many GPCR-mediated ERK activation
(Daaka et al. 1998; Pierce et al. 2000). As shown in
Fig. 1(e), a 10-min exposure to DPDPE led to a signi®cant
increase in ERK phosphorylation above the control level
Fig. 3 Elution of bound b-arrestins from CT and I3L with increasing
concentrations of NaCl. Puri®ed b-arrestin 1 (a) or b-arrestin 2 (b)
was incubated with CT or I3L immobilized to glutathione±Sepharose
4B. The proteins bound to the beads were eluted with the same
buffer containing increasing concentration of NaCl and analyzed by
western blotting with b-arrestin antibodies. Data are expressed as
percentages of basal and means ^ SE of three independent
experiments.
(without DPDPE stimulation) in cells cotransfected with
b-arrestin 1 and DOR (WT) or DOR (D15) (Fig. 1e). and
compared with cells transfected with DOR (WT) or DOR
(D15) alone (data not shown), a modest increase in ERK
phosphorylation was observed in cells cp-expressing
b-arrestin 1. Whereas the basal expression of ERK was
unchanged in the same cells (data not shown).
Binding of b-arrestins in cell cytosolic fractions and
recombinant b-arrestins to immobilized DOR I3L and
CT
Studies show that the carboxyl terminus and the third
intracellular loop of receptor are key sites for initiation and
termination of signals mediated by GPCRs. To determine if
the carboxyl terminal and the third intracellular loop
domains are capable of binding b-arrestins, the carboxyl
terminus (Gln331-Ala372) and the third intracellular loop
(Leu235-Ile259) of DOR were expressed in bacteria as GST
fusion proteins (designated as CT and I3L, respectively) and
puri®ed (Fig. 2). As shown in Fig. 2(b), the endogenous
b-arrestins in cytosolic fractions of THP-1 and NG108-15
cells (both cell lines express b-arrestin 1 and b-arrestin 2 but
the majority of them is b-arrestin 1 according to our analysis
with reverse transcription PCR) were retained by CT and
I3L immobilized to glutathione±sepharose beads. Further
experiments using puri®ed b-arrestin 1 or b-arrestin 2
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Interaction of d opioid receptor with b-arrestins
Fig. 4 Real-time SPR detection of b±arrestin 1 interaction with CT
and I3L. (a) Saturating level of GST, CT, or I3L (200 mM) was
injected over the immobilized b-arrestin 1 surface. (b and c) Increasing concentrations (6.5, 9.8, 13, 20 and 26 mM) of CT or I3L were
injected over immobilized b-arrestin 1 surface.
con®rm that the carboxyl tail and the third intracellular loop
domains of DOR were both capable of interacting with
b-arrestins in the cytoplasmic extracts from THP-1 and
NG108-15 cells as well as puri®ed b-arrestins (Fig. 2b). As
shown in Fig. 3, CT and I3L were able to bind b-arrestin 1
and b-arrestin 2 subtypes. Parallel control experiments show
that neither b-arrestin 1 nor b-arrestin 2 bound to
glutathione±Sepharose 4B (data not shown) or GST
(Fig. 2b) and p42/44 MAPK in the cytosol fractions was
not associated with CT or I3L under such conditions (data
not shown). The association of b-arrestin/CT was inhibited
by the a2AR-I3L peptide which could bind b-arrestin (Wu
et al. 1997). In contrast, the association of the b-arrestin/I3L
was not signi®cantly inhibited by this peptide under the
same conditions, further implying the existence of distinct
binding sites on b-arrestin (Fig. 2c). These results provide
clear evidence that the carboxyl terminal and the third
intracellular loop domains are able to speci®cally mediate
interaction of DOR with b-arrestins independently.
Binding of DOR CT and I3L to immobilized b-arrestin 1
The characteristics of association of b-arrestins with the
1891
Fig. 5 Binding of CT and I3L to b-arrestin 1 at distinct sites. Consecutive saturating levels of CT and I3L were injected sequentially
over immobilized b-arrestin 1: (a) CT was injected before I3L. (b)
I3L was injected before CT. (c) Saturating levels of CT and I3L were
injected either alone or together over immobilized b-arrestin 1.
third intracellular loop and the carboxyl terminal domains of
DOR were further investigated via a series of SPR
measurements using a BIAcore-1000 with the puri®ed
b-arrestin 1 immobilized on the sensor chip. As shown in
Fig. 4(a), injection of saturating level of CT or I3L resulted
in remarkable increase of the SPR signal, re¯ecting binding
of CT and I3L to the b-arrestin 1. Whereas no binding was
detected during the injection of GST under the same
conditions (Fig. 4a).
Our results show that binding of CT and I3L to b-arrestin
1 occurs rapidly and exhibits a concentration-dependent
manner (Fig. 4). The kinetic rate constants and dissociation
constants of interaction of b-arrestin with CT and I3L of
DOR were calculated by analysis of the SPR data obtained
from injection of 6.5, 9.8, 13, 20 and 26 mm of CT or I3L
over immobilized b-arrestin 1 surface with BIAevaluation
software version 2.1 (Pharmacia Biosensor AB). Analysis
indicates that binding of CT to b-arrestin was best ®tted
with one-site model and that of I3L with two-site model.
The disassociation equilibrium constant (KD) of CT from
b-arrestin 1 was estimated as 2.9 mm and those of I3L were
7.6 and 80 mm. These results from SPR measurements
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1892 B. Cen et al.
clearly demonstrate the ability of the carboxyl terminal
domain and the third intracellular loop of DOR to associate
with b-arrestin 1.
Binding of CT and I3L to distinct sites of b-arrestin
To examine whether CT and I3L occupy the distinct binding
sites on b-arrestin, saturating levels of CT and I3L (200 mm
each) were injected consecutively over the surface of
b-arrestin 1 chip. b-Arrestin 1 was still able to bind I3L
after it was saturated by CT (Fig. 5a) and the saturating
binding of I3L to b-arrestin did not interfere the subsequent
binding of CT either (Fig. 5b). Furthermore, simultaneous
application of CT and I3L resulted in an additive response
curve equivalent to summation of the signals produced from
individual injections of CT and I3L (Fig. 5c). Experiment
using anti-GST antibody-linked chip did not detect any
signi®cant binding between CT and I3L (data not shown)
therefore it can be reasoned that CT and I3L bind to
b-arrestin 1 at distinct sites and without considerable spatial
hindrance.
Discussion
The physiological importance of b-arrestins in modulating
the function of opioid receptors in vivo has been demonstrated recently by the fact that functional deletion of
b-arrestin 2 gene in mice results in remarkable potentiation
and prolongation of the analgesic effect of morphine (Bohn
et al. 1999). Therefore, elucidation of interaction between
opioid receptor and b-arrestin and underlying mechanisms
would greatly bene®t the study and treatment of pain and
narcotic tolerance. The data from the current study
demonstrated for the ®rst time that b-arrestins directly
interact with opioid receptor and thus provide a molecular
mechanism for functional regulation of opioid receptor by
b-arrestins shown by many laboratories (Kovoor et al. 1997;
Cheng et al. 1998; Appleyard et al. 1999; Li et al. 1999;
Zhang et al. 1999). Our results also demonstrated that either
of the two distinct functional domains of DOR, the carboxyl
terminus or the third intracellular loop, is suf®cient to bind
to b-arrestins.
Surface plasmon resonance analysis is an optical detection technique in which the reactants are monitored in real
time without the use of labels, thus permitting the
determinations of kinetic rate constants, binding af®nities,
and binding site characterization (Calakos et al. 1994;
Vales-Gomez et al. 1999; Hoff et al. 2000). The use of SPR
techniques to study arrestin interactions with receptor
elements is a relatively novel approach. Employment of
the SPR technique in this study enabled us not only to
con®rm but also distinguish the interactions of b-arrestin
with the two distinctly different functional domains of DOR.
Signal transduction mediated by opioid receptors is
regulated by b-arrestins through promoting receptor
desensitization and internalization (Kovoor et al. 1997;
Zhang et al. 1998; Appleyard et al. 1999; Li et al. 1999;
Zhang et al. 1999). Studies indicate that the carboxyl
terminus is involved in b-arrestin-mediated opioid receptor
desensitization. Deletion of the carboxyl terminus of d and k
opioid receptor attenuates G protein-coupled receptor
kinase- and b-arrestin-mediated receptor desensitization
(Kovoor et al. 1997; Appleyard et al. 1999). These results
suggest functional interaction between b-arrestin and
carboxyl terminus of opioid receptors. The present study
clearly established that b-arrestins directly interact with the
carboxyl terminus of DOR.
The direct interaction between the third intracellular loop
of GPCR and b-arrestin has been reported (Wu et al. 1997;
Gelber et al. 1999). Our earlier research (Cheng et al. 2000)
indicates the existence of several different structural
elements on chemokine receptor CXCR4 contributing to
its binding to b-arrestin, suggesting the possibility of
different functional domains of the receptor to bind to
distinct sites on b-arrestin simultaneously. Studies also
suggest the involvement of cytoplasmic domains other than
the carboxyl terminus in desensitization of opioid receptors.
Results from d/k chimeric opioid receptors indicate that at
least two domains, including the carboxyl terminal tail,
determine the receptor type speci®city of agonist-stimulated
opioid receptor internalization (Chu et al. 1997). Koch et al.
showed that eliminating putative calmodulin-dependent
protein kinase II (CaMK II) sites in the third intracellular
loop of m opioid receptor confers resistance to CaMK
II-induced receptor desensitization (Koch et al. 2000). These
data suggest that the third intracellular loop of the opioid
receptor family may very likely interact directly with protein
kinases and other intracellular proteins and serve a role in
regulation of opioid signaling, in addition to its established
role in G protein activation (Merkouris et al. 1996;
Georgoussi et al. 1997; Wang 1999). The present work
shows that two functional domains of DOR, the carboxyl
terminus and the third intracellular loop, are capable of
binding to b-arrestins. Furthermore, the saturation binding
of the third intracellular loop and the C-terminal domains of
DOR to b-arrestin is not exclusive but additive to each
other, suggesting that the two domains bind to distinct sites
on b-arrestin 1. The current results indicate that b-arrestins
physically interact with more than one functional domains
of DOR and suggest that the interaction of b-arrestins with
distinct functional domains of DOR may play differential
roles in the regulation of opioid receptor-mediated signaling
as in the case of CXCR4 (Cheng et al. 2000).
Acknowledgements
The authors wish to thank Ying Xiong, Ping Wang and Wen-Bo
Zhang for their technical assistance, Dr Tian-Hua Zhou and
Ping Wang for their helpful discussion, and Xu-Min Zhang,
q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1887±1894
Interaction of d opioid receptor with b-arrestins
Pei-Hua Wu, Shun-Mei Xin and Hai-Lian Xiao for their
help. This work was supported by the grants from the
National Natural Science Foundation of China (39625015
and 39825110), Chinese Academy of Sciences (KJ951-B1),
Ministry of Science and Technology (G1999053907 and
G1999054003), National Laboratory of Medical Neurobiology
and the German Max-Planck Society.
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