Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M500171200 on January 18, 2005

J. Biol. Chem., Vol. 280, Issue 12, 11152-11164, March 25, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/12/11152    most recent
M500171200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Law, P.-Y.
Right arrow Articles by Loh, H. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Law, P.-Y.
Right arrow Articles by Loh, H. H.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Heterodimerization of µ- and {delta}-Opioid Receptors Occurs at the Cell Surface Only and Requires Receptor-G Protein Interactions*

Ping-Yee Law{ddagger}§, Laurie J. Erickson-Herbrandson, Qin Q. Zha, Jon Solberg, Ji Chu, Aili Sarre, and Horace H. Loh

From the Department of Pharmacology, Medical School, University of Minnesota, Minneapolis, Minnesota 55455-0217

Received for publication, January 6, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Homo- and heterodimerization of the opioid receptors with functional consequences were reported previously. However, the exact nature of these putative dimers has not been identified. In current studies, the nature of the heterodimers was investigated by producing the phenotypes of the 1:1 heterodimers formed between the constitutively expressed µ-opioid receptor (MOR) and the ponasterone A-induced expression of {delta}-opioid receptor (DOR) in EcR293 cells. By examining the trafficking of the cell surface-located MOR and DOR, we determined that these two receptors endocytosed independently. Using cell surface expression-deficient mutants of MOR and DOR, we observed that the corresponding wild types of these receptors could not rescue the cell surface expression of the mutants, whereas the antagonist naloxone could. Furthermore, studies with constitutive or agonist-induced receptor internalization also indicated that MOR and DOR endocytosed independently and could not "drag in" the corresponding wild types or endocytosis-deficient mutants. Additionally, the heterodimer phenotypes could be eliminated by the pretreatment of the EcR293 cells with pertussis toxin and could be modulated by the deletion of the RRITR sequence in the third intracellular loop that is involved in the receptor-G protein interaction and activation. These data suggest that MOR and DOR heterodimerize only at the cell surface and that the oligomers of opioid receptors and heterotrimeric G protein are the bases for the observed MOR-DOR heterodimer phenotypes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The ability of G protein-coupled receptors (GPCRs)1 to homo- or heterodimerize has implications in the functions of the receptors. Dimerization of the receptors has been reported for the class A GPCRs such as the adenosine (1), adrenergic (2–5), angiotensin (6), dopamine (7, 8), muscarinic (9), vasopressin (2, 10), and opioid (11–15) receptors and the class C GPCRs such as the calcium-sensing (16), the metaboropic glutamate receptors (17), and the {gamma}-amino-n-butyric acid type B (GABAB) receptors (18–20). The homo- and heterodimerization of these receptors have been demonstrated by co-immunoprecipitation experiments (11, 21) and subsequently by the fluorescence resonance energy transfer or bioluminescence resonance energy transfer techniques (3, 12, 23). The heterodimerization of the GPCRs was shown to be selective, with formation of heterodimers with some but not all subtypes of the receptors (13, 24, 25). Most importantly, there are functional differences between the monomers and the homo- and heterodimers of the GPCRs. The classic example is the inability of individual GABAB1 and GABAB2 subunit to form a functional receptor (18–20). Alteration in the GPCR function or expression was also observed with the heterodimerization of 5HT1B and -1D (26), dopamine D1 and adenosine A1 (27), muscarinic M2 and M3 (28), or dopamine and somatostatin (29) receptors. Heterooligomerization of the GPCRs with other receptor types, such as the ionotropic GABAA receptor, has been observed, resulting in the alteration in the ion-gating properties of the channels (30). As for the opioid receptors, which belong to the rhodopsin subfamily of the GPCRs, homo- and heterodimerization of the receptors have been reported. Homodimerization of the {delta}-opioid receptor (DOR) was reported with immunoprecipitation, and agonist-induced receptor internalization appeared to be related to the dimers formation (11). The agonist-induced change in DOR oligomerization was not observed with bioluminescence resonance energy transfer experiments (12). The {kappa}-opioid receptor is reported to exist in homodimers and could heterodimerize with DOR but not with the µ-opioid receptor (MOR) (13). The heterodimerization of DOR and {kappa}-opioid receptor resulted in the decrease in the affinities of receptor-selective ligands (13). The DOR and {kappa}-opioid receptor could also heterodimerize with the {beta}2-adrenergic receptor, resulting in an alteration of {beta}2-adrenergic receptor functionality (31). The most interesting of the heterodimers is the reported heterodimerization of the MOR and DOR receptors. Using different epitope-tagged receptors, both George et al. (15) and Gomes et al. (14) reported the ability of MOR and DOR to heterodimerize. Both groups reported a change in functionality of the receptor, but George et al. (15) reported that the heterodimer function appeared to be insensitive to pertussis toxin pretreatment, implying coupling to G proteins other than Gi/Go (15).

The exact nature of the opioid receptor homo- and heterodimerization has not been defined. Two general schemes for the dimerization of GPCRs have been proposed by Reynolds and co-workers (32); 1) the 1:1 stoichiometric molecular complexes of the receptors or contact dimers and 2) the swapping of the transmembrane domains of the GPCRs. The domain swapping was initially suggested by Gouldson et al. (5) to explain the ability of adrenergic-muscarinic receptor fragments 1–5 and 6–7 to function as autonomous folding units when co-expressed in the same cells (33, 34). Using computational three-dimensional models of the opioid receptors, Filizola and Weinstein (35) suggested the most likely interfaces between the opioid receptor homodimers are TM4-TM4, TM4-TM5, and TM5-TM5 for DOR, TM1-TM1 for MOR, and TM5-TM5 for {kappa}-opioid receptor. These hypotheses do not consider the possible roles intracellular domains could have, as in the case of GABAB receptors heterodimerization (36, 37) and of adrenergic-muscarinic receptor heterodimerization (34). Hence, to investigate the nature of the putative MOR and DOR heterodimers, DOR expression in the EcR293 cells constitutively expressing MOR was controlled by the ecdysone-inducible mammalian expression system. The role of the intracellular domains in the formation of the MOR and DOR heterodimers, particularly those domains within the third intracellular loop that are involved in G protein coupling, was investigated. It could be demonstrated from our current studies that the observed heterodimer phenotypes are the results of opioid receptors interaction with the G proteins.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Expression of MOR and DOR in the EcR293 Cells—The mouse MOR tagged with the human hemagglutinin (HA) epitope (YPYDVPDYA) and mouse DOR tagged with the FLAG epitope (DYKDDDDK) were subcloned in pCDNAmp and in the pINDsp1 vectors, respectively. EcR293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15 mM glucose, 0.43 M NaHCO3, 100 units/ml penicillin, 100 µg/ml streptomycin, 0.1 mg/ml Zeocin, and 10% fetal calf serum (DMEM growth medium) in a 10% CO2 incubator. These EcR293 cells, derived from HEK293 cells stably expressing the heterotrimic ecdysone receptor and the retinoid X receptor and obtained from the manufacturer (Invitrogen), were transfected with 10 µg of the above plasmids in a 4:1 ratio (MOR:DOR). The colonies surviving the antibiotic Geneticin (G418) selection (1 mg/ml) were combined. The EcR293 cell mixture constitutively expressing MOR was isolated by fluorescence flow cytometry (FACS) using the monoclonal antibodies 16B12 for HA and the AlexaTM488-conjugated anti-mouse antibodies. Subsequently, the population of the EcR293 cells that expressed DOR within the MOR-expressing population was isolated by treating the cells with 1 µM ponasterone A for 48 h, and the DOR-expressing cells were identified by FACS using the monoclonal antibodies M2 for FLAG (Sigma) and the Alexa488TM-conjugated anti-mouse antibodies. The expression of MOR and DOR in the cells selected by FACS was substantiated with the [3H]diprenorphine receptor binding assays with intact cells. The EcR293 cells expressing both receptors were maintained in the DMEM medium supplemented with 0.2 mg/ml G418.

Membrane Preparation and Radioligand Binding—Competition binding studies were carried out with membranes isolated from EcR293 cells expressing the MOR and DOR receptors. EcR293 cells were treated with 0 or 2 µM ponasterone A (PA) for 48 h before harvesting by phosphate-buffered saline-EDTA (0.1 M NaCl, 0.01 M NaH2PO4, 0.04% EDTA, pH 7.4). The cell pellets were then homogenized in a sucrose-HEPES solution (0.32 M sucrose, 10 mM HEPES, pH 7.7) with a glass Dounce homogenizer. After centrifuging at 1000 x g for 10 min to remove the unbroken cell and nuclei, the membranes were isolated by centrifuging at 100,000 x g for 60 min. The final pellets were resuspended in the sucrose-HEPES solution. After the protein concentrations of the pellets were determined by the Lowry method, [3H]diprenorphine (2 nM) binding in the presence or absence of 10 µM naloxone were carried out so as to determine the specific binding. The percentages of MOR and DOR in the total specific binding were determined by carrying out the [3H]diprenorphine binding in the presence of 1 µM CTOP (selective antagonist for MOR) or 1 µM TIPP{Psi} (selective antagonist for DOR), respectively. Competition binding assays were performed by varying the concentrations of agonists and antagonists from 10–11 to 10–4 M. Prism 3 program (GraphPad, San Diego, CA) was used to analyze and determine the relative Ki values from the competition binding experiments. All binding experiments were performed in triplicate, and 3–6 competition binding assays were carried out for individual agonist or antagonists.

Intracellular cAMP Measurements—The ability of opioid agonists to inhibit the forskolin-stimulated adenylyl cyclase activity was determined by measuring the intracellular cAMP level in the EcR293 expressing either MOR alone or MOR and DOR together. The amplified luminescent proximity homogenous assays (AlphaScreenTM) for cAMP supplied by PerkinElmer Life Sciences (Boston, MA) were used to determine the cAMP levels. Twenty-four hours before the assays EcR293 cells treated with 0 or 2 µM PA for 24 h were plated into 96-well plates. Various concentrations of opioid agonists and antagonists were diluted with Krebs-Ringer·Hepes·buffer containing 10 µM forskolin and 0.5 mM isobutylmethylxanthine. After removal of the growth medium, the 96-well plates were placed on ice, and 100 µl of the drug solution was added to the well, with 4 wells per drug concentration. After sealing the plates with HotSealTM (Diversified Biotech, Boston, MA), the plates were incubated at 37 °C for 15 min. Afterward, the plates were placed in a water bath at 85–90 °C for 5 min so as to lyse the cells and to release the intracellular cAMP. After centrifuging the plates at 500 x g for 2 min, the amount of cAMP in 4 µl of the supernatant was determined using the donor beads coated with streptavidin, acceptor beads coated with anti-cAMP antibodies, and biotinylated cAMP in the AlphaScreenTM assay system. The cAMP concentrations, from 10–11 to 10–4 M, were used to construct the standard curve. The donor beads (final 20 µg/ml), the acceptor beads (final 15 µg/ml), and biotinylated cAMP (final 10 nM) were diluted in the 1x control buffer (5 mM HEPES, pH 7.4, and 0.3% Tween 20 (60%) and Hanks' buffered salts saline (40%)). 4 µl of the reaction supernatant or standard cAMP solutions were pipetted into duplicate wells of a 384-well Opti-plate (PerkinElmer Life Sciences) with a Biomek 2000 (Beckman-Coulter, Fullerton, CA) in a dimly lit room at 4 °C. The plate was then sealed with TopSealTM adhesive sealing films (PerkinElmer Life Sciences) and incubated in the dark at 4 °C for 2 h. Then 12 µl of the donor beads were pipetted into the wells, and the mixtures were incubated in the dark at 4 °C for 18–24 h. After equilibrating to room temperature in the dark (4 h), the content of the cAMP in each well was determined by exciting the donor beads at 680 nm, generating a singlet O2, resulting in the fluorescence emission of the acceptor beads at 520–620 nm. The fluorescence of the wells was measured with the FusionTM (PerkinElmer Life Sciences) plate reader, and the amount of cAMP in each sample was extrapolated from the standard curve. The dose response curves of the opioid agonists were constructed accordingly. The IC50 value and the maximal inhibition level for individual agonist were obtained from Prism 3 program analyses of 3–12 dose-response curves.

Fluorescence Flow Cytometry—The opioid receptors located on the plasma membrane was quantified by assaying the cell surface immunofluoresence using FACS. Briefly, EcR293 cells were treated with 0 or 2 µM PA for 48 h. Then cells were treated with 1 µM etorphine for different time periods or with various concentrations of etorphine for 60 min so as to induce receptor internalization. For the experiments in which the turnover of the cell surface-located receptors was measured, media containing PA were removed, and the cells were washed and refed with fresh medium. Before the addition of antibodies, EcR293 cells were rinsed twice with serum-free DMEM. Then the cells were incubated at 4 °C for 60 min in serum-free DMEM with either anti-HA (1:500 dilution) or anti-FLAG (1:300) antibody. Afterward, the cells were washed twice with serum-free DMEM and incubated with Alexa488TM-labeled goat anti-mouse IgG secondary antibody (1:400 dilution) at 4 °C for an additional 1 h. Then the cells were washed and fixed with 3.7% formaldehyde before quantitating the receptor immunoreactivity with fluorescence flow cytometry (FACScan, BD Biosciences). Fluorescence intensity of 10,000 cells was collected for each sample. Cell Quest software (BD Biosciences) was used to calculate the mean fluorescence intensity of the cells population. All FACS analyses were conducted three times with triplicate in each experiment.

Surface Biotinylation—To examine the constitutive internalization of MOR and DOR, the cell surface-located opioid receptors were biotinylated. EcR293 cells were grown in 10-cm dishes and treated with 0 or 2 µM PA for 48 h. Normally, biotinylation was carried out with cells reaching 90% confluency in the dishes. After washing twice with ice-cold phosphate-buffered saline, the cell surface proteins were biotinylated by incubating with 300 µg/ml sulfo-NHS-SS-biotin in phosphate-buffered saline for 30 min at 4 °C. Unreacted biotin was quenched and washed twice with Tris-buffered saline (10 mM Tris, pH 7.4, 154 mM NaCl) at 4 °C. Then pre-warmed DMEM medium (37 °C) was added to the cells for 1 h. Afterward, the remaining cell surface biotin was stripped with a solution containing 50 mM glutathione, 0.3 M NaCl, 75 mM NaOH, and 1% fetal bovine serum at 4 °C for 30 min. For the control, the cells were not treated with such stripping solution. Cells were then extracted with cell lysis buffer (0.2% (v/v) Triton X-100, 10 mM Tris-HCl, pH 7.5, 120 mM NaCl, 25 mM KCl, protease inhibitor mixture tablet Complete (Roche Applied Science; 1 tablet/50 ml)). Cellular debris was removed by centrifuging at 12,000 x g for 10 min at 4 °C. Clarified cell extracts were immunoprecipitated with immobilized streptavidin at 4 °C overnight. After washing five times with cell lysis buffer, the beads were extracted with SDS-PAGE sample buffer, and eluted proteins were resolved by SDS-PAGE. Resolved proteins were transferred to polyvinylidene difluoride membranes in transfer buffer (48 mM Tris-HCl, 39 mM glycine, 20% methanol) and blocked for 60 min in a blocking solution (10% dry milk, 0.1% Tween 20 in Tris-buffered saline). Receptors were detected by subsequent incubation with mouse anti-HA (1:2000) for MOR or rabbit anti-DOR (1:500) and goat anti-mouse or anti-rabbit antibodies conjugated to alkaline phosphatase (1:5000), both 60-min incubations. Proteins bands were detected by the addition of the ECF substrate, and fluorescence of the bands was determined with Storm 840 (Amersham Biosciences). Band intensities were quantitated and analyzed using ImageQuant (Amersham Biosciences).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The ability of MOR and DOR to heterodimerize has been reported previously (14, 15). In those studies the binding properties and functions of various ligands were examined in cells expressing one type of the receptors and compared with those in cells expressing both receptors. Because cellular background could affect the opioid responses, we decided to re-evaluate the possible interaction between MOR and DOR in the same population of cells that express one type of the receptors and, by induction, express both types of the receptors. This was accomplished by selecting EcR293 cells constitutively expressing MOR and additionally culturing these cells in the presence of PA to stimulate the expression of both MOR and DOR. As summarized in Fig. 1A, in the absence of PA, 0.46 ± 0.025 pmol/mg of MOR was expressed in the EcR293 cells, as defined by the amount of [3H]diprenorphine binding competed by 1 µM CTOP, a MOR-selective antagonist. Meanwhile, 0.057 ± 0.003 pmol/mg of DOR was detected in these EcR293 cells, as defined by specific binding in the presence of 1 µM TIPP{Psi}, a DOR-selective antagonist. The low level or absence of DOR expression in the absence of PA was substantiated by the absence of DOR protein in the Western analyses as shown in the left-hand panel of Fig. 1B. However, when the EcR293 was treated with various concentrations of PA for 48 h, there was a concentration-dependent increase in the DOR-specific binding, with a slight decrease in the MOR binding to a plateau level of ~0.35 pmol/mg of protein. At 2 µM PA, the amount of DOR (0.43 ± 0.039 pmol/mg of protein) statistically was not different from the MOR level (0.37 ± 0.033 pmol/mg of protein) expressed in these EcR293 cells. The presence of DOR protein was also demonstrated with the Western analyses (Fig. 1B). Interestingly, the constitutively expressed MOR could be co-immunoprecipitated with anti-FLAG M2 monoclonal antibodies only when the FLAG-DOR was induced by PA treatment and was PA concentration-dependent (Fig. 1B). Thus, in subsequent experiments EcR293 cells were treated with 2 µM PA for 48 h to achieve a 1:1 MOR:DOR ratio and apparent receptor heterodimerization.



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 1.
Ponasterone A-dependent increase in DOR expression. A, MOR ({blacktriangleup}) and DOR (•) levels expressed in EcR293 cells were determined by [3H]diprenorphine binding assays as described under "Experimental Procedures." The EcR293 cells were cultured at various concentrations of PA for 48 h before harvesting and determining the receptor levels. The values represent the average ±S.E. of three separate 60-mm plates thus treated. B, membranes from EcR293 cells cultured at various concentrations of PA were isolated and extract as described (15). The FLAG-DOR expressed were immunoprecipitated (IP) with M2 anti-FLAG monoclonal antibody, and Western analyses (IB) were carried out first with guinea pig polyclonal antibodies developed against DOR C-terminal sequence, and the presence of MOR was subsequently detected by probing the same blot with anti-HA monoclonal antibodies 16B12.

 
In addition to co-immunoprecipitation, one of the evidences in support of MOR/DOR heterodimerization was the differences in the affinities of the ligands between the receptor heterodimers and homodimers or monomers (14, 15). In current studies the affinities of several MOR-selective agonists for the homodimers or monomers were compared with those for the heterodimers. To limit the differences in cellular background, EcR293 expressing only DOR was used in our comparison. As summarized in Table I, the MOR-selective agonists, endomorphin-2 and PL017, exhibited low or no affinities for the DOR. Interestingly, although endomorphin-1 and -2 have been reported previously to be highly selective agonists for MOR (39), endomorphin-1 exhibited some affinity for DOR, whereas endomorphin-2 did not. Endomorphin-2 has similar selectivity for MOR and DOR as PL017 (Table I). The affinities of these two endomorphins for MOR were very similar, with the high affinity binding lower than those observed with DAMGO and morphine.


View this table:
[in this window]
[in a new window]
  		TABLE I
Affinities and potencies of various opioid ligands for MOR and DOR expressed in EcR293 or HEK293 cells

The relative affinities of various opioid agonists and antagonists for MOR alone (0 PA) or for MOR in the presence of DOR (2 µM PA) were determined by competition binding studies with [3H]diprenorphine (2 nM) and compared to the affinities for DOR alone. The values represent the averages from a minimum of three separate competition binding experiments analyzed by the Prism program. The values in parentheses represent the percentages of receptor in the high affinity states. As for the potencies of various agonists to inhibit 10 µM forskolin-stimulated intracellular cAMP production, the IC50 values and the maximal inhibition levels (values in parentheses) were determined and were averages of a minimum of 3 (PL017) to 13 (endomorphin-1) dose-response curves analyzed by the Prism program.

 
However, when the DOR level was increased to that of MOR in these EcR293 cells, a decrease in the binding affinity was observed. For DAMGO, morphine, and endomorphin-1, there were 3–10-fold decreases in the high affinity binding (Table I). There was a 3-fold decrease in PL017 binding affinity. In contrast, in the presence of DOR the high affinity binding for endomorphin-2 increased 10-fold. Because both endomorphin-2 and PL017 did not exhibit affinity for DOR, the observed alterations in the binding affinities for MOR exhibited by these agonists could not be accounted by their affinities for DOR. Such changes in these ligand affinities for MOR could reflect the alteration in the binding domains within the receptor as a result of heterodimerization.

Apparently, the changes in the ligand affinities for MOR in the presence of DOR were limited to agonists. When the affinities of naloxone and naltrexone were determined, these two antagonists exhibited selectivity for MOR over DOR with a ratio of 1:13 to 1:15 (Table I). Interestingly, when the DOR level was increased to that of MOR in EcR293 cells, these two antagonists exhibited multiple affinity states binding. The affinities of these two antagonists for RH and RL paralleled the affinities for MOR and DOR, respectively. The percentage of receptor in RH (~40%) approached the 1:1 ratio of MOR:DOR when specific binding was determined with CTOP and TIPP{Psi} (Fig. 1A). Thus, the putative heterodimerization did not affect the antagonists binding affinities.

The activation of DOR has been reported to have a synergistic effect on the MOR functional activities (14, 40). We reexamined the abilities of the MOR-selective agonists to inhibit forskolin-stimulated adenylyl cyclase activity in our model system. The abilities of the agonists to control intracellular cAMP levels were measured first in EcR293 cells cultured in the absence of PA. The same agonist abilities to control cAMP levels in EcR293 cells cultured in the presence of 2 µM PA were measured again. This current paradigm should reflect any difference in the opioid responses in similar backgrounds with only the level of DOR changed. As summarized in Table I, all opioid agonists tested exhibited adenylyl cyclase inhibition with IC50 values much lower than their respective affinities for the RH. With the exception of endomorphin-2, which showed increased potency paralleling the increase in this peptide affinity in the presence of DOR, the presence of DOR did not affect other opioid agonists potencies tested. This is not unexpected for as agonist such as PL017, which has no affinity for DOR. The inhibition of intracellular cAMP levels by PL017 should reflect the activation of MOR. However, the absence of changes in the IC50 values for agonists such as DAMGO and morphine, which have been shown to activate DOR in various in vitro cell lines, suggests there is no measurable synergism in the MOR and DOR activation. Nevertheless, the observed increase in endomorphin-2 potency in the presence of DOR suggested the putative heterodimers of MOR/DOR could have different functional properties than the corresponding homodimers or monomers.

The presence of DOR did not alter the opioid receptor-G protein coupling. Earlier studies indicated that the presence of DOR allowed the coupling of the heterodimers to pertussis toxin (PTX)-insensitive G proteins. However, as shown in Fig. 2, this does not appear to be the case. DAMGO inhibited the forskolin-stimulated intracellular cAMP production in EcR293 cells expressing MOR alone or MOR/DOR heterodimers in a dose-dependent manner. Pretreating the cells with 100 ng/ml PTX for 24 h blocked the DAMGO inhibition. At 100 µM, DAMGO inhibited the cAMP production by 13 ± 3.6% in EcR293 cells cultured at 0 PA after PTX treatment, whereas the same concentration of DAMGO did not inhibit the cAMP production in cells cultured in 2 µM PA for 48 h (Fig. 2). Hence, the putative MOR/DOR heterodimers control intracellular cAMP levels via the Gi/Go proteins, as in the case of the homodimers or monomers.



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 2.
Effect of PTX on DAMGO inhibition of forskolin-stimulated intracellular cAMP production. EcR293cells were cultured in the presence or absence of 2 µM PA for 48 h. Eighteen hours before the assays the cells were treated with 100 ng/ml PTX. Then the abilities of DAMGO to inhibit 10 µM forskolin-stimulated intracellular cAMP production in cells cultured without PA ({circ}) and treated with PTX (•) were compared with the cells cultured in the presence of 2 µM PA ({triangleup}) for 48 h and treated with PTX ({blacktriangleup}). The values represent the averages ±S.E. of four separate dose-response curves.

 
The existence of unique binding and function phenotypes when MOR was expressed in the presence of DOR supports the notion of heterodimers for these receptors. Heterodimers could form during the trafficking from endoplasmic reticulum (ER), facilitating the transport of the receptors to the plasma membrane. For example, GABAB1 is retained in the ER during homomeric expression, and GABAB2 is responsible for the trafficking of GABAB1 to the cell surface and the formation of high affinity GABA binding complexes (41, 42). Truncation mutants of D2-dopamine receptor could inhibit the cell surface expression of the wild type, thus suggesting the formation of receptor dimers within the cells (43). MOR and DOR likewise could form heterodimers during the trafficking from ER. To examine such a possibility, the traffic-deficient mutants of MOR were utilized. Our previous studies indicated that deletion of a MOR sequence within the third intracellular loop ({Delta}258RLSKV262, i3–1 mutant) or a sequence residing at the proximal carboxyl tail ({Delta}344KRCFR348, C-2 mutant) resulted in the retention of the mutants at ER (44). As in the case with wild type DOR, where the cell surface expression could be increased with ligands (45), the cell surface expression of these mutants could be rescued by incubating with hydrophobic ligands such as naloxone. If MOR and DOR heterodimerize during the receptor export, it is reasonable to surmise that the wild type DOR should be able to rescue these traffic-deficient mutants of MOR.

When MOR C-2 mutant was transiently expressed in HEK293 cells, the cell surface expression was monitored with FACS analyses using the anti-HA antibodies. As expected, there was 64 ± 7% increase in the cell surface fluorescence when compared with the cells that were mock-transfected (Fig. 3C). Treatment of the HEK293 cells with 10 µM naloxone for 48 h after transfection resulted in a 3.6 ± 0.01-fold increase in cell surface fluorescence when compared with mock transfection. This naloxone-induced increase in cell surface expression of MOR C-2 mutant was similar to those previously reported with HEK293 cells stably expressing the mutant (44). On the other hand, co-transfection of the MOR C-2 mutant with wild type DOR did not increase the cell surface expression of MOR C-2. As shown in Fig. 3C, there was no significant difference in cell surface fluorescence between the cells transfected with receptors and the mock control. Naloxone treatment increased the cell surface fluorescence by 2.1 ± 0.07-fold. The failure to increase MOR C-2 cell surface expression was not due to the absence of DOR expression. When DOR expression was monitored with the FLAG M-2 antibodies, co-expression of wild type DOR with MOR C-2 mutant resulted in a 9.3 ± 0.04-fold increase in cell surface fluorescence when compared with mock control (Fig. 3D). This level of DOR cell surface expression was significantly lower than that observed when HEK293 cells were transfected with DOR alone (12.3 ± 0.7-fold increase in fluorescence). Because culturing of the cells transfected with MOR C-2 mutant and DOR or with DOR alone with naloxone resulted in a similar increase (~15-fold) in the cell surface fluorescence (Fig. 3D), the reduced level of cell surface DOR expression in the presence of MOR C-2 could be due to the intracellular retention of wild type DOR by the traffic-deficient MOR. However, similar reduction in the cell surface expression of the wild type receptors was observed when both MOR and DOR were transiently transfected into the HEK293 cells. Tagging was reversed to demonstrate receptor trafficking was unaffected by the epitope used. As shown in Fig. 3, A and B, co-expression of the MOR and DOR in the same cells together reduced the level of cell surface expression of these opioid receptors when they were expressed in the HEK293 cells separately. Similar to earlier reports (45), naloxone treatment could increase the levels of DOR or MOR expressed in the cell surface. Hence, the observed decrease in the wild type DOR cell surface expression in the presence of MOR C-2 mutant was not due to the retention of the wild type receptor by the traffic-deficient mutant.



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 3.
Inability of wild type opioid receptor to rescue traffic-deficient mutants. MOR or DOR wild type receptor tagged with FLAG epitope was co-transfected into HEK293 cells with the MOR i3–1 ({Delta}258RLSKV262, HA-MOR{Delta}RLKSV) or the MOR C-2 ({Delta}344KRCFR348, HA-MOR{Delta}KRCFR) as described under "Experimental Procedures." The cell surface expressions of the receptors after culturing the cells for 48 h after transfection were determined by FACS analyses (open bars) and compared with those cells cultured for 48 h in the presence of 10 µM naloxone (filled bars). The cell surface fluorescence of these cells was also compared with those that were transfected with the plasmid pCDNA3, mock transfection (hatched bars). In panels A, C, and E, the cell surface expressions of HA-tagged receptors were determined, whereas in panels B, D, and F, the cell surface expression of FLAG-tagged receptors were determined. In panels A and B, HEK293 cells were transfected with FLAG-tagged MOR and HA-tagged DOR. In panels C and D, HEK293 cells were transfected with HA-MOR{Delta}KRCFR and FLAG-tagged DOR. In panels E and F, the cells were transfected with HA-MOR{Delta}RLKSV and FLAG-tagged DOR. The values represent the averages ±S.D. of three separate transfections and FACS analyses. RFU, relative fluorescence units.

 
Wild type DOR could not rescue the traffic-deficient i3–1 mutant of MOR also. As shown in Fig. 3E, there was a basal level of cell surface expression of MOR i3–1 (2.3 ± 0.09-fold increase from mock transfection) that could be increased by culturing the HEK293 cells in the presence of 10 µM naloxone (4.1 ± 0.13-fold). Co-transfection of MOR i3–1 with wild type DOR did not result in the rescuing of the cell surface expression of the traffic-deficient mutant. In contrast, the cell surface fluorescence of the cells thus transfected was similar to that of mock-transfected cells. Naloxone treatment increased the cell surface fluorescence of MOR i3–1 in these cells by 2.0 ± 0.1-fold over mock control. Again, the failure to rescue the traffic-deficient mutant was not due to the absence of wild type DOR expression, as indicated by the cell surface fluorescence detected and summarized in Fig. 3F. Similar deletion of the third intracellular loop sequence of DOR, {Delta}239RLRS242 (DOR i3–1), also resulted in a traffic-deficient mutant of DOR. This suggests that the cell surface expression of this mutant could be rescued by naloxone but not by co-expression with wild type MOR (data not shown). Thus, the inability of wild type receptor to enhance the export of the traffic-deficient mutant suggests that the observed heterodimer phenotypes must be the results of MOR and DOR oligomerization after receptor insertion in the plasma membrane.

Previous studies with DOR and its carboxyl tail-truncated mutant (11) and with MOR and the MOR/DOR receptor chimera (46) have suggested that the opioid receptors internalize as dimers. If the model of domain swapping is the basis for the heterodimers formation, then it is reasonable to assume that MOR and DOR will internalize as a complex. As shown in Fig. 4A, removal of the insect hormone PA from the culturing medium will result in the time-dependent decrease in the receptor level. For the EcR293 cells expressing DOR under the inducible promoter, t1/2 for the disappearance of the cell surface binding site was determined to be 6.1 ± 0.9 h, whereas t1/2 for MOR was determined to be 9.7 ± 1.6 h. Thus, by inducing the expression of either MOR or DOR in the presence of 2 µM PA for 48 h followed by culturing the EcR293 cells in the absence of PA for additional 24 h, we examined whether the turnover of the induced receptors at the cell surface influences the receptor level that is constitutively expressed. Fig. 4, B–E, summarized the data obtained with the FACS analyses experiments. In Fig. 4, B and C, the EcR293 cells stably expressing the PA-inducible HA-tagged DOR (HA-DOR) were transiently transfected with either FLAG-tagged DOR or FLAG-tagged MOR. As shown in these figures, removal of PA in cells transfected with FLAG-DOR resulted in the reduction the HA-DOR level to 28 ± 1.0% that observed in cells treated with 2 µM PA for 48 h. In cells transfected with FLAG-MOR, the HA-DOR level was reduced to 46 ± 1.0% of the initial receptor level. Such data suggest that FLAG-MOR increased the retention time of HA-DOR at the cell surface after PA removal. However, the >50% decrease in the cell surface HA-DOR did not affect the amount of FLAG-DOR or FLAG-MOR detected at the cell surface (Fig. 4, B and C). It was striking to observe that the amount of FLAG-MOR transiently expressed at the cell surface was reduced in the presence of HA-DOR-induced expression. The cell surface amount of FLAG-MOR was increased after the induced level of HA-DOR was decreased upon PA removal (Fig. 4C). Similar observations were obtained with the EcR293 cells expressing inducible HA-MOR and transiently transfected FLAG-DOR or FLAG-MOR. Fig. 4, D and E, indicated that the removal of PA after 48 h of induction resulted in a 70% reduction of the initial HA-MOR cell surface level. Again, the reduction in HA-MOR level did not decrease the FLAG-MOR or FLAG-DOR steady-state level at the cell surface. Thus, these data suggested that the internalization of the HA-DOR or HA-MOR leading to the turnover of these receptors did not affect the steady-state levels of FLAG-DOR or FLAG-MOR that were constitutively expressed. Hence, MOR and DOR endocytosis do not appear to require the formation of homo- or heterodimers.



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 4.
MOR and DOR do not endocytose as dimers. Whether or not MOR and DOR endocytose as dimers was examined by determining the turnover of the cell surface receptors. In A, EcR293 cells expressing PA-inducible HA-DOR ({blacktriangleup}) or inducible HA-MOR ({circ}) were cultured in the presence of 2 µM PA for 48 h. Then the cells were washed to remove the PA, and the amount of receptor remained on the cell surface at different time periods after PA removal was determined with [3H]diprenorphine binding. The values represent the averages from 3 separate 60-mm plates of cells thus treated. In panels B and C, EcR293 cells expressing the PA-inducible HA-DOR were transfected with either FLAG-DOR or FLAG-MOR in pCDNA3, cultured in the presence of 2 µM PA for 48 h, and then cultured in the absence of PA for 24 h. The cell surface receptors were then determined by FACS analyses. The open bars represent the averages ± S.D. of three separate FACS analyses using anti-HA, whereas the filled bars represent the averages of three separate FACS analyses using anti-FLAG. In panels D and E, EcR293 cells expressing PA-inducible HA-MOR were transfected with either FLAG-MOR or FLAG-DOR and were treated with PA as described above. Again, the open bars represent the averages of three separate FACS analyses using anti-HA, and the filled bars represent the averages ±S.D. of three separate FACS analyses using anti-FLAG.

 
It is possible that the measurement of the steady-state cell surface receptor levels does not reflect the internalization kinetics of MOR and DOR heterodimers. Thus, the endocytosis of the cell surface receptor pools was measured with biotinylation studies. Previous studies indicated that DOR could internalize constitutively, whereas MOR could not (47). Thus, if MOR and DOR form domain-swapping heterodimers, such dimers should internalize as complexes. As shown in Fig. 5, the EcR293 cell surface-located MOR could be biotinylated, and there were similar amounts of receptor detected in cells cultured in the presence of 0 or 2 µM PA by Western analyses. However, when the cells were incubated at 37 °C for 1 h after biotinylation and the cell surface biotin labels were stripped before immunoprecipitation, a minimal amount of receptor was detected (2.1 ± 0.5%). These data suggest that the majority of cell surface-located MOR did not internalize during incubation. Similar levels of biotinylated MOR (1.4 ± 0.2%) were detected after stripping the biotin labels from EcR293 cells that were cultured in the presence of 2 µM PA for 48 h (Fig. 5). In the presence of PA, DOR was induced as shown by the Western analyses summarized in Fig. 5. After incubating for 1 h at 37 °C and stripping the cell surface biotin, 82 ± 3.9% of the labels remained. These data suggest that most of the biotin-labeled cell surface-located DOR internalized during the 1 h of incubation. These data also suggest that the cell surface-located MOR was not endocytosed with the constitutively internalized DOR.



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 5.
MOR does not constitutively endocytose and is not affected by DOR constitutive internalization activity. The constitutive internalization of MOR and DOR was examined. The EcR293 cells expressing MOR and inducible DOR were cultured in the absence or presence of 2 µM PA for 48 h. The cell surface located receptors were biotinylated as described. After biotinylation, the cells were incubated at 37 °C for 1 h, and then the cell surface biotin labels were stripped. The receptors were extracted and immunoprecipitated with streptavidin, and the amount of receptors remaining biotinylated were determined by Western analyses using the HA epitope and DOR-specific antibodies as described under "Experimental Procedures."

 
Although turnover studies and constitutive endocytosis studies have indicated that MOR and DOR do not internalize as heterodimers, earlier studies suggested that receptor oligomerization was a prerequisite for agonist-induced receptor internalization (11). Therefore, the ability of etorphine, an opioid receptor non-selective agonist to induce MOR internalization in the presence or absence of DOR, was examined. As reported in earlier studies (46–48), agonist induced rapid internalization of DOR, but slower MOR endocytosis rate was observed due to the recycling of MOR. When the kinetics of etorphine-induced HA-MOR internalization were measured in EcR293 cells cultured without PA, a rate of t1/2 = 8.0 ± 3.4 min was observed, with a maximal level of 46 ± 4.2% receptor internalized (Fig. 6A). Inducing the expression of FLAG-DOR by culturing in the presence of 2 µM PA for 48 h did not significantly alter the rate or maximal level of HA-MOR internalized in the presence of etorphine (t1/2 = 9.1 ± 1.1 minimum, and maximal level = 52 ± 1.8% receptor internalized). At the same time, etorphine induced the FLAG-DOR to internalize 85 ± 12% maximally with a t1/2 = 22 ± 10 min (Fig. 6A). The absence of DOR effect on agonist-induced MOR internalization was not due to the high concentration of etorphine used in the studies. Etorphine EC50 to induce MOR endocytosis in the absence or presence of DOR was determined to be 0.56 ± 0.16 and 0.71 ± 0.17 nM respectively, with maximal receptor internalized at 47 ± 1.8 and 40 ± 1.4% (Fig. 6B). Meanwhile, the EC50 value of etorphine to induce DOR internalization was determined to be 2.2 ± 0.03 nM, with the maximal receptor internalized to be 74 ± 1.4%. Because both the concentration-dependent and the endocytosis kinetics studies did not reveal any changes in agonist-induced MOR internalization in the presence of DOR, it is likely MOR and DOR endocytosed separately.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 6.
Agonist-induced internalization of MOR is independent of the presence of DOR. The ability of etorphine to induce the internalization of MOR and DOR was examined in EcR293 cells constitutively expressing the HA-MOR and the PA-inducible FLAG-DOR. In panel A, the kinetics of 1 µM etorphine to induce the internalization of HA-MOR in cells cultured at 0 µM PA ({circ}) or 2 µM PA ({blacktriangleup}) were determined and compared with the kinetics of FLAG-DOR in cells cultured in the presence of 2 µM PA ({triangleup}). In panel B, EcR293 cells were treated for 1 h with various concentrations of etorphine so as to induce the internalization of HA-MOR in cells cultured in the absence ({circ}) or in the presence ({blacktriangleup}) of 2 µM PA or to induce the internalization of FLAG-DOR in cells cultured at 2 µM PA ({triangleup}). The values in both panels are the averages ±S.D. from three separate FACS analyses.

 
A traffic-deficient mutant of MOR was used to further demonstrate the failure of DOR to promote the internalization of MOR in the presence of agonist. Deletion of the amino acid sequence in the third intracellular loop (iL3) of MOR, {Delta}276RRITR280, resulted in a receptor mutant ({Delta}i3–5) that could not be down-regulated by agonist treatment (49). As shown in Fig. 7, FACS analyses of HEK293 cells transiently transfected with the HA-MOR {Delta}i3–5 mutant and treated with 1 µM etorphine did not result in the reduction of the cell surface fluorescence. Expression of this traffic-deficient mutant in EcR293 cells expressing the FLAG-MOR did not result in an agonist-induced endocytosis of the mutant (Fig. 7). Meanwhile, the rate (t1/2 = 18 ± 6.3 min) and the maximal receptor internalized level (32 ± 7.7%) observed with the wild type FLAG-MOR appeared to be attenuated by the presence of this mutant. Again, the expression of this MOR {Delta}i3–5 mutant in EcR293 cells expressing the FLAG-DOR did not result in the etorphine-induced internalization of the mutant (Fig. 7). The rate (t1/2 = 10 ± 0.3 min) and maximal level of DOR internalized (75 ± 7.3%) were similar to those observed when DOR was co-expressed with wild type MOR (Fig. 6). Parallel results were obtained with the {Delta}257RRITR261 deletion of the iL3 sequence of DOR ({Delta}i3–5). Co-expression of wild type MOR with DOR {Delta}i3–5 mutant receptor did not result in the agonist-induced endocytosis of the mutant (data not shown). Hence, the failure to rescue the traffic-deficient mutant by wild type receptor suggests that agonist-induced receptor internalization does not involve the heterodimerization of the receptors.



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 7.
Inability of wild type opioid receptors to rescue the endocytosis-deficient MOR mutant. HA-MOR{Delta}i3–5 ({Delta}276RRITR280) mutant receptor was co-transfected with wild type FLAG-MOR or FLAG-DOR into HEK293 cells. Forty-eight hours after transfection the kinetics of etorphine-induced receptor endocytosis were determined by FACS analyses. The HA-MOR{Delta}i3–5 levels in cells transfected with the HA-MOR{Delta}i3–5 alone (•), transfected with HA-MOR{Delta}i3–5 and FLAG-DOR ({circ}), or transfected with HA-MOR{Delta}i3–5 and FLAG-MOR ({diamondsuit}) were determined by FACS analyses with the anti-HA 16B12 antibodies. In parallel, the FLAG-DOR level in cells expressing both HA-MOR{Delta}i3–5 and FLAG-DOR ({triangleup}) and FLAG-MOR levels in cells expressing both HA-MOR{Delta}i3–5 and FLAG-MOR ({blacktriangleup}) were determined with the anti-FLAG M2 antibodies. The values represent the averages ±S.D. of three FACS analyses.

 
The absence of MOR and DOR trafficking together likely indicates that the observed heterodimers phenotypes in the binding assays could not be the consequences of domain swapping. Furthermore, the trafficking data suggest that the putative heterodimers only exist at the plasma membrane. Interestingly, the heterodimer phenotypes were observed only with the agonists tested. For antagonists such as naloxone and naltrexone, the binding affinities for MOR in the EcR293 cells when DOR was induced reflected the affinities of the ligands when these two receptors were expressed alone (Table I). These data suggest that the MOR and DOR heterodimers could be the contact dimers formed involving G proteins, i.e. receptor-G protein interactions are required for the formation of dimers. If this is the case, then pretreatment of PTX, i.e. ADP-ribosylating the Gi/Go {alpha}-subunits and uncoupling the receptors from the G proteins, should attenuate the formation of heterodimers. To demonstrate that PTX pretreatment might affect the MOR/DOR heterodimer formation and to minimize the co-immunoprecipitation artifacts, an N terminus His6 epitope-tagged MOR was co-expressed with FLAG-DOR in HEK293 cells. After the receptor complex was extracted with Triton X-100 as described (14), the heterodimers were retained with a Ni2+ column and eluted from the column with 10 mM imidazole, pH 8.0. As shown in Fig. 8A, similar to the co-immunoprecipitation experiments, FLAG-DOR could be retained by the Ni2+ resin in the presence of His6-MOR. Interestingly, a similar Ni2+ column retained drastically lower amounts of FLAG-DOR from HEK293 cells expressing both opioid receptors but previously treated with 100 ng/ml PTX for 24 h. Such a reduction in the amount of DOR retained was not due to the difference in the level of His6-MOR bound to the Ni2+ resin or the amount of FLAG-DOR expressed in the HEK293 cells (Fig. 8A). When membranes were prepared from the PTX-treated cells, the induction of DOR by 2 µM PA did not decrease the morphine affinity for the opioid receptor to the same extent as with membranes isolated from control cells (Fig. 8, B and C). When the affinities of other opioid agonists were determined, the induction of DOR expression in EcR293 cells did not alter the affinities of these agonists significantly also (Table II). With the exception of morphine, all agonists tested exhibited a single affinity state binding after PTX treatment. Instead of a 10-fold decrease in the high affinity binding observed in control membrane (Table I), the induction of DOR expression in cells with MOR only reduced the DAMGO affinity 2-fold. The affinities after PTX treatment for endomorphin-2 and PL017 in membranes containing both MOR and DOR remained similar to those expressing MOR alone (Table II). All these data suggested that the observed MOR-DOR heterodimer phenotypes required the coupling of the receptors with G proteins.



View larger version (47K):
[in this window]
[in a new window]
  		FIG. 8.
PTX pretreatment attenuated the formation of MOR/DOR heterodimers. A, Triton X-100 extracts from HEK293 cells co-expressing His6-MOR and FLAG-DOR or similar cells pretreated with 100 ng/ml PTX for 24 h were partially purified with the Ni2+ columns. Opioid receptors were eluted from the columns with 250 mM imidazole, pH 8.0. Western analyses were carried out with guinea pig antibodies against the carboxyl tail domain of DOR and with rabbit antibodies against the carboxyl tail domain of MOR to detect the opioid receptors retained by the Ni2+ columns. Total amounts of DOR expressed in these cells were determined by immunoprecipitation (IP) of FLAG-DOR using monoclonal anti-FLAG M2 and immunoblotting (IB) with guinea pig (GP) anti-DOR. In B,[3H]diprenorphine competition binding studies with morphine were carried out with membrane prepared from EcR293 cells treated with 0 PA ({circ}) and with membranes isolated from cells treated with 2 µM PA (•) for 48 h. In C, similar competition binding studies were carried out with membranes isolated from EcR293 cells treated with (•) or without ({circ}) 2 µM PA that were pretreated with 100 ng/ml PTX 24 h before harvesting the cells. The values represent the averages of three separate competition binding experiments.

 


View this table:
[in this window]
[in a new window]
  		TABLE II
Agonists affinities for the opioid receptors expressed in EcR293 cells pretreated with pertussis toxin

EcR293 cells constitutively expressing MOR were cultured with 0 or 2 µM PA for 48 h. 24 h before harvesting the cells were treated with 100 ng/ml PTX. Membranes were prepared, and competition binding studies were carried out with 2 nM [3H]diprenorphine. The values represent the average of 3 separate competition curves determined with 15 concentrations of agonists and analyzed with Graphpad Prism 3 program.

 
The involvement of the opioid receptor iL3 domain in the activation of G proteins has been well documented with mutational analyses and peptide competition studies (50). Deletion of the 276RRITR280 sequence within the iL3 domain of MOR resulted in the complete blockade of the agonist effect (49). If interaction and subsequent activation of G proteins are prerequisites for the observed heterodimer phenotypes, then the disruption of G protein interaction by the deletion of RRITR sequence should also result in the blunting of the heterodimers formation. Hence, we subcloned the {Delta}257RRITR261 DOR mutant receptor (DOR{Delta}i3–5) into the pINDsp1 vector system and established a stable EcR293 cell line that expressed the MOR constitutively and the inducible DOR {Delta}i3–5 mutant receptor. As shown in Fig. 9A, the clone we identified expressed a low level of DOR in the absence of PA, and the level of DOR{Delta}i3–5 increased in the presence of PA. Thus, the EcR293 cells expressing the MOR and DOR{Delta}i3–5 were cultured in the presence of PA for 48 h, and the agonist competition binding studies were carried out at various ratios of DOR{Delta}i3–5:MOR. As shown in Fig. 9, B and C, at the low or equal level of DOR{Delta}i3–5 to MOR, the DAMGO or PL017 affinities were similar. When the cells were cultured in the absence of PA, i.e. the DOR{Delta}i3–5: MOR ratio was ~1:6, DAMGO interacted with MOR with multiple affinities (KH = 1.5 ± 0.5 nM (37 ± 3.2%), and KL = 280 ± 76 nM), whereas PL017 exhibited single affinity binding (KD = 110 ± 17 nM). These values were similar to those obtained with EcR293 cells expressing the HA-MOR only (Table I). When the EcR293 cells expressing inducible DOR{Delta}i3–5 were cultured in the presence of 0.2 µM PA for 48 h (resulting in an ~1:1 ratio of DOR{Delta}i3–5:MOR), the affinities of DAMGO (KH = 0.51 ± 0.40 nM, and KL = 150 ± 31 nM) and PL017 (KD = 130 ± 15 nM) were similar to those observed in the absence of PA. A reduction of the percentage of receptor (11 ± 3.6%) existing in the high affinity binding was observed with DAMGO binding. Only when the cells were cultured in the presence of 2 µM PA for 48 h, resulting in an ~3:1 ratio of DOR{Delta}i3–5:MOR, were there reductions in the affinities of DAMGO (KD = 570 ± 100 nM) and PL017 (KD = 480 ± 100 nM).



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 9.
Agonist competition binding studies in EcR293 cells constitutively expressing MOR and inducible DOR{Delta}i3–5. Stable EcR293 cell clones were established to express constitutively MOR and PA-inducible DOR{Delta}i3–5 ({Delta}257RRITR261). In panel A, the amount of MOR and DOR{Delta}i3–5 expressed in cells cultured in the presence of 0 PA (open bars), 0.2 µM PA (hatched bars), or 2 µM PA (filled bars) for 48 h were determined by [3H]diprenorphine binding. In panels B and C, the ability of DAMGO and PL017, respectively, to compete for [3H]diprenorphine binding in the membranes isolated from these EcR293 cells cultured at 0 PA ({circ}), 0.2 µM PA (•), or 2 µM PA ({triangleup}) were determined. The values represent the averages ±S.E. of three separate competition binding assays.

 
The presence of heterodimer phenotypes at the high ratio of DOR{Delta}i3–5:MOR suggests that, unlike the MOR{Delta}i3–5 deletion mutant, this DOR mutant could interact and activate the Gi/Go. The ability of DOR{Delta}i3–5 to interact with the G proteins was demonstrated by agonist competition binding experiments. As expected, the DOR agonist DPDPE exhibited multiple affinity states binding to the wild type receptor expressed in HEK293 cells (Table III). When the competition binding assays were carried out in the presence of 100 mM Na+ and 5 µM GTP{gamma}S, resulting in the uncoupling of the receptor from G proteins, DPDPE was observed to exhibit single low affinity binding to the receptor. Although there was a reduction in the DPDPE affinity for the mutant receptor, a similar decrease in this agonist binding to DOR{Delta}i3–5 in the presence of Na+ and GTP{gamma}S was observed (Table III). As a matter of fact, in the presence of 100 mM Na+ and 5 µM GTP{gamma}S, which uncouple the receptor from G protein, the DPDPE affinity for the DOR{Delta}i3–5 mutant was similar to that of wild type DOR. This suggests that deletion of the RRITR sequence from the iL-3 domain did not affect the binding domain of this opioid peptide. The ability of DOR{Delta}i3–5 to interact and activate Gi/Go could be demonstrated further by examining the ability of DPDPE to inhibit the forskolin-stimulated increase in the intracellular cAMP level. As shown in Table III, the potency and the maximal inhibition level of DPDPE were similar in EcR293 cells expressing either the wild type DOR or DOR{Delta}i3–5. The potency of DPDPE to inhibit the forskolin-stimulated increase of intracellular cAMP production was lower in the DORi3–5 cells. Such a decrease in the agonist potency reflected the parallel decrease in the agonist affinity for the mutant receptor (Table III).


View this table:
[in this window]
[in a new window]
  		TABLE III
Comparison of DPDPE affinity and potency for the wild type and {Delta}257RRITR261 mutant {delta}-opioid receptor

DPDPE affinity for the receptor and its ability to inhibit the forskolin-stimulated intracellular cAMP production in EcR293 cells expressing either wild type FLAG-DOR or PA-inducible HA-DOR{Delta}257RRITR261 (DOR{Delta}i3-5) were carried out as described under "Experimental Procedures." The values represent the average ±S.E. from three separate competition curves or dose-response curves. In competition binding studies, either 5 mM Mg2+ or 100 mM Na+ and 5 µM GTP{gamma}S were present in the assay mixtures. The values in parentheses for the receptor affinity determinations represent the percentage of receptor in the high affinity states. The values in parentheses for the potency determinations represent the calculated maximal inhibition levels.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the current studies, by controlling the expression of DOR with the mammalian ecdysone-inducible expression system, the functions of MOR were measured at different DOR levels in the same cells. Similar to previous reports (14, 15) in which the functions of MOR when expressed independently were compared with different cells co-expressing MOR and DOR, the presence of DOR reduced the affinities of various MOR-selective agonists for the receptor (Table I). However, such a reduction in affinities was restricted to agonists and was not uniform with all the agonists tested. To our surprise, the MOR-selective agonist, endomorphin-2, actually exhibited an increase in its affinity for the receptor when DOR was present. This is in contrast with another MOR-selective agonist, PL017, which exhibited a reduction in its affinity for the receptor. Because these peptide agonists have minimal affinities for DOR, such alterations in the agonists' affinities suggest an interaction between MOR and DOR; hence, the probable existence of the receptor heterodimers. The physical interaction between MOR and DOR was demonstrated in our expression system by the co-immunoprecipitation studies (Fig. 1B), similar to the studies reported earlier (14, 15). In contrast to previous reports (14, 40), the presence of DOR did not potentiate MOR activities. With the exception of endomorphin-2, the abilities of the MOR-selective agonists to inhibit the forskolin-stimulated adenylyl cyclase activity were the same in the presence or absence of DOR expression. In the presence of DOR, the potency of endomorphin-2 was increased 5-fold (Table I), which was within the range of increase in the agonist affinity for the receptor (10-fold). Furthermore, regardless of the agonists used in the assays, pretreatment of EcR293 cells expressing MOR alone or MOR and DOR together with PTX resulted in a complete loss of opioid agonist-mediated inhibition of adenylyl cyclase activity (Fig. 2). This is in direct contrast to the previous report by George et al. (15) in which the ability of MOR/DOR heterodimers to activate PTX-insensitive G proteins was suggested. The induction of DOR in the same cells expressing MOR did not alter the coupling of MOR with respective G proteins. Because the agonists tested have selectivity toward MOR, it is not obvious from our current studies whether the DOR activities could be affected by the presence of MOR.

The prevailing models for heterodimers are either the domain-swapping dimers or the contact dimers (32–35). In either case, the individual receptors within the heterodimers should have similar cellular trafficking patterns. With other GPCRs, effects of one type of the receptor monomer on the trafficking of the other monomer type have been documented (41–43). The dimerization of DOR as the prerequisite for agonist-induced receptor internalization had been suggested (11). The ability of morphine to induce the endocytosis of the wild type MOR in the presence of a MOR/DOR receptor chimera was reported (46). These studies suggested that opioid receptor could internalize as homo- or heterodimers. However, from our current studies on the trafficking of MOR and DOR, the results do not support such hypothesis. Using the traffic-deficient mutants, such as the MOR C-2, MOR i3–1, or DOR i3–1, the corresponding wild types or wild types of other opioid receptors could not rescue the cell surface expression of the these mutant receptors, whereas the opioid antagonist naloxone could (Fig. 3). Although the co-expression of the wild type and mutant receptors has resulted in a decrease in the cell surface expression of the wild type receptors, this was not due the intracellular retention of the wild type receptors as a consequence of the heterodimerization between the wild type and traffic-deficient mutant receptors. Parallel decreases in the cell surface expression of the wild type receptors were observed when both MOR and DOR were co-expressed in the same cells. Similarly, by monitoring the rapid constitutive endocytosis of the receptors (Fig. 5) and by determining the turnover rate of the cell surface receptor content with the removal of the insect hormone inducer ponasterone A (Fig. 4), we demonstrated that MOR and DOR do not endocytose constitutively as homo- or heterodimers. Furthermore, our data also suggest that agonists can induce MOR and DOR to internalize as monomers. In addition, the presence of DOR did not alter the internalization kinetics of MOR (Fig. 6), and wild type receptors could not rescue the endocytosis-deficient mutants (Fig. 7). All these data together suggest that the observed heterodimers phenotypes in the binding assays exist only when MOR and DOR are localized at the cell surface.

In some of our receptor trafficking studies, mutant receptors with deletion in the intracellular domains were used. One could argue that the failure of wild type receptors to rescue the trafficking of these mutant receptors is due to the involvement of intracellular domains in the formation of the opioid receptor heterodimers. The participation of the intracellular domains of the two GABAB receptors in the signaling of the agonist has been reported (36, 37). The carboxyl tail of GABAB2 interacts with the carboxyl tail of GABAB1, thus masking the ER retention signals within the GABAB1 and resulting in the proper trafficking of the receptors (51–53). The heterodimerization between the adrenergic and muscarinic receptors appeared to involve the third intracellular loop (34). Thus, the deletion of amino acid residues within the intracellular loops or the carboxyl tail domains of the opioid receptors could prevent the dimerization of these receptors. Failure to dimerize could contribute to the inability of wild type to rescue the traffic-deficient mutants (i3–1 and C-2) or the endocytosis-deficient mutants (i3–5). However, this could not be the case for the putative MOR and DOR heterodimers. If iL3 domain participates in the homo- or heterodimerization of these opioid receptors, then the respective wild type receptors should be able to rescue the MOR C-2 deletion mutant. The converse is true if the carboxyl tail domain participates in the dimers formation. An argument against such a conclusion could be made if both iL3 and carboxyl domains are involved in the opioid receptor heterodimerization. Such a scenario is not supported by the rhodopsin model. Furthermore, the observation that the presence of wild type DOR does not affect the agonist-dependent and -independent endocytosis of MOR also argues against the notion that iL3 domain (in particular, the i3–5 (RRITR) sequence or carboxyl tail domain, i.e. the C-2 (KRCFR) sequence) is essential for the formation of homo- or heterodimers.

If indeed the formation of the heterodimers of MOR and DOR could be observed only at the cell surface, probably other cellular structures or proteins (such as the cytoskeleton or proteins with multiple protein interaction motifs) could serve as the scaffold for the oligomers. An excellent example is the ability of the third loop of D2- or D3-dopamine receptor to interact with filamin A (ABP-280) of actin, thereby causing an increase in the clustering of receptors at cell surface and the efficiency of effector coupling (54, 55). Also, cellular proteins such as {beta}-arrestin that are recruited to the vicinity of the GPCR could serve as scaffolding proteins (56). Because the alterations in the ligand binding affinities were observed only with the opioid agonists and not the antagonists, we surmise that such alteration is due to the interaction of the receptors with the G protein molecules. As indicated by our PTX studies, the uncoupling of the MOR and DOR from the G proteins attenuated the observed heterodimers phenotypes. George et al. (15) reported similar observations with their binding studies. We further demonstrated with the DOR{Delta}i3–5 deletion mutant, that the putative heterodimer phenotypes were the results of receptors-G protein interactions. The DOR{Delta}i3–5 deletion receptor mutant has a phenotype distinct from that of MOR{Delta}i3–5. With the deletion of the RRITR sequence from the iL3 domain of MOR, the µ-opioid agonists could not inhibit adenylyl cyclase activity, and the MOR receptor mutant exists in a low affinity G protein-uncoupled state (49). However, the deletion of the same sequence from DOR resulted in a reduction of the high affinity agonist binding, with the retention of the ability of the agonist DPDPE to inhibit the adenylyl cyclase activity (Table III). These data suggest that DOR remained coupled to the G protein after the deletion of the RRITR sequence from the iL3 domain, albeit with a lower affinity. Thus, it was not surprising to observe that at a high DOR{Delta}i3–5 to MOR ratio the heterodimer phenotypes persisted, whereas at the equal DOR{Delta}i3–5 to MOR ratio, the heterodimer phenotypes were not observed (Fig. 9). These data together with the PTX pretreatment data strongly suggest that the observed heterodimers phenotypes were due to the interactions between the opioid receptors and their respective G proteins.

If the formation of MOR/DOR heterodimers requires interaction with G proteins, then what could be a possible model that involves the participation of G proteins? In two recent reports, using atomic force microscopy, which allows vertical resolution of ±2 Å or higher in the imaging of the rhodopsin in native disks, Fotiadis et al. (57) and Liang et al. (58) demonstrate that native rhodopsin or opsin proteins are densely packed in the membranes of rod outer segment as rows of oligomers. The individual units of rhodopsin or opsin exists as dimeric structures, with interactions between transmembrane domains 4 and 5 constituting the strongest interaction between the monomers (58). The dimers form rows stemming from the contact between iL3 and transmembrane domains 1 and 2 of the adjacent dimers (58). An interesting observation derived from these studies is that the rhodopsin dimers, in contrast to the monomers with cytoplasmic surfaces that are too small to anchor both the {alpha}- and {beta}{gamma}-subunits of the Gt, provide sufficiently large platforms for such interactions. Hence, a model of 2:1 rhodopsin-Gt interaction is proposed (58). A similar model could apply for the MOR/DOR heterodimers or their respective homodimers. Similar to the reported rhodopsin dimers, computational analyses of putative structures of the opioid receptors dimers have suggested transmembrane domains 4 and 5 as possible contact sites (35). Thus, similar to rhodopsin, a 2:1 receptor-G protein interaction could exist for the opioid receptors. Then, the interaction between the Gi/Go heterotrimers and the platforms formed by the receptor dimers could offer an explanation for our current observations. Tight interaction between opioid receptor and G proteins was demonstrated by the co-immunoprecipitation of the G proteins with receptor-specific antibodies (59). Because motifs within the G{alpha} subunits serve as receptor recognition sites (60, 61), the distinct interaction between receptor and G proteins could define the GPCR function. For the homodimeric receptors, the individual receptor unit has equal affinity for the G{alpha} subunit, and agonist binding would only reflect the high affinity state (the G protein interacting unit) and the low affinity state (the G protein non-interacting unit). However in the case of the MOR/DOR heterodimers, in which DOR has been shown to remain coupled to G proteins after PTX treatment, whereas MOR does not (62), the receptor selective agonist binding should reflect whether the receptor interacts with the G{alpha} subunit or not. The G protein higher affinity for DOR would dictate the decrease of the MOR-selective agonist binding to the receptor with increasing DOR concentration, i.e. the increase level of heterodimers. This is exactly what our data and others (15) have indicated. Our data further appear to indicate that the interaction with the G proteins precedes the formation of the heterodimers. The deletion of the RRITR sequence from the IL3 domain of DOR resulted in the reduction in the DPDPE affinity and potency (Table III). Hence, a higher DOR{Delta}i3–5 mutant receptor level as compared with wild type was needed to reduce agonist affinities, indicative of heterodimer phenotypes. Furthermore, the failure to observe heterodimer phenotypes after PTX pretreatment supports the interaction with the G proteins as the critical step in formation of heterodimers. Such a model is also in agreement with the data in which receptor monomers and not heterodimers are the units in the constitutive or agonist-induced receptor endocytosis. The absolute requirement for {beta}-arrestin for opioid receptor internalization has been demonstrated (22, 63). The replacement of G protein molecule with {beta}-arrestin within the binding platform of the dimers should be the initial step for the endocytosis process. Such binding of {beta}-arrestin could have a similar effect on the stability of the heterodimers as the PTX pretreatment.

Regardless of whether the interaction with G proteins precedes the formation of heterodimers or vice versa, our data clearly suggest that the model of domain swapping cannot be the basis for the opioid receptor heterodimerization. If domain swapping is the basis, then we should be able to detect an alteration in the trafficking of MOR proteins in the presence of DOR. Under all the experimental paradigms used in current studies, the MOR trafficking was not affected by the presence of DOR or the activation of DOR. Thus, the MOR/DOR heterodimers are contact dimers, with transmembrane domains 4 and 5 the most likely candidates for interaction sites as suggested by the modeling studies. The interaction between the G proteins and receptors stabilizes such contact dimers.


   FOOTNOTES
 
* This research is supported in part by National Institutes of Health Grants DA007339, DA015091, DA016674, and DA011806. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Recipient of National Institutes of Health Grant K05 DA00513. Back

Recipient of National Institutes of Health Grant K05 DA70544. Back

§ To whom all correspondence should be addressed: Dept. of Pharmacology, Medical School, University of Minnesota, 6-120 Jackson Hall, 321 Church St. S. E., Minneapolis, MN 55455-0217; Tel.: 612-626-6497; E-mail: lawxx001{at}umn.edu.

1 The abbreviations used are: GPCR, G protein-couple receptor; HA, the hemagglutinin epitope, YPTDVPDYA; FLAG epitope, DYKDDDDKDOR; MOR, µ-opioid receptor; DOR, {delta}-opioid receptor; PA, ponasterone A; CTOP, D-Phe-c[Cys-Tyr-D-Trp-Orn-Thr-Pen]-Thr-NH2; TIPP{Psi}, Tyr-Tic{Psi}[CH2NH]Phe-Phe-OH; DAMGO, Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol; PL017, Tyr-Pro-N-Me-Phe-D-Pro-NH2; endomorphin-1, Tyr-Pro-Trp-Phe-NH2; endomorphin-2, Tyr-Pro-Phe-Phe-NH2; DPDPE, [D-Peu215]enkephalin; iL3, intracellular loop 3; TM, transmembrane domain; DMEM, Dulbecco's modified Eagle's medium; FACS, fluorescence flow cytometry; GABA, {gamma}-amino-n-butyric acid; GTP{gamma}S, guanosine 5'-3-O-(thio)triphosphate; PTX, pertussis toxin; ER, endoplasmic reticulum. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Carl Herbrandson for critical reading of this manuscript. Mouse MOR epitope tagged with FLAG in pCDNA3 vector and with HA in PEAK-10 vector were generous gifts from Dr. Chris Evans (UCLA) and Dr. Roberto D. Polakiewicz (Cell Signalling). Mouse DOR epitope tagged with FLAG in pCDNA3 was a generous gift from Dr. Mark von Zastrow (University of California, San Francisco).



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ciruela, F, Casado, V., Mallol, J., Canela, E.I., Lluis, C., and Franco, R. (1995) J. Neurosci. Res. 42, 818–828[CrossRef][Medline] [Order article via Infotrieve]
  2. Herbert, T. E., Moffett, S., Morello, J. P., Loisel, T. P., Bichet, D. G., Barret, C., and Bouvier, M. (1996) J. Biol. Chem. 271, 16384–16392[Abstract/Free Full Text]
  3. Angers, S., Salahpour, A., Joly, E., Hilairet, S., Chelsky, D., Dennis, M., and Bouvier, M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 3684–3689[Abstract/Free Full Text]
  4. Gouldson, P. R., Snell, C. R., and Reynolds, C. A. (1997) J. Med. Chem. 40, 3871–3886[CrossRef][Medline] [Order article via Infotrieve]
  5. Gouldson, P. R., Snell, C. R., Bywater, R. P., Higgs, C., and Reynolds, C. A. (1998) Protein Eng. 11, 1181–1193[Abstract/Free Full Text]
  6. Monnot, C., Bihoreau, C., Conchon, S., Curnow, K. M., Corvol, P., and Clauser, E. (1996) J. Biol. Chem. 271, 1507–1513[Abstract/Free Full Text]
  7. Ng, G. Y., O'Dowd, B. F., Lee, S. P., Chung, H. T., Brann, M. R., Seeman, P., and George, S. R. (1996) Biochem. Biophys. Res. Commun. 227, 200–204[CrossRef][Medline] [Order article via Infotrieve]
  8. Zawarynski, P., Tallerico, T., Seeman, P., Lee, S. P., O'Dowd, B. F., and George, S. R. (1998) FEBS Lett. 441, 383–386[CrossRef][Medline] [Order article via Infotrieve]
  9. Zeng, F. Y., and Wess, J. (1999) J. Biol. Chem. 274, 19487–19497[Abstract/Free Full Text]
  10. Zhu, X., and Wess, J. (1998) Biochemistry 37, 15773–15784[CrossRef][Medline] [Order article via Infotrieve]
  11. Cvejic, S., and Devi, L. A. (1997) J. Biol. Chem. 272, 26959–26964[Abstract/Free Full Text]
  12. McVey, M., Ramsay, D., Kellett, E., Rees, S., Wilson, S., Pope, A. J., and Milligan, G. (2001) J. Biol. Chem. 276, 14092–14099[Abstract/Free Full Text]
  13. Jordan, B. A., and Devi, L. A. (1999) Nature 399, 697–700[CrossRef][Medline] [Order article via Infotrieve]
  14. Gomes, I., Jordan, B. A., Gupta, A., Trapaidze, N., Nagy, V., and Devi, L. A. (2000) J. Neurosci. 20, RC110: 1–5[Abstract/Free Full Text]
  15. George, S. R., Fan, T., Xie, Z., Tse, R., Tam, V., Varghese, G., and O'Dowd, B. F. (2000) J. Biol. Chem. 275, 26128–26135[Abstract/Free Full Text]
  16. Bai, M., Trivedi, S., and Brown, E. M. (1998) J. Biol. Chem. 273, 23605–23610[Abstract/Free Full Text]
  17. Kunishima, N., Simada, Y., Tsuji, Y., Sato, T., Yamamoto, M., Kumasaka, T., Nakanishi, S., Jingami, H., and Morikawa, K. (2000) Nature 407, 971–977[CrossRef][Medline] [Order article via Infotrieve]
  18. Romano, C., Yang, W. L., and O'Malley, K. L. (1996) J. Biol. Chem. 271, 28612–28616[Abstract/Free Full Text]
  19. Kaupmann, K., Malitschek, B., Schuler, V., Heid, J., Froestl, W., Beck, P., Mosbacher, J., Bischoff, S., Kulik, A., Shigemoto, R., Karschin, A., and Bettler, B. (1998) Nature 396, 683–687[CrossRef][Medline] [Order article via Infotrieve]
  20. Jones, K. A., Borowsky, B., Tamm, J. A., Craigm D. A., Durkin, M. M., Dai, M., Yao, W. J., Johnson, M., Gunwaldsen, C., Huang, L. Y., Tang, C., Shen, Q., Salon, J. A., Morse, K., Laz, T., Smith, K. E., Nagarathnam, D., Noble, S. A., Branchek, T. A., and Gerald, C. (1998) Nature 396, 674–679[CrossRef][Medline] [Order article via Infotrieve]
  21. Salim, K., Fenton, T., Bacha, J., Urien-Rodriguez, H., Bonnert, T., Skynner, H. A., Watts, E., Kerby, J., Heald, A., Beer, M., McAllister, G., and Guest, P. C. (2002) J. Biol. Chem. 277, 15482–15485[Abstract/Free Full Text]
  22. Whistler, J. L., and von Zastrow, M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9914–9919[Abstract/Free Full Text]
  23. Ramsay, D., Kellett, E., McVey, M., Rees, S., and Milligan, G. (2002) Biochem. J. 365, 429–440[CrossRef][Medline] [Order article via Infotrieve]
  24. Mellado, M., Rodriguez-Frade, J. M., Vila-Coro, A. J., Fernandez, S., Martin de Ana, A., Jones, D. R., Toran, K. L., and Martinez-A, C. (2001) EMBO J. 20, 2497–2507[CrossRef][Medline] [Order article via Infotrieve]
  25. Rocheville, M., Lange, D. C., Kumar, U., Sasi, R., Patel, R. C., and Patel, Y. C. (2000) J. Biol. Chem. 275, 7862–7869[Abstract/Free Full Text]
  26. Xie, Z., Lee, S. P., O'Dowd, B. F., and George, S. R. (1999) FEBS Lett. 456, 63–67[CrossRef][Medline] [Order article via Infotrieve]
  27. Ginés, S., Hillion, J., Torvinen, M., Le Crom, S., Casado, V., Canela, E. I., Rondin, S., Lew, J. Y., Watson, S., Zoli, M., Agnati, L., Vernier. P., Lluis, C., Ferré, S., Fuxe, K., and Franco, R. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8606–8611[Abstract/Free Full Text]
  28. Sawyer, G. W., and Ehlert, F. J. (1999) J. Pharmacol. Exp. Ther. 289, 464–476[Abstract/Free Full Text]
  29. Rocheville, M., Lange, D., Kumar, U., Patel, S. C., Patel, R. C., and Patel, Y. C. (2000) Science 288, 154–157[Abstract/Free Full Text]
  30. Liu, F., Wang, Q., Pristupa, Z. B., Yu, X. M., Wang, Y. T., and Niznik, H. B. (2000) Nature 403, 274–280[CrossRef][Medline] [Order article via Infotrieve]
  31. Jordan, B. A., Trapaidze, N., Gomes, I., Nivarthi, R., and Devi, L. A. (2000) Proc. Natl. Acad. Sci. U. S. A. 98, 343–348
  32. Dean, M. K., Higgs, C., Smith, R. E., Bywater, R. P., Snell, C. R., Scott, P. D., Upton, G. J. G., Howe, T. J., and Reynolds, C. A. (2001) J. Med. Chem. 44, 4595–4614[CrossRef][Medline] [Order article via Infotrieve]
  33. Maggio, R., Vogel, Z., and Wess, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3103–3107[Abstract/Free Full Text]
  34. Maggio, R., Barbier, P., Fornai, F., and Corsini, G. U. (1996) J. Biol. Chem. 271, 31055–31060[Abstract/Free Full Text]
  35. Filizola, M., and Weinstein, H. (2002) Biopolymers 66, 317–325[CrossRef][Medline] [Order article via Infotrieve]
  36. Robbins, M. J., Calver, A. R., Filippov, A. K., Hirst, W. D., Russell, R. B., Wood, M. D., Nasir, S., Couve, A., Brown, D. A., Moss, S. J., and Pangalos, M. N. (2001) J. Neurosci. 21, 8043–8052[Abstract/Free Full Text]
  37. Galvez, T., Duthey, B., Kniazeff, J., Blahos, J., Rovelli, G., Bettler, B., Prézeau, L., and Pin, J.-P. (2001) EMBO J. 20, 2152–2159[CrossRef][Medline] [Order article via Infotrieve]
  38. Deleted in proof
  39. Zadina, J. E., Hackler, L., Ge, L. J., and Kastin, A. J. (1997) Nature 386, 499–502[CrossRef][Medline] [Order article via Infotrieve]
  40. Martin, N. A., and Prather, P. L. (2001) Mol. Pharmacol. 59 774–783[Abstract/Free Full Text]
  41. Couve, A., Filippov, A. K., Connolly, C. N., Bettler, B., Brown, D. A., Moss, S. J. (1998) J. Biol. Chem. 273, 26361–26367[Abstract/Free Full Text]
  42. Filippov, A. K., Couve, A., Pangalos, M. N., Walsh, F. S., Brown, D. A., and Moss, S. J. (2000) J. Neurosci. 20, 2867–2874[Abstract/Free Full Text]
  43. Lee, S. P., O'Dowd, B. F., Ng, G. Y. K., Varghese, G., Akil, H., Mansour, A., Ngugen, T., and George, S. R. (2000) Mol. Pharmacol. 58, 120–128[Abstract/Free Full Text]
  44. Chaipatikul, V., Erickson-Herbrandson, L. J., Loh, H. H., and Law, P. Y. (2003) Mol. Pharmacol. 64, 32–41[Abstract/Free Full Text]
  45. Petaja-Repo, U. E., Hogue, M., Bhalla, S., Laperriere, A., Morello, J. P., and Bouvier, M. (2002) EMBO J. 21, 1628–1637[CrossRef][Medline] [Order article via Infotrieve]
  46. He, L., Fong, J., von Zastrow, M., and Whistler, J. L. (2002) Cell 108 271–282[CrossRef][Medline] [Order article via Infotrieve]
  47. Wang, W., Loh, H. H., and Law, P. Y. (2003) J. Biol. Chem. 278, 36848–36858[Abstract/Free Full Text]
  48. Whistler, J. L., Chuang, H. H., Chu, P., Jan, L. Y., and von Zastrow, M. (1999) Neuron 23 737–746[CrossRef][Medline] [Order article via Infotrieve]
  49. Chaipatikul, V., Loh, H. H., and Law, P. Y. (2003) J. Pharmacol. Exp. Ther. 305, 909–918[Abstract/Free Full Text]
  50. Georgoussi, Z., Merkouris, M., Mullaney, I., Megaritis, G., Carr, C., Zioudrou, C., and Milligan, G. (1997) Biochim. Biophys. Acta 1359, 263–274[Medline] [Order article via Infotrieve]
  51. Pagano, A., Rovelli, G., Mosbacher, J., Lohmann, T., Duthey, B., Stauffer, D., Ristig, D., Schuler, V., Meigel, I., Lampert, C., Stein, T., Prezeau, L., Blahos, J., Pin, J., Froestl, W., Kuhn, R., Heid, J., Kaupmann, K., and Bettler, B. (2001) J. Neurosci. 21, 1189–1202[Abstract/Free Full Text]
  52. Calver, A. R., Robbins, M. J., Cosio, C., Rice, S. Q., Babbs, A. J., Hirst, W. D., Boyfield, I., Wood, M. D., Russell, R. B., Price, G. W., Couve, A., Moss, S. J., and Pangalos, M. N. (2001) J. Neurosci. 21, 1203–1210[Abstract/Free Full Text]
  53. Margeta-Mitrovic, M., Jan, Y. N., and Jan, L. Y. (2000) Neurons 27, 97–106[CrossRef][Medline] [Order article via Infotrieve]
  54. Li, M., Bermak, J. C., Wang, W., and Zhou, Q. Y. (2000) Mol. Pharmacol. 57, 446–452[Abstract/Free Full Text]
  55. Lin, R., Karpa, K., Kabbani, N., Goldman-Rakic, P., and Levenson, R. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 5258–5263[Abstract/Free Full Text]
  56. Tohgo, A., Pierce, K. L., Choy, E. W., Lefkowitz, R. J., and Luttrell, L. M. (2002) J. Biol. Chem. 277, 9429–9436[Abstract/Free Full Text]
  57. Fotiadis, D., Liang, Y., Filipek, S., Saperstein, D. A., Engel, A., and Palczewski, K. (2003) Nature 421, 127–128[CrossRef][Medline] [Order article via Infotrieve]
  58. Liang, Y., Fotiadis, D., Filipek, S., saperstein, D. A., Palczewski, K., and Engel, A. (2003) J. Biol. Chem. 278, 21655–21662[Abstract/Free Full Text]
  59. Chalecka-Franaszek, E., Weems, H. B., Crowder, A. T., Cox, B. M., and Cote, T. E. (2000) J. Neurochem. 74, 1068–1078[CrossRef][Medline] [Order article via Infotrieve]
  60. Conklin, B. R., Farfel, Z., Lustig, K. D., Julius, D., and Bourne, H. R. (1993) Nature 363, 274–276[CrossRef][Medline] [Order article via Infotrieve]
  61. Tsu, R. C., Ho, M. K., Yung, L. Y., Joshi, S., and Wong, Y. H. (1997) Mol. Pharmacol. 52, 38–45[Abstract/Free Full Text]
  62. Chakrabarti, S., Yang, W., Law, P. Y., and Loh, H. H. (1997) Mol. Pharmacol. 52, 105–113[Abstract/Free Full Text]
  63. Zhang, J., Ferguson, S. S., Law, P. Y., Barak, L. S., and Caron, M. G. (1999) J. Recept. Signal Transduct. Res. 19 301–313[Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Mol. Pharmacol.Home page
W. Walwyn, S. John, M. Maga, C. J. Evans, and T. G. Hales
{delta} Receptors Are Required for Full Inhibitory Coupling of {micro} Receptors to Voltage-Dependent Ca2+ Channels in Dorsal Root Ganglion Neurons
Mol. Pharmacol., July 1, 2009; 76(1): 134 - 143.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
X. Ge, H. H. Loh, and P.-Y. Law
{micro}-Opioid Receptor Cell Surface Expression Is Regulated by Its Direct Interaction with Ribophorin I
Mol. Pharmacol., June 1, 2009; 75(6): 1307 - 1316.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Tan, W. M. Walwyn, C. J. Evans, and C.-W. Xie
p38 MAPK and {beta}-Arrestin 2 Mediate Functional Interactions between Endogenous {micro}-Opioid and {alpha}2A-Adrenergic Receptors in Neurons
J. Biol. Chem., March 6, 2009; 284(10): 6270 - 6281.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
F. M. Decaillot, R. Rozenfeld, A. Gupta, and L. A. Devi
Cell surface targeting of {micro}-{delta} opioid receptor heterodimers by RTP4
PNAS, October 14, 2008; 105(41): 16045 - 16050.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
M. Grant, H. Alturaihi, P. Jaquet, B. Collier, and U. Kumar
Cell Growth Inhibition and Functioning of Human Somatostatin Receptor Type 2 Are Modulated by Receptor Heterodimerization
Mol. Endocrinol., October 1, 2008; 22(10): 2278 - 2292.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. C. Y. Choi, J. Simon, K. W. K. Tsim, and E. A. Barnard
Constitutive and Agonist-induced Dimerizations of the P2Y1 Receptor: RELATIONSHIP TO INTERNALIZATION AND SCAFFOLDING
J. Biol. Chem., April 18, 2008; 283(16): 11050 - 11063.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
K. Y. Song, C. K. Hwang, C. S. Kim, H. S. Choi, P.-Y. Law, L.-N. Wei, and H. H. Loh
Translational repression of mouse mu opioid receptor expression via leaky scanning
Nucleic Acids Res., March 12, 2007; 35(5): 1501 - 1513.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
L. A. Snook, G. Milligan, B. L. Kieffer, and D. Massotte
{micro}-{delta} Opioid Receptor Functional Interaction: Insight Using Receptor-G Protein Fusions
J. Pharmacol. Exp. Ther., August 1, 2006; 318(2): 683 - 690.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. U. Gehret, A. Bajaj, F. Naider, and M. E. Dumont
Oligomerization of the Yeast {alpha}-Factor Receptor: IMPLICATIONS FOR DOMINANT NEGATIVE EFFECTS OF MUTANT RECEPTORS
J. Biol. Chem., July 28, 2006; 281(30): 20698 - 20714.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/12/11152    most recent
M500171200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Law, P.-Y.
Right arrow Articles by Loh, H. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Law, P.-Y.
Right arrow Articles by Loh, H. H.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement