QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

HOME  |   SEARCH  |   ARCHIVE  |   SUBSCRIBE  |   CONTACT  |   HELP

This Article Abstract Full Text (PDF) Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gomes, I. Articles by Devi, L. A. Search for Related Content PubMed PubMed Citation Articles by Gomes, I. Articles by Devi, L. A.

 Previous Article  |  Next Article 

The Journal of Neuroscience, 2000, 20:RC110:1-5

RAPID COMMUNICATION
Heterodimerization of µ and  Opioid Receptors: A Role in Opiate Synergy

I. Gomes, B. A. Jordan, A. Gupta, N. Trapaidze, V. Nagy, and L. A. Devi

Departments of Pharmacology and Anesthesiology, New York University School of Medicine, New York, New York 10016

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Opiate analgesics are widely used in the treatment of severe pain. Because of their importance in therapy, different strategies have been considered for making opiates more effective while curbing their liability to be abused. Although most opiates exert their analgesic effects primarily via µ opioid receptors, a number of studies have shown that  receptor-selective drugs can enhance their potency. The molecular basis for these findings has not been elucidated previously. In the present study, we examined whether heterodimerization of µ and  receptors could account for the cross-modulation previously observed between these two receptors. We find that co-expression of µ and  receptors in heterologous cells followed by selective immunoprecipitation results in the isolation of µ- heterodimers. Treatment of these cells with extremely low doses of certain -selective ligands results in a significant increase in the binding of a µ receptor agonist. Similarly, treatment with µ-selective ligands results in a significant increase in the binding of a  receptor agonist. This robust increase is also seen in SKNSH cells that endogenously express both µ and  receptors. Furthermore, we find that a  receptor antagonist enhances both the potency and efficacy of the µ receptor signaling; likewise a µ antagonist enhances the potency and efficacy of the  receptor signaling. A combination of agonists (µ and  receptor selective) also synergistically binds and potentiates signaling by activating the µ- heterodimer. Taken together, these studies show that heterodimers exhibit distinct ligand binding and signaling characteristics. These findings have important clinical ramifications and may provide new foundations for more effective therapies.

Key words: receptor subtypes; agonist; antagonist; enkephalin; Deltorphin II; DAMGO; DPDPE; TIPP; G-protein-coupled receptor; oligomerization; MAP kinase

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Opioid receptors can regulate several biological effects, including analgesia, miosis, bradycardia, general sedation, feeding, and hypothermia (Herz, 1993). Morphine, a prototype opioid agonist, binds to µ and  opioid receptors and inhibits neurotransmitter release (MacDonald and Nelson, 1978; Yaksh, 1993). Studies with transgenic animals lacking µ receptors show that morphine functions primarily via µ receptors (Matthes et al., 1996). Interestingly,  ligand-mediated analgesia is altered in these animals, suggesting an interaction between the two receptors (Sora et al., 1997).

A number of pharmacological studies have suggested that µ and  receptors interact and influence each other's properties (Traynor and Elliot, 1993). For example, mice treated with  antagonists exhibit diminished development of morphine tolerance and dependence (Abdelhamid et al., 1991; Zhu et al., 1999). Selective reduction of  receptors by antisense oligonucleotides attenuates the development of morphine dependence (Sanchez-Blazquez et al., 1997). In  receptor knockout animals, the extent of dependence attributable to morphine administration is also selectively altered (Zhu et al., 1999). Ligand binding studies show that µ-selective ligands inhibit the binding of -selective ligands in both competitive and noncompetitive manners (Rothman and Westfall, 1981; Rothman et al., 1983). Findings such as these led to the proposal that  receptors exist as two subtypes: those that are associated with µ receptors and those that are not. However, the biochemical basis for this association was not explored.

Early studies using radioligand binding and electrophysiology suggested that both µ and  receptors colocalize to cells in the dorsal root ganglia (Fields et al., 1980; Egan and North, 1981; Zieglgansberger et al., 1982). Immunohistochemical studies of the opioid receptor distribution in the CNS have shown that µ and  receptors colocalize to the same axonal terminals of the superficial dorsal horn (Arvidsson et al., 1995). Ultrastructural analysis of the dorsal horn neurons also revealed colocalization of  receptors with µ receptors in the plasmallema (Cheng et al., 1997). In addition, several neuroblastomas have been shown to co-express these two opioid receptors (Yu et al., 1986; Kazmi and Mishra, 1987; Baumhaker et al., 1993; Palazzi et al., 1996). Taken together, these studies provide evidence for colocalization of µ and  receptors and suggest that the existence of  and µ receptor complexes is physically possible.

Receptor dimerization is a potential mechanism for modulation of their function (Salahpour et al., 2000). Early studies showing that dimeric analogs of oxymorphone and enkephalin exhibit higher affinity and potency than their monomeric forms suggested that µ receptors could function as dimers (Hazum et al., 1982). We have previously shown that  receptors exist as homodimers and undergo agonist-mediated monomerization (Cvejic and Devi, 1997). Furthermore,  receptors heterodimerize with  receptors, and heterodimerization affects their ligand binding and signaling properties (Jordan and Devi, 1999). Because  receptors show significant sequence homology with µ receptors at the amino acid level (Miotto et al., 1995), we examined whether  receptors physically associate with µ receptors and whether this interaction alters their properties. Here we show that µ and  receptors associate to form a ~150 kDa heterodimer, and these heterodimers exhibit distinct ligand binding and signaling properties. Therefore, µ- heterodimerization represents a novel mechanism that could modulate the function of these receptors.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials. Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO), Tyr-D-Ala-Phe-Glu-Val-Val-Gly (Deltorphin II), and diprenorphine were from RBI (Natick, MA) and Sigma (St. Louis, MO). Naltriben (NTB), benzylidenenaltrexone (BNTX), and SNC 80 were from Tocris Cookson. D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP) and [D-Pen², Pen⁵]enkephalin (DPDPE) were from Peninsula Inc. ³H-DAMGO and ³H-deltorphin II were from NEN (DuPont). Anti-myc, anti-Flag, and anti-tubulin antibodies were from Sigma. Monoclonal antibody against pMAPK (E10) was from Cell Signaling Technologies (New England Biolabs). Tyr-Tic(CH₂NH)-Phe-Phe (TIPP) was a gift from Dr. Peter Schiller (Institut de Reserches Cliniques de Montreal, Canada).

Cell culture and transfection. Human embryonic kidney (HEK)-293 cells expressing wild-type mouse myc- receptors alone or wild-type mouse Flag-µ receptors alone, or co-expressing wild-type myc- with wild-type Flag-µ receptors or wild-type myc- with C-terminally truncated Flag-µ receptors were generated as described previously (Jordan et al., 2000; Trapaidze et al., 2000). Chinese hamster ovary (CHO) cells stably expressing wild-type Flag-µ and wild type myc- receptors were generated using Lipofectamine reagent (Life Technologies) as described (Jordan and Devi, 1999). SKNSH cells that express endogenous µ and  receptors were grown in DMEM containing 10% FBS.

Coimmunoprecipitation and Western blotting. Immunoprecipitation and Western blotting analysis of receptors expressed in HEK-293 cells were essentially as described previously (Jordan and Devi, 1999). Briefly, cells were lysed for 1 hr in buffer G (1% Triton X-100, 10% glycerol, 300 mM NaCl, 1.5 mM MgCl₂, and 1 mM CaCl₂ and 50 mM Tris-Cl, pH 7.4) containing 10-100 mM iodoacetamide and a protease inhibitor mixture (Jordan and Devi, 1999). For immunoprecipitation, 100-200 µg of protein was incubated with 1-2 µg of polyclonal anti-myc antibody overnight at 4°C. Immunocomplexes were isolated by incubation with 10% v/v protein A-Sepharose for 2-3 hr. The beads were washed three times with buffer G, resolved on a nonreducing 8% SDS-PAGE, and subjected to Western blotting as described using M1, monoclonal anti-Flag antibody.

Whole-cell binding assays. The binding assay was performed essentially as described (Gomes et al., 2000). Briefly, cells were incubated with indicated concentrations of ³H-DAMGO or ³H-Deltorphin II in 50 mM Tris-Cl buffer, pH 7.4, for 2 hr at 37°C in the absence or presence of various ligands (at 10 nM). Under these conditions the level of agonist-mediated receptor internalization is insignificant (I. Gomes and L. A. Devi, unpublished observations). Cells were washed three times with cold buffer, and the radioactivity was determined after solubilization as described (Gomes et al., 2000). Concentrations of ³H-DAMGO or ³H-Deltorphin II were from 0.1 to 10 nM for saturation analysis, and ³H-DAMGO was 3 nM for the determination of EC₅₀ values. Nonspecific binding was determined with 100 nM DAMGO, Deltorphin II, or Diprenorphine.

Functional assays. The opioid-induced increase in MAP kinase phosphorylation in SKNSH cells or CHO cells co-expressing µ and  receptors was essentially as described previously (Jordan et al., 2000; Trapaidze et al., 2000). Briefly, cells were treated for 5 min at 37°C with indicated concentrations of either DAMGO ± 10 nM TIPP or Deltorphin II ± 10 nM CTOP. The level of phosphorylated MAPK (p44/42 MAPK; Erk1/2) was determined by Western blotting using anti-phospho-MAP kinase antibody and the levels of tubulin using anti-tubulin antibody.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

µ and  receptors associate to form detergent-stable heterodimers

A number of pharmacological studies have provided indirect evidence for the interaction between µ and  receptors (Traynor and Elliot, 1993). We directly examined the association between these two classic receptors by co-expressing myc-tagged  receptors with Flag-tagged µ receptors. Myc-tagged  receptors in cell lysates were immunoprecipitated with polyclonal anti-myc antibodies and the Flag-tagged µ receptors in the immunoprecipitate were visualized with monoclonal anti-Flag antibody (Cvejic and Devi, 1997). We find that µ receptors interact with  receptors to form a ~150 kDa heterodimer only in cells co-expressing both receptors (Fig. 1). We also see the presence of higher molecular weight forms representing oligomers only in cells co-expressing both receptors. Pretreatment of cells with a reducing agent (1 mM DTT) results in the destabilization of dimers (Fig. 1). These heterodimers are not induced during solubilization/immunoprecipitation conditions because they are not seen in immunoprecipitates from a mixture of cells individually expressing µ and  receptors (Fig. 1). Interestingly, when a mutant µ receptor lacking C-terminal 42 amino acids is co-expressed with wild-type  receptors, a band representing µ- heterodimer is seen; the decrease in the size of the band is consistent with the size of the truncated µ receptor (Fig. 1), suggesting that the C terminus of µ receptors does not play an important role in the heterodimerization of these two receptors.

View larger version (67K): [in this window] [in a new window]   Figure 1.   µ and  receptors interact with each other to form heterodimers. Immunoprecipitation of cell lysates from HEK-293 cells individually expressing either myc- (Myc) or Flag-µ (Flagµ) receptors, mixed cells individually expressing myc- or Flag-µ receptors (Myc + Flagµ), or cells co-expressing myc- and Flag-µ (Myc-Flagµ) was performed using anti-myc antibodies. Western blotting of these immunocomplexes using anti-Flag antibodies shows a ~150 kDa protein representing the µ- heterodimer only in cells co-expressing both myc- and Flag-µ receptors. Pretreatment of cells co-expressing µ and  receptors with 1 mM DTT (Myc-Flagµ + DTT) results in the destabilization of dimers. µ- heterodimers are also seen in cells co-expressing wild-type  receptors and C-terminally truncated µ receptors (Myc-Flagµ42).

ligands are able to uncover a population of µ receptors

To explore whether heterodimerization affects the ligand binding properties of the receptors, we examined the ability of a  antagonist (TIPP) to modulate the binding of a µ agonist, ³H-DAMGO. We find a substantial increase (~100%) in the number of µ binding sites in the presence of TIPP only in cells expressing both µ and  receptors (Fig. 2A, Table 1). This effect is also seen in SKNSH cells, a neuroblastoma cell line that expresses endogenous µ and  receptors (Fig. 2A, Table 1). An increase (~60%) in ligand binding is also observed when the binding assay is performed at 4°C for 5 hr (Gomes and Devi, unpublished observations), suggesting that one of the mechanisms for the observed drug effect could be the modulation of the µ binding site by direct interaction with  receptors. Taken together, these results support the notion that TIPP is able to synergize with ³H-DAMGO to reveal a hidden population of receptors in heterologous cells expressing µ and  receptors as well as in neuroblastoma cells endogenously expressing these two receptors.

View larger version (27K): [in this window] [in a new window]   Figure 2.   A, Binding of µ agonist to CHO cells stably co-expressing µ and  receptors (µ-) or neuroblastoma cells expressing endogenous µ and  receptors (SKNSH) in the absence or presence of  antagonist. Cells were incubated with indicated concentrations of 3H-DAMGO in the absence (Control) or presence of 10 nM TIPP (+TIPP) as described above. In both cell types, the population of µ receptors increased in the presence of TIPP; this increase is not seen in cells expressing only µ receptors (B, µ). Data represent mean ± SEM from seven independent experiments performed in triplicate. Similar results were obtained in additional clones expressing a lower number of µ and  receptors. B, µ agonist binding in the presence of  agonists, Deltorphin II, and DPDPE. CHO cells stably expressing µ- heterodimers (µ-) or µ receptors alone (µ) were incubated with indicated concentrations of 3H-DAMGO in the absence (Control) or presence of 10 nM Deltorphin II (+Delt II) or 10 nM DPDPE (+DPDPE) as described. The population of µ receptors is increased in the presence of Deltorphin II but not DPDPE; this increase is not seen in cells expressing only µ receptors (µ). Data represent mean ± SEM from three to five independent experiments performed in triplicate.

View this table: [in this window] [in a new window]   Table 1.   Ligand binding properties

We next examined whether two agonists are able to synergize in cells expressing µ- heterodimers and whether this would lead to an increase in µ binding sites. We find that treatment with Deltorphin II, but not DPDPE, leads to a significant increase in µ agonist binding; this increase is not seen in cells expressing only µ receptors (Fig. 2B, Table 1). Taken together, these results show that both the -selective antagonist and the agonist can synergize with the µ receptor agonist and uncover a population of receptors with unique binding properties. This property of the µ- heterodimer is distinct from that of the - heterodimer because in the latter case the  antagonist was not able to synergize with the  agonist; only two agonists or two antagonists were able to show synergistic effects (Jordan and Devi, 1999).

µ ligands can uncover a population of  receptors

To examine whether µ receptor-selective ligands are able to modulate the binding of a  agonist, we examined the effect of a µ-selective antagonist (CTOP) on the binding of a -selective agonist, ³H-Deltorphin II. We find that there is a significant increase in the number of  binding sites in the presence of CTOP only in cells expressing µ- heterodimers and not in cells expressing only  receptors (Fig. 3, Table 1). Furthermore, a µ-selective agonist, DAMGO, is also able to increase the number of  binding sites as seen in SKNSH cells (Table 1). These results suggest that µ-selective ligands are able to uncover a hidden population of  receptors in cells expressing µ and  receptors.

View larger version (31K): [in this window] [in a new window]   Figure 3.   A, Binding of a  agonist, 3H-Deltorphin II, to CHO cells stably co-expressing µ and  receptors (µ-) or  receptor alone () in the absence or presence of µ antagonist, CTOP. Cells were incubated with indicated concentrations of 3H-Deltorphin II in the absence (Control) or presence of 10 nM CTOP (+CTOP), and the extent of 3H-Deltorphin II binding to cells was measured as described. The population of  receptors is increased in the presence of CTOP; this increase is not seen in cells expressing only  receptors (). Data representing mean ± SEM (n = 3) are shown. B, Functional properties of µ- heterodimer. SKNSH cells endogenously expressing µ and  receptors were treated with indicated concentrations of DAMGO in the absence or presence of 10 nM TIPP or 10 nM Deltorphin II. Alternatively, cells were treated with indicated concentrations of Deltorphin II in the absence or presence of 10 nM CTOP. The extent of MAP kinase phosphorylation was determined using Western blot analysis as described. The extent of phospho-MAP kinase (pMAPK) in cells treated with individual or a combination of ligands at a fixed concentration is shown in the top panel. pMAPK/Tubulin refers to the ratio of phospho-MAPK levels to the tubulin levels; the level in untreated cells is taken as zero. Data represent mean ± SEM (n = 4). Statistically significant differences (Dunnett's test) from the control values are indicated by *p = <0.005. EC50 = dose that gives 50% of maximum response.

µ- heterodimers have unique ligand binding properties

To further characterize the binding properties of the µ- heterodimers, we took advantage of the availability of various  receptor-selective ligands. Among the ligands tested, treatment with a relatively low dose (picomole) of TIPP or Deltorphin II results in a substantial increase (approximately twofold) in ³H-DAMGO binding (data not shown). At a higher dose (nanomole), BNTX is also able to increase ³H-DAMGO binding albeit to a lower extent (~1.3-fold). Interestingly, neither DPDPE nor a structurally unrelated agonist, SNC 80, is effective even at relatively high doses (1 µM). These results suggest that the synergistic binding exhibits ligand selectivity and that this cooperative binding requires very low doses of a subset of selective ligands.

µ- heterodimers represent functional receptors

We also examined whether the heterodimer represents a functional receptor and whether the synergistic binding leads to a potentiation of effector function. The activation of opioid receptors results in an increase in the level of phosphorylated MAP kinase (Trapaidze et al., 2000). We examined whether the  antagonist (TIPP) or agonist (Deltorphin II) could potentiate the µ agonist (DAMGO)-induced phosphorylation of p-42/44 MAP kinases (Erk1/2). Treatment with TIPP or Deltorphin II leads to a significant increase in the potency (~6- or 90-fold, respectively) and efficacy (approximately twofold) of MAP kinase phosphorylation by DAMGO (Fig. 3B). In a reciprocal experiment, we examined whether the µ antagonist (CTOP) could potentiate the  agonist (Deltorphin II)-induced signaling. As shown in Figure 3B, treatment with CTOP significantly increases the potency and efficacy of MAP kinase phosphorylation by Deltorphin II (Fig. 3B). These results imply that µ- heterodimers represent functional receptors and that the selective ligands are able to enhance signaling by opioid agonists.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

µ receptors associate with  receptors to form µ- heterodimers

In this paper we provide biochemical, pharmacological, and functional evidence for dimerization between µ and  opioid receptors. Previous studies using moderately selective ligands provided indirect evidence for the interaction between these two receptors (Traynor and Elliot, 1993). This led to alternate explanations of the findings because these ligands could interact with both receptors. In the present study, we used highly selective ligands and heterologous cells expressing both  and µ receptors (or individually expressing these receptors) to demonstrate direct physical and functional interactions between these two classic opioid receptors.

Does the µ- heterodimer represent a  receptor subtype?

The complexities of opioid receptor pharmacology have often been attributed to two different phenomena: receptor-receptor interactions and opioid receptor subtypes. A number of investigators have proposed that receptor-receptor interactions could form the basis for some of the opioid receptor subtypes (Porreca et al., 1992; Xu et al., 1993; Jordan and Devi, 1999). It was shown that a subset of -selective ligands enhanced µ receptor-mediated analgesia; these ligands were thus proposed to bind to  receptors complexed with µ receptors (Traynor and Elliot, 1993). Two independent studies demonstrated that the µ--complexed receptors were sensitive to characteristic 2 selective ligands, suggesting that they represented the 2 receptor subtype (Porreca et al., 1992; Xu et al., 1993). Interestingly, recent work with antisense oligonucleotides to  receptors and  receptor knockout mice showed a blockade of analgesia induced by a 2-selective ligand (Deltorphin II) but not that induced by a 1-selective ligand, DPDPE (Bilsky et al., 1996; Zhu et al., 1999). Remarkably, no DPDPE or Deltorphin II binding could be observed in either case. These results would then suggest that the cloned  receptor may represent the 2 subtype and not the previously thought 1 subtype. However, when expressed in heterologous cells, the cloned  receptor is able to bind both 1- and 2-selective ligands with high affinity. It is therefore unclear as to what represents a 2 (and a 1) binding site. We have shown previously that heterodimers of  and  receptors reveal ligand binding sites that are virtually identical to previously described 2 subtypes (Jordan and Devi, 1999). It is therefore likely that the heterodimers described here represent a novel  receptor subtype. Consistent with this notion, we find that the µ- heterodimer is able to bind some 1 (BNTX)- and 2 (Deltorphin II)-selective ligands and not others (DPDPE, NTB).

Direct interactions and G-proteins in receptor dimerization

One interesting observation is that only a subset of selective ligands including antagonists are able to enhance µ agonist binding. This effect is also seen (to a lesser extent) when the binding assay is performed at 4°C. These findings strongly point to a phenomenon occurring at the level of ligand binding. Because most, if not all, cellular processes arrest at low temperatures and given the ability of antagonists to synergize with agonists, it is apparent that neither downstream effects nor receptor activation are required for this phenomenon to occur. The simplest explanation is that heterodimerization alters the binding pocket of both receptors and that the binding of one ligand can "restore" the binding site of the other.

The possibility that G-proteins may be involved in the interactions cannot be ignored. It is possible that G-protein switching between receptors may cause these alterations in affinity and the number of binding sites. In such a model, the binding of receptor selective ligands (agonists and antagonists) could cause the selective uncoupling of G-proteins that, if in limiting conditions, may significantly affect the binding of ligands to a nearby receptor that uses a similar G-protein. It is therefore possible that G-proteins could play a role not only in determining ligand selectivities but also in mediating the synergistic effects on downstream signaling observed as a potentiation of function.

In summary, heterodimerization between µ and  represents a novel mechanism that could modulate receptor function and provides a new strategy for the development of novel therapies.

	FOOTNOTES

Received Aug. 17, 2000; accepted Aug. 30, 2000.

This work was supported in part by National Institutes of Health Grants DA 08863 and DA 00458 to L.A.D. and National Research Service Award Grant DA05885 to B.A.J. We thank Dr. Peter Schiller for the gift of TIPP.

Correspondence should be addressed to Dr. Lakshmi A. Devi, Department of Pharmacology, New York University School of Medicine, MSB 411, 550 First Avenue, New York, NY 10016. E-mail: lakshmi.devi{at}med.nyu.edu.

This article is published in The Journal of Neuroscience, Rapid Communications Section, which publishes brief, peer-reviewed papers online, not in print. Rapid Communications are posted online approximately one month earlier than they would appear if printed. They are listed in the Table of Contents of the next open issue of JNeurosci. Cite this article as: JNeurosci, 2000, 20:RC110 (1-5). The publication date is the date of posting online at www.jneurosci.org.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

• Abdelhamid E, Sultana M, Portoghese P, Takemori A (1991) Selective blockage of delta opioid receptors prevents the development of morphine tolerance and dependence in mice. J Pharmacol Exp Ther 258:299-303.
• Arvidsson U, Riedl M, Chakrabarti S, Lee J, Nakano A, Dado R, Loh H, Law P, Wessendorf M, Elde R (1995) Distribution and targeting of a mu-opioid receptor (MOR1) in brain and spinal cord. J Neurosci 15:3328-3341.
• Baumhaker Y, Wollman Y, Goldstein M, Sarne Y (1993) Evidence for mu-, delta-, and kappa-opioid receptors in a human neuroblastoma cell line. Life Sci 52:205-210.
• Bilsky EJ, Bernstein RN, Hruby VJ, Rothman RB, Lai J, Porreca F (1996) zensCharacterization of antinociception to opioid receptor selective agonists after antisense oligodeoxynucleotide-mediated "knock-down" of opioid receptor in vivo. J Pharmacol Exp Ther 277:491-501.
• Cheng P, Liu-Chen L, Pickel V (1997) Dual ultrastructural immunocytochemical labeling of mu and delta opioid receptors in the superficial layers of the rat cervical spinal cord. Brain Res 778:367-380.
• Cvejic S, Devi L (1997) Dimerization of the  opioid receptor: implication for a role in receptor internalization. J Biol Chem 272:26959-26964.
• Egan T, North R (1981) Both mu and delta opiate receptors exist on the same neuron. Science 214:923-924.
• Fields H, Emson P, Leigh B, Gilbert R, Iversen L (1980) Multiple opiate receptor sites on primary afferent fibres. Nature 284:351-353.
• Gomes I, Trapaidze N, Turndorf H, Devi LA, Bansinath M (2000) Acute ethanol treatment modulates delta opioid receptors in N18TG2 cells. Anesthesiology 92:1789-1798.
• Hazum E, Chang K, Leighton H, Lever Jr O, Cuatrecasas P (1982) Increased biological activity of dimers of oxymorphone and enkephalin: possible role of receptor crosslinking. Biochem Biophys Res Commun 104:347-353.
• Herz A (1993) In: Opioids, Vol 1. Berlin: Springer.
• Jordan B, Devi L (1999) G-protein coupled receptor heterodimerization modulates receptor function. Nature 399:697-700.
• Jordan B, Cvejic S, Devi L (2000) Kappa opioid receptor endocytosis by dynorphin peptides. DNA Cell Biol 19:19-27.
• Kazmi S, Mishra R (1987) Comparative pharmacological properties andfunctional coupling of mu and delta opioid receptor sites in human neuroblastoma SH-SY5Y cells. Mol Pharmacol 32:109-118.
• MacDonald RL, Nelson PG (1978) Specific-opiate-induced depression of transmitter release from dorsal root ganglion cells in culture. Science 199:1449-1451.
• Matthes H, Maldonado R, Simonin F, Valverde O, Slowe S, Kitchen I, Befort K, Dierich A, Le Meur M, Dolle P, Tzavara E, Hanoune J, Roques B, Kieffer B (1996) Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the mu-opioid-receptor gene. Nature 383:819-823.
• Miotto K, Magendzo K, Evans CJ (1995) Molecular characterization of opioid receptors. In: The pharmacology of opioid peptides, Ch 3 (Tseng L, ed), pp 57-71. Location: Harwood Academy Publishers.
• Palazzi E, Ceppi E, Guglielmetti F, Catozzi L, Amoroso D, Groppetti A (1996) Biochemical evidence of functional interaction between mu- and delta-opioid receptors in SK-N-BE neuroblastoma cell line. J Neurochem 67:138-144.
• Porreca F, Takemori A, Sultana M, Portoghese P, Bowen W, Mosberg H (992) Modulation of mu-mediated antinociception in the mouse involves opioid delta-2 receptors. J Pharmacol Exp Ther 263:147-152.
• Rothman R, Westfall T (1981) Allosteric modulation by leucine-enkephalin of [3H]naloxone binding in rat brain. Eur J Pharmacol 72:365-368.
• Rothman R, Bowen W, Schumacher U, Pert C (1983) Effect of beta-FNA on opiate receptor binding: preliminary evidence for two types of mu receptors. Eur J Pharmacol 95:147-148.
• Salahpour A, Angers S, Bouvier M (2000) Functional significance of oligomerization of G protein-coupled receptors. Trends Endocrinol Metab 11:163-168.
• Sanchez-Blazquez P, Garcia-Espana A, Garzon J (1997) Antisense oligodeoxynucleotides to opioid mu and delta receptors reduced morphine dependence in mice: role of delta-2 opioid receptors. J Pharmacol Exp Ther 280:1423-1431.
• Sora I, Funada M, Uhl G (1997) The mu-opioid receptor is necessary for [D-Pen2, D-Pen5]enkephalin-induced analgesia. Eur J Pharmacol 324:2-3.
• Trapaidze N, Gomes I, Cvejic S, Bansinath M, Devi LA (2000) Opioid receptor endocytosis and activation of MAP kinase pathway. Mol Brain Res 76:220-228.
• Traynor J, Elliot J (1993) -opioid receptor subtypes and cross talk with µ-receptors. Trends Pharmacol Sci 14:84-86.
• Xu H, Partilla J, de Costa B, Rice K, Rothman R (1993) Differential binding of opioid peptides and other drugs to two subtypes of opioid delta ncx binding sites in mouse brain: further evidence for delta receptor heterogeneity. Peptides 14:893-907.
• Yaksh T (1993) In: The spinal actions of opioids. Berlin: Springer.
• Yu V, Richards M, Sadee W (1986) A human neuroblastoma cell line expresses mu and delta opioid receptor sites. J Biol Chem 261:1065-1070.
• Zhu Y, King M, Schuller A, Nitsche J, Reidl M, Elde R, Unterwald E, Pasternak G, Pintar J (1999) Retention of supraspinal delta-like analgesia and loss of morphine tolerance in delta opioid receptor knockout mice. Neuron 24:243-252.
• Zieglgansberger W, French E, Mercuri N, Pelayo F, Williams J (1982) Multiple opiate receptors on neurons of the mammalian central nervous system. Life Sci 31:2343-2346.

---

Copyright © 2000 Society for Neuroscience  0270-6474/00/$05.00/0

This article has been cited by other articles:

				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]

				R. Guan, X. Feng, X. Wu, M. Zhang, X. Zhang, T. E. Hebert, and D. L. Segaloff Bioluminescence Resonance Energy Transfer Studies Reveal Constitutive Dimerization of the Human Lutropin Receptor and a Lack of Correlation between Receptor Activation and the Propensity for Dimerization J. Biol. Chem., March 20, 2009; 284(12): 7483 - 7494. [Abstract] [Full Text] [PDF]

				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]

				K. T. Sykes, S. R. White, R. W. Hurley, H. Mizoguchi, L. F. Tseng, and D. L. Hammond Mechanisms Responsible for the Enhanced Antinociceptive Effects of {micro}-Opioid Receptor Agonists in the Rostral Ventromedial Medulla of Male Rats with Persistent Inflammatory Pain J. Pharmacol. Exp. Ther., August 1, 2007; 322(2): 813 - 821. [Abstract] [Full Text] [PDF]

				R. Rozenfeld and L. A. Devi Receptor heterodimerization leads to a switch in signaling: {beta}-arrestin2-mediated ERK activation by {micro}-{delta} opioid receptor heterodimers FASEB J, August 1, 2007; 21(10): 2455 - 2465. [Abstract] [Full Text] [PDF]

				M. Roumy, C. Lorenzo, S. Mazeres, S. Bouchet, J.-M. Zajac, and C. Mollereau Physical Association between Neuropeptide FF and {micro}-Opioid Receptors as a Possible Molecular Basis for Anti-opioid Activity J. Biol. Chem., March 16, 2007; 282(11): 8332 - 8342. [Abstract] [Full Text] [PDF]

				B. H. Meyer, J.-M. Segura, K. L. Martinez, R. Hovius, N. George, K. Johnsson, and H. Vogel FRET imaging reveals that functional neurokinin-1 receptors are monomeric and reside in membrane microdomains of live cells PNAS, February 14, 2006; 103(7): 2138 - 2143. [Abstract] [Full Text] [PDF]

				W. Guo, L. Shi, M. Filizola, H. Weinstein, and J. A. Javitch From The Cover: Crosstalk in G protein-coupled receptors: Changes at the transmembrane homodimer interface determine activation PNAS, November 29, 2005; 102(48): 17495 - 17500. [Abstract] [Full Text] [PDF]

				J. A. Javitch The Ants Go Marching Two by Two: Oligomeric Structure of G-Protein-Coupled Receptors Mol. Pharmacol., November 1, 2004; 66(5): 1077 - 1082. [Abstract] [Full Text] [PDF]

				I. Gomes, A. Gupta, J. Filipovska, H. H. Szeto, J. E. Pintar, and L. A. Devi A role for heterodimerization of {micro} and {delta} opiate receptors in enhancing morphine analgesia PNAS, April 6, 2004; 101(14): 5135 - 5139. [Abstract] [Full Text] [PDF]

				F. Trettel, S. Di Bartolomeo, C. Lauro, M. Catalano, M. T. Ciotti, and C. Limatola Ligand-independent CXCR2 Dimerization J. Biol. Chem., October 17, 2003; 278(42): 40980 - 40988. [Abstract] [Full Text] [PDF]

				M. C. Dinger, J. E. Bader, A. D. Kobor, A. K. Kretzschmar, and A. G. Beck-Sickinger Homodimerization of Neuropeptide Y Receptors Investigated by Fluorescence Resonance Energy Transfer in Living Cells J. Biol. Chem., March 14, 2003; 278(12): 10562 - 10571. [Abstract] [Full Text] [PDF]

				A. C. Hanyaloglu, R. M. Seeber, T. A. Kohout, R. J. Lefkowitz, and K. A. Eidne Homo- and Hetero-oligomerization of Thyrotropin-releasing Hormone (TRH) Receptor Subtypes. DIFFERENTIAL REGULATION OF beta -ARRESTINS 1 AND 2 J. Biol. Chem., December 20, 2002; 277(52): 50422 - 50430. [Abstract] [Full Text] [PDF]

				J. F. Nitsche, A. G. P. Schuller, M. A. King, M. Zengh, G. W. Pasternak, and J. E. Pintar Genetic Dissociation of Opiate Tolerance and Physical Dependence in delta -Opioid Receptor-1 and Preproenkephalin Knock-Out Mice J. Neurosci., December 15, 2002; 22(24): 10906 - 10913. [Abstract] [Full Text] [PDF]

				M. D. Hayward, J. E. Pintar, and M. J. Low Selective Reward Deficit in Mice Lacking beta -Endorphin and Enkephalin J. Neurosci., September 15, 2002; 22(18): 8251 - 8258. [Abstract] [Full Text] [PDF]

				C. Lavoie, J.-F. Mercier, A. Salahpour, D. Umapathy, A. Breit, L.-R. Villeneuve, W.-Z. Zhu, R.-P. Xiao, E. G. Lakatta, M. Bouvier, et al. beta 1/beta 2-Adrenergic Receptor Heterodimerization Regulates beta 2-Adrenergic Receptor Internalization and ERK Signaling Efficacy J. Biol. Chem., September 13, 2002; 277(38): 35402 - 35410. [Abstract] [Full Text] [PDF]

				J. D. Lowe, J. P. Celver, V. V. Gurevich, and C. Chavkin {micro}-Opioid Receptors Desensitize Less Rapidly than delta -Opioid Receptors Due to Less Efficient Activation of Arrestin J. Biol. Chem., May 3, 2002; 277(18): 15729 - 15735. [Abstract] [Full Text] [PDF]

				H. Ni, J. Capodici, G. Cannon, D. Communi, J.-M. Boeynaems, K. Kariko, and D. Weissman Extracellular mRNA Induces Dendritic Cell Activation by Stimulating Tumor Necrosis Factor-alpha Secretion and Signaling through a Nucleotide Receptor J. Biol. Chem., April 5, 2002; 277(15): 12689 - 12696. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gomes, I. Articles by Devi, L. A. Search for Related Content PubMed PubMed Citation Articles by Gomes, I. Articles by Devi, L. A.

Home  |   Search  |   Archive  |   Subscribe  |   Contact  |   Help