Heterodimerization of Μ and Δ Opioid Receptors: a Role in Opiate Synergy

The Journal of Neuroscience, 2000, Vol. 20 RC110 1of5 Heterodimerization of ␮ 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 Opiate analgesics are widely used in the treatment of severe ␮-selective ligands results in a significant increase in the bind- pain. Because of their importance in therapy, different strate- ing of a ␦ receptor agonist. This robust increase is also seen in gies have been considered for making opiates more effective SKNSH cells that endogenously express both ␮ and ␦ recep- while curbing their liability to be abused. Although most opiates tors. Furthermore, we find that a ␦ receptor antagonist en- exert their analgesic effects primarily via ␮ opioid receptors, a hances both the potency and efficacy of the ␮ receptor signal- number of studies have shown that ␦ receptor-selective drugs ing; likewise a ␮ antagonist enhances the potency and efficacy can enhance their potency. The molecular basis for these find- of the ␦ receptor signaling. A combination of agonists (␮ and ␦ ings has not been elucidated previously. In the present study, receptor selective) also synergistically binds and potentiates we examined whether heterodimerization of ␮ and ␦ receptors signaling by activating the ␮–␦ heterodimer. Taken together, could account for the cross-modulation previously observed these studies show that heterodimers exhibit distinct ligand between these two receptors. We find that co-expression of ␮ binding and signaling characteristics. These findings have im- and ␦ receptors in heterologous cells followed by selective portant clinical ramifications and may provide new foundations immunoprecipitation results in the isolation of ␮–␦ het- for more effective therapies. erodimers. Treatment of these cells with extremely low doses of Key words: receptor subtypes; agonist; antagonist; enkepha- certain ␦-selective ligands results in a significant increase in the lin; Deltorphin II; DAMGO; DPDPE; TIPP⌿; G-protein-coupled binding of a ␮ receptor agonist. Similarly, treatment with receptor; oligomerization; MAP kinase Opioid receptors can regulate several biological effects, including are associated with ␮ receptors and those that are not. However, analgesia, miosis, bradycardia, general sedation, feeding, and hy- the biochemical basis for this association was not explored. pothermia (Herz, 1993). Morphine, a prototype opioid agonist, Early studies using radioligand binding and electrophysiology binds to ␮ and ␦ opioid receptors and inhibits neurotransmitter suggested that both ␮ and ␦ receptors colocalize to cells in the release (MacDonald and Nelson, 1978; Yaksh, 1993). Studies with dorsal root ganglia (Fields et al., 1980; Egan and North, 1981; transgenic animals lacking ␮ receptors show that morphine func- Zieglgansberger et al., 1982). Immunohistochemical studies of tions primarily via ␮ receptors (Matthes et al., 1996). Interest- the opioid receptor distribution in the CNS have shown that ␮ ingly, ␦ ligand-mediated analgesia is altered in these animals, and ␦ receptors colocalize to the same axonal terminals of the suggesting an interaction between the two receptors (Sora et al., superficial dorsal horn (Arvidsson et al., 1995). Ultrastructural 1997). analysis of the dorsal horn neurons also revealed colocalization of A number of pharmacological studies have suggested that ␮ ␦ receptors with ␮ receptors in the plasmallema (Cheng et al., and ␦ receptors interact and influence each other’s properties 1997). In addition, several neuroblastomas have been shown to (Traynor and Elliot, 1993). For example, mice treated with ␦ co-express these two opioid receptors (Yu et al., 1986; Kazmi and antagonists exhibit diminished development of morphine toler- Mishra, 1987; Baumhaker et al., 1993; Palazzi et al., 1996). Taken ance and dependence (Abdelhamid et al., 1991; Zhu et al., 1999). together, these studies provide evidence for colocalization of ␮ Selective reduction of ␦ receptors by antisense oligonucleotides and ␦ receptors and suggest that the existence of ␦ and ␮ receptor attenuates the development of morphine dependence (Sanchez- complexes is physically possible. Blazquez et al., 1997). In ␦ receptor knockout animals, the extent Receptor dimerization is a potential mechanism for modula- of dependence attributable to morphine administration is also tion of their function (Salahpour et al., 2000). Early studies selectively altered (Zhu et al., 1999). Ligand binding studies show that ␮-selective ligands inhibit the binding of ␦-selective ligands in both competitive and noncompetitive manners (Rothman and This article is published in The Journal of Neuroscience, Rapid Westfall, 1981; Rothman et al., 1983). Findings such as these led Communications Section, which publishes brief, peer-reviewed to the proposal that ␦ receptors exist as two subtypes: those that papers online, not in print. Rapid Communications are posted online approximately one month earlier than they would ap- Received Aug. 17, 2000; accepted Aug. 30, 2000. This work was supported in part by National Institutes of Health Grants DA 08863 pear if printed. They are listed in the Table of Contents of the and DA 00458 to L.A.D. and National Research Service Award Grant DA05885 to next open issue of JNeurosci. Cite this article as: JNeuro- B.A.J. We thank Dr. Peter Schiller for the gift of TIPP␺. sci, 2000, 20:RC110 (1–5). The publication date is the date of Correspondence should be addressed to Dr. Lakshmi A. Devi, Department of Pharmacology, New York University School of Medicine, MSB 411, 550 First posting online at www.jneurosci.org. Avenue, New York, NY 10016. E-mail: [email protected]. Copyright © 2000 Society for Neuroscience 0270-6474/00/200001-05$15.00/0 http://www.jneurosci.org/cgi/content/full/4736 2of5 J. Neurosci., 2000, Vol. 20 Gomes et al. • ␮–␦ Opioid Receptor Heterodimers showing that dimeric analogs of oxymorphone and enkephalin exhibit higher affinity and potency than their monomeric forms suggested that ␮ 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 heterodimer- ize with ␬ receptors, and heterodimerization affects their ligand binding and signaling properties (Jordan and Devi, 1999). Be- cause ␦ receptors show significant sequence homology with ␮ receptors at the amino acid level (Miotto et al., 1995), we examined whether ␦ receptors physically associate with ␮ receptors and whether this interaction alters their properties. Here we show that ␮ and ␦ receptors associate to form a ϳ150 kDa heterodimer, and these heterodimers exhibit distinct ligand binding and signaling properties. Therefore, ␮–␦ heterodimerization represents a novel mechanism that could modulate the function of these receptors. Figure 1. ␮ and ␦ receptors interact with each other to form heterodimers. Immunoprecipitation of cell lysates from HEK-293 cells individually expressing either myc-␦ (Myc␦) or Flag-␮ (Flag␮) receptors, MATERIALS AND METHODS mixed cells individually expressing myc-␦ or Flag-␮ receptors (Myc␦ ϩ Flag␮), or cells co-expressing myc-␦ and Flag-␮ (Myc␦–Flag␮) was per- Materials. Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO), Tyr-D-Ala-Phe- formed using anti-myc antibodies. Western blotting of these immunocom- Glu-Val-Val-Gly (Deltorphin II), and diprenorphine were from RBI plexes using anti-Flag antibodies shows a ϳ150 kDa protein representing (Natick, MA) and Sigma (St. Louis, MO). Naltriben (NTB), benzyliden- the ␮–␦ heterodimer only in cells co-expressing both myc-␦ and Flag-␮ ␮ ␦ enaltrexone (BNTX), and SNC 80 were from Tocris Cookson. D-Phe- receptors. Pretreatment of cells co-expressing and receptors with 1 2 5 mM DTT (Myc␦–Flag␮ ϩ DTT) results in the destabilization of dimers. Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) and [D-Pen , Pen ] ␮ ␦ ␦ enkephalin (DPDPE) were from Peninsula Inc. 3H-DAMGO and 3H- – heterodimers are also seen in cells co-expressing wild-type receptors and C-terminally truncated ␮ receptors (Myc␦–Flag␮⌬42). 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 phospho-MAP kinase antibody and the levels of tubulin using anti- ␺ ␺ tubulin antibody. Biolabs). Tyr-Tic (CH2NH)-Phe-Phe (TIPP ) was a gift from Dr. Peter Schiller (Institut de Reserches Cliniques de Montreal, Canada). RESULTS Cell culture and transfection. Human embryonic kidney (HEK)-293 cells expressing wild-type mouse myc-␦ receptors alone or wild-type ␮ and ␦ receptors associate to form mouse Flag-␮ receptors alone, or co-expressing wild-type myc-␦ with detergent-stable heterodimers wild-type Flag-␮ receptors or wild-type myc-␦ with C-terminally truncated Flag-␮ receptors were generated as described previously (Jordan et A number of pharmacological studies have provided indirect al., 2000; Trapaidze et al., 2000). Chinese hamster ovary (CHO) cells evidence for the interaction between ␮ and ␦ receptors (Traynor stably expressing wild-type Flag-␮ and wild type myc-␦ receptors were and Elliot, 1993). We directly examined the association between generated using Lipofectamine reagent (Life Technologies) as described ␦ ␮ ␦ these two classic receptors by co-expressing myc-tagged recep- (Jordan

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