Review
Oligomerization of opioid receptors: generation of novel signaling units

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Abstract

G-protein-coupled receptors such as opioid receptors exist in oligomeric complexes. Homo-oligomerization of μ, δ, and κ opioid receptors has been demonstrated, suggesting a critical functional role for receptor–receptor interactions. Hetero-oligomerization creates δ–κ and μ–δ receptor complexes, which display novel pharmacology, coupling to effector systems and signaling regulation properties. Exciting new opportunities are arising from the recent characterization of these oligomeric receptors, with the potential to revolutionize opioid research and therapeutic targeting.

Introduction

Oligomerization of G-protein-coupled receptors (GPCRs) is a burgeoning field of research that is significantly advancing and remodeling our understanding of GPCR regulation and function 1••., 2.. Crucial to this progress is the characterization of opioid receptor homo-oligomerization and hetero-oligomerization and this is vital to a detailed understanding of the endogenous opioid system. This review documents the features of opioid receptor homo-oligomerization and hetero-oligomerization. At each stage, the consequences and implications of opioid receptor–receptor interactions are addressed, such as drug design, in vivo opioid system regulation and function and the relevance to development of analgesic tolerance. Additionally, the specificity of opioid receptor oligomer formation is discussed, particularly in relation to hetero-oligomerization with other GPCRs.

Section snippets

Opioid receptor homo-oligomers

The homo-oligomeric association of opioid receptors has been documented by numerous reports. Using Western blot analysis of heterologous cell expression systems, we and others have demonstrated immunoreactive bands corresponding to monomers, dimers, and higher-order oligomers for the μ [3••], δ 3••., 4., 5•., and κ [4] opioid receptors (MOR, DOR and KOR). In addition, MOR antibodies labeled receptor complexes in mouse brain corresponding to receptor dimers [6], suggesting that oligomerization

DOR homo-oligomerization

The properties of the DOR homo-oligomer have been most extensively examined. Differentially epitope-tagged DOR constructs coimmunoprecipitated when expressed in COS, CHO, or HEK293 cells 7., 8••. and DOR oligomers were detected in HEK293 cells using bioluminescence resonance energy transfer (BRET) and time-resolved fluorescence resonance energy transfer (FRET) assays [8••]. BRET and time-resolved FRET are relatively new approaches to examining GPCR oligomerization that rely on energy transfer

Dynamic regulation?

The existence of dynamic regulation of DOR oligomerization is a contentious issue. Using Western blots, cross-linker mediated dimerization of the DOR was reported to be attenuated by agonists [7]. A dose-dependent relative increase in monomers was observed with DOR-selective and non-selective agonists (DADLE, DSLET, DPDPE, etorphine; Box 1), but not following treatment with MOR-selective agonists (morphine, DAMGO) or non-selective antagonists (naloxone) [7]. In contrast, using both BRET and

Structure

A general structural basis for GPCR oligomerization has yet to be established. It is commonly thought that multiple factors, such as transmembrane domain hydrophobicity, extracellular disulfide bonds and intracellular coiled-coil interactions, cooperatively mediate oligomerization [1••]. Other factors could additionally account for receptor–receptor interactions, such as cross-linking through accessory proteins or leucine zipper interactions. Sensitivity to reducing agents implicate disulfide

Physiological role

Homo-oligomerization of opioid receptors raises questions regarding its physiological role. Oligomerization may be necessary for signal amplification, such that agonist activation of a limited number of receptors triggers activation of other associated receptors, resulting in increased effector coupling, receptor phosphorylation, G-protein uncoupling and internalization of an entire receptor complex. Interestingly, a role for oligomerization in trafficking of the dopamine D2 receptor to the

Hetero-oligomerization between opioid receptors

Overall, the three opioid receptors share approximately 60–65% amino acid identity, with the transmembrane domains being among the most highly conserved regions (Fig. 1). Given that homo-oligomerization had been demonstrated and given the expectation that transmembrane interactions exist in these complexes, hetero-oligomerization between opioid receptors was hypothesized.

DOR–KOR hetero-oligomers

Heterodimerization between DOR and KOR was the first opioid receptor complex identified [4]. Differentially epitope-tagged DOR and KOR expressed in HEK293 or COS cells coimmunoprecipitated and this receptor complex displayed many novel characteristics [4]. The DOR–KOR heterodimer had an altered ligand-binding profile, such that KOR-selective agonists (U69593, dynorphin A) and antagonists (norbinaltorphimine) and DOR-selective agonists (DPDPE) and antagonists (TIPPΨ, BNTX; Box 1) had decreased

MOR–DOR hetero-oligomers

Interaction between MOR and DOR receptors has long been postulated and is extensively documented [15]. We recently demonstrated direct interaction between these two receptors. Differentially epitope-tagged MOR and DOR also coimmunoprecipitated from COS cells and this hetero-oligomeric complex also displayed a host of unique features [3••]. A novel binding site was revealed by an altered rank order of potency: selective synthetic agonists (DADLE, DPDPE, DAMGO, morphine) had a reduced affinity

MOR–KOR interaction?

The remaining possible hetero-oligomeric complex that could be formed between opioid receptors would be as a result of MOR–KOR interaction. Coexpression of these receptors did not result in coimmunoprecipitation [4]; however, cell-surface expression of the two receptors was not confirmed. If MOR and KOR do not hetero-oligomerize, the opposing physiology of the two systems may provide a rationale. The KOR generally antagonizes MOR-mediated actions such as analgesia, tolerance, reward, learning

Hetero-oligomerization between opioid receptors and other GPCRs

If different opioid receptors hetero-oligomerize, do they also directly interact with other GPCRs? Recent studies show that DOR and KOR are capable of interacting with the β2-adrenoceptor 8••., 27., however the significance of this interaction remains uncertain. Coimmunoprecipitation of the β2-adrenoceptor with the KOR [27], and the DOR 8••., 27. was demonstrated in heterologous cell systems. To address concerns that this interaction may be an artifact, the interaction of these two receptors

Opioid receptor oligomerization: questions, implications and opportunities

The organization of opioid receptors into oligomeric complexes raises many intriguing questions. For example, what is the functional receptor unit and do monomers, dimers and higher order oligomers coexist on the cell surface? Multiple studies have demonstrated opioid receptor species corresponding to all of these receptor states, but how accurately does Western blot analysis reflect cell-surface expression? Interestingly, MOR and DOR homo-oligomers were observed following coimmunoprecipitation

Conclusions

Oligomerization adds a previously unappreciated level of complexity to opioid receptor function and is a mechanism by which the products of a limited number of receptor genes may give rise to a greater diversity of signaling units with unique properties (Fig. 2). New approaches to therapeutic drug design and discovery directly arise from the identification of opioid receptor hetero-oligomers. Numerous questions surrounding opioid receptor homo-oligomerization and hetero-oligomerization remain,

Update

Chronic morphine treatment was recently demonstrated to induce recruitment of the DOR from intracellular stores to the plasma membrane in both cultured cortical neurons and rat spinal cord [36]. DOR upregulation was solely mediated through morphine activation of coexpressed MORs [36], suggesting a possible role for MOR–DOR hetero-oligomerization in DOR trafficking.

Acknowledgements

BF O'Dowd and SR George receive support from the Canadian Institutes of Health Research and the National Institute on Drug Abuse. The authors thank Samuel PLee for his graphical design expertise.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

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