2.2. The importance of opioid receptor complexes
Opioid systems are key systems for the control of pain. Importantly, opioid receptors are expressed throughout nociceptive processing circuitry and present multiple sites for inhibiting nociception. As a consequence, local application of opioid drugs induces analgesia via mu opioid receptors in the following areas: In the periphery where neurons from the dorsal root ganglia (DRG) sense painful stimuli; in the spinal cord where DRG neurons synapse with brain-bound nociceptive neurons; in the brain-stem structures, such as the Periaqueductal Gray that process nociceptive input; in the cortex and areas of the reward system that are responsible for higher processing of pain. These brain regions contain opioid receptors that, when activated, diminish the sensation of pain.
Animal, cellular and molecular models over the past decade have begun to reveal features of the “opioid receptor” that give hope for the optimization of drugs targeting opioid receptors for pain-relief without the detrimental effects of respiratory depression, addiction, constipation. The view of the opioid receptor as a signaling on/off switch has changed. The current view of the receptor is a component of a dynamic protein complex within the membrane. The receptor is capable of orchestrating the interaction of many different proteins and the final complexes formed dependent upon; the available proteins for interacting with the receptor, the history of the local environment of the receptor, and the ligand occupying the receptor (Evans, 2004
). The signaling cascades that are activated depend upon the complex formed, which also dictates the trafficking and desensitization mechanisms of the receptor (Kelly et al., 2008
). shows the proteins that have been implicated in mu opioid receptor complexes leading to signaling, trafficking and functional regulation of the receptor and indeed other receptors that are associated with the complex.
Fig. 2 Mu receptor signaling complexes. (A) Mu receptor signaling complexes. Agonists of the mu receptor bind to extracellular regions of this GPCR. This leads to dissociation of the cognate G-proteins from the receptor which, either through a conformational (more ...)
So why should the view of the receptor as a dynamic complex change how we view opioid therapeutics? There are two key issues: The first is that different ligands can induce the formation of different complexes that result in different signaling and trafficking cascades. The second is that different receptor environments, perhaps in different areas of brain or parts of the cell, can signal and traffic receptors in different ways in response to the same ligand. This opens up the exciting possibility for individual ligands to have distinct effects on the array of opioid behaviors and adaptive processes—effects ultimately dependent upon signaling and regulatory pathways activated by specific complexes. The on/off switch as the image of an opioid receptor has transformed to a sophisticated sensor that responds in different ways, depending on the local environment and how the sensor is manipulated.
Here we will focus on what has been learned from behavioral studies and cellular studies on dorsal root ganglia neurons from animals lacking β-arrestins, key molecules in the formation of signaling and trafficking opioid receptor complexes (Reiter and Lefkowitz, 2006
). β-arrestin 1 and β-arrestin 2 are molecules named for one of their many functions, namely to bind to receptors following agonist activation and “arrest” further signaling via G-proteins. As indicated in , β-arrestins are implicated both in the signaling and trafficking of opioid receptors. β-Arrestins bind multiple signaling, trafficking, and regulatory proteins in addition to receptors. Thus, β-arrestins provide a hub for the formation of receptor complexes. In a classical sequence of events, agonist binding to a G-protein coupled receptor (GPCR), such as the mu receptor, promotes G-Protein activation by GDP/GTP exchange and the conformational rearrangements ultimately result in β-arrestin 1 or 2 binding to the receptor. β-Arrestin binding is often facilitated by kinases such as G-protein receptor kinases or GRK’s that phosphorylate intracellular components of the receptor (generally the C-terminal tail), and thereby increase affinity of the receptor for the β-arrestins. β-arrestins act as linkers for a series of other proteins including kinases (such as cSrc and JNK3, a c-Jun N-Terminal Kinase) and scaffolding proteins involved in trafficking (such as alpha-adaptin-2). The agonist interaction with the receptor generates a cluster of proteins in close proximity of the receptor to accomplish signaling (), regulatory and trafficking () events.
Multiple studies now implicate different opioid drugs inducing different receptor-containing protein complexes. One of the initial observations was found in cell lines containing the delta opioid receptor whereby treatment with several opioid peptides and alkaloid agonists but not morphine induced loss of surface opioid binding (Von Zastrow et al., 1993
). Subsequently, many agonist-selective receptor-mediated effects have been documented, including receptor phosphorylation, receptor trafficking, receptor signaling and receptor desensitization (Evans, 2004
). Research indicating that the same drug can induce different complexes in different environments is less well documented, but has been implicated in a study of mu receptor trafficking in dendrites and cell bodies. In this study, morphine was found to induce effective internalization in processes but not in cell bodies (Haberstock-Debic et al., 2003
Multiple studies have shown that activation of or even just the presence of other GPCR’s can influence the pharmacology, function, and trafficking of mu receptors (Alfaras-Melainis et al., 2009
). One example from our studies is the interaction of mu receptors and α2A
adrenergic receptors in DRG cells (Tan et al., 2009
). Primary cultures of mouse DRG neurons express multiple opioid receptors (Mu, delta and kappa) as well as α2A
adrenergic receptors. Activation of α2A
adrenergic receptors with an agonist such as clonidine is able to cause desensitization and internalization of α2A
adrenergic receptors and mu opioid receptors. Likewise, activation of mu opioid receptors with a peptide mimetic of enkephalin ([D-Ala2
]enkephalin or DAMGO) causes internalization and desensitization of both Mu and α2A
adrenergic receptors. Clonidine or DAMGO-induce receptor cross-regulation can be disrupted by p38 MAP kinase inhibition. p38 inhibition also blocks DAMGO-induced mu receptor desensitization and internalization, although α2A
adrenergic receptor desensitization and internalization by clonidine is unaffected. Like p38, β-Arrestin 2 also appears to be required for cross-regulation of Mu and α2A
adrenergic receptors. However, unlike p38 inhibition, β-Arrestin 2 is required for α2A
adrenergic receptor desensitization and not desensitization of mu receptors (Tan et al., 2009
). The data clearly demonstrate that the action at one receptor can markedly influence the responsivity and trafficking of a different receptor. The hypothesized complex following DAMGO activation of mu opioid receptors in DRG neurons is indicated in .
Fig. 3 Morphine vs DAMGO receptor signaling complexes. The differential internalization, pharmacology of desensitization and signaling of the DAMGO-treated receptor (A) and morphine-treated mu receptor (B) in DRG neurons (see text) can be explained by ligand-specific (more ...)
Treatment with either morphine or DAMGO shows that different agonists lead to different signaling pathways and trafficking in DRG neurons (Tan et al., 2009
). Firstly, DAMGO is found to cause internalization of mu receptors in the DRG neurons, but morphine does not. Secondly, morphine does not cause cross-desensitization or internalization of α2A
adrenergic receptors as does DAMGO. And thirdly as might be anticipated, morphine does not activate p38 kinase that is observed with DAMGO treatment. However, mu receptor desensitization still occurs after treatment with morphine, and morphine-induced desensitization does not depend upon activation of p38 suggestive of different mechanisms for desensitization via morphine and DAMGO. This has been explored in other systems where morphine desensitization is shown to be PKC-dependent but not GRK-dependent, and DAMGO desensitization GRK-dependent but not PKC-dependent (Kelly et al., 2008
). In DRG neurons, morphine desensitization does not appear to be PKC-dependent (unpublished observation) and it is anticipated that different cells will have different desensitization mechanisms for the same ligand activating the receptor. A hypothesis for a morphine-induced complex in DRG neurons is depicted in .
The bottom line from the experiments described above in DRG neurons as well as analogous studies in other systems, is that protein complexes that form around the mu receptor are ligand-dependent, dependent on cell type and compartment within the cell, and regulated by recent history of the receptor environment. Though experiments are still in the early stages of research, it is clear that the differential formation of complexes will contribute to individual differences between drugs targeting opioid receptors and other GPCRs. It is probable that optimal complex formation for a designated treatment will become a future criteria for searching therapeutic targets and add to an already long list of requirements, including receptor specificity, receptor potency, receptor efficacy, low organ toxicity, blood brain barrier permeability, and metabolic stability.
2.3. Upregulation of Mu receptor constitutive activity
Mice lacking β-arrestin 2 have revealed another important characteristic of mu receptor trafficking. mu opioid receptors can be constitutively active and activate G-proteins in the absence of agonist ligands. In DRG neurons we have shown that constitutively active receptors are efficiently removed from the cell surface, a process that appears dependent upon cSrc and β-arrestin 2 (Walwyn et al., 2007
). Thus in DRG neurons from β-arrestin 2 knockout mice or cells from wild-type mice in the presence of c-Src inhibitors, there is an increased level of surface constitutive activity of mu opioid receptors that constantly inhibits Ca2+
channels in a similar (but not identical) fashion to agonists. This constant constitutive activity appears not to have desensitized mu opioid receptor signaling, since mu agonist dose–response curves in DRG neurons from wild-type and β-arrestin 2 knockout mice are indistinguishable. Given that constitutive activity of mu receptors is enhanced in β-arrestin 2 knockout DRG neurons, we have determined if this would result in mu mediated analgesia in the absence of agonists. β-arrestin 2 knockout mice were found to have an increased nociceptive threshold in the tail immersion assay supporting previously published data (Bohn et al., 1999
). Recent unpublished data from our laboratory support the notion that this opioid-mediated basal analgesia or increased nociceptive threshold in the β-arrestin 2 knockout mice is indeed due to mu-receptor constitutive activity and not due to activation of opioid receptors by endogenous opioid ligands. This finding is of potential clinical relevance since it presents a novel therapeutic target, namely the interference of mu-receptor interaction with β-arrestin 2, as a mechanism to develop analgesia that appears to not be susceptible to complete desensitization. Furthermore, the β-arrestin 2 knockout mice do not appear to have an elevated basal hedonic tone, based upon indistinguishable mu-antagonist-induced aversion in wild-type and β-arrestin 2 KO mice. Currently, we are screening for allosteric regulators and neutral antagonists that disrupt arrestin interactions yet retain constitutive activity of mu opioid receptors in hopes to discover new-non-agonist ligands of the mu receptor that use constitutive activity to produce an analgesic response.
2.5. Optimizing opioid actions by targeting other systems
One strategy to optimize opioid therapeutics that has not been sufficiently explored at the clinical level and has been revealed by rodent models using antagonists and knockout approaches is that several systems appear to work with the opioid system circuitry to control reward and opioid adaptive processes (for review see Bryant et al., 2005
). Thus, the cannabinoid CB1 receptor appears to be required for opioid reward but not opioid analgesia and the substance P receptor, NK1, is required both for full morphine-reward and morphine-induced hyperalgesia that emerges during withdrawal (King et al., 2005a
). The application of mixed opioid-NK1 or opioid-CB1 antagonists could be useful; especially in pain patients that are high-risk for addiction and that require opioid analgesics. However, the side effects in humans of the CB1 antagonist Rimonabant in promoting depression and suicidal behaviors (Lee et al., 2009
) and the lack of a suitable NK1 antagonist has not facilitated the testing of opioid combinations with CB1 or NK1 antagonists at the clinical level. Interestingly, agents that prevent hyperalgesia also reduce tolerance demonstrating similar underlying cellular adaptations to chronic opioids. Exciting work by De-Yong Liang and colleagues suggests that different strains of mice have a greater susceptibility to develop hyperalgesia after opioid administration and polymorphisms of the β2 adrenergic receptor (β2-AR) gene were linked to these differences (Liang et al., 2006
). Using the selective β2-AR antagonist butoxamine, the investigators observed a dose-dependent reversal of OIH. This study holds promise that the addition of a β2-AR antagonist to a chronic opioid regiment in humans might improve the long-term analgesic efficacy (Liang et al., 2006
). Similar promising results have been found with antagonists of different components of the system such as CCK and NK1 (review; Bryant et al., 2005
). The development of non-addictive opioid formulas should be a goal of pharmaceutical companies despite the lost revenue that would occur by negating abuse liability of their drug products.
2.7. Opioids and the immune system
Opioid-mediated analgesia may also be influenced by the peripheral release of opioid peptides, β-endorphin, met-enkephalin, and dynorphin, from specific immune cells during inflammation. These are the granulocytes, lymphocytes, and monocytes, each population releasing opioids at different stages of the inflammatory process. The granulocytes are the first to be recruited to the site of inflammation and require an intact chemokine cascade, series of adhesion molecules, and other chemo-attractants to direct their migration. Once at the site, they increase the release of opioid peptides in a p38 and PI3K dependent manner (review; Rittner et al., 2008
). These opioids bind to the peripheral nerve terminals of the primary afferent neurons, which express the delta, mu, and kappa opioid receptors. This initiates the opioid signaling cascade to increase hyperpolarization and inhibit neurotransmitter release, thus reducing the sensation of pain. Such peripherally restricted release of opioids from immune cells appears not to induce tolerance, and certainly is not associated with the central side effects of the centrally acting opioids (Smith, 2008
). This suggests that peripherally-restricted opioids may be more attractive alternative compounds than their centrally acting counterparts for peripheral pain.