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A cell biologist’s perspective on physiological adaptation to opiate drugs
Neuropharmacology 47 (2004) 286–292 www.elsevier.com/locate/neuropharm
A cell biologist’s perspective on physiological adaptation to opiate drugs Mark von Zastrow Departments of Psychiatry and Pharmacology, University of California, San Francisco, Room N212E Genentech Hall, 600 16th Street, San Francisco, CA 94143-2140, USA Received 20 April 2004; accepted 14 May 2004
Abstract Opiate drugs such as morphine and heroin are among the most effective analgesics known but are also highly addictive. The clinical utility of opiates is limited by adaptive changes in the nervous system occurring after prolonged or repeated drug administration. These adaptations are believed to play an important role in the development of physiological tolerance and dependence to opiates, and to contribute to additional changes underlying the complex neurobehavioral syndrome of drug addiction. All of these adaptive changes are initiated by the binding of opiate drugs to a subfamily of G protein-coupled receptors that are also activated by endogenously produced opioid neuropeptides. It is increasingly evident that opiate-induced adaptations occur at multiple levels in the nervous system, beginning with regulation of opioid receptors themselves and extending to a complex network of direct and indirect modifications of ‘‘downstream’’ signaling machinery. Efforts in my laboratory are directed at understanding the biochemical and cell biological basis of opiate adaptations. So far, we have focused primarily on adaptations occurring at the level of opioid receptors themselves. These studies have contributed to defining a set of membrane trafficking mechanisms by which the number and functional activity of opioid receptors are controlled. The role of these mechanisms in affecting adaptation of ‘‘downstream’’ neurobiological substrates, and in mediating opiate-induced changes in whole-animal physiology and behavior, are exciting questions that are only beginning to be explored. # 2004 Elsevier Ltd. All rights reserved. Keywords: Opiate; Opioid; Receptor; Addiction; Mechanism; Tolerance; Dependence; Plasticity
1. Introduction Opiate drugs mediate their physiological effects by binding to a subset of G protein-coupled receptors that are also activated by endogenously produced opioid neuropeptides. Three pharmacologically distinct types of mammalian opioid receptor (mu, delta and kappa) are encoded by separate structural genes (Zaki et al., 1996). Elegant studies using receptor-knockout mice, together with an extensive set of pharmacological studies, support the hypothesis that mu opioid receptors are of primary importance for mediating analgesic and addictive effects of clinically important opiate drugs (Kieffer, 1999). Early studies conducted using native tissue explants and neuroblastoma-derived cell cultures
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established evidence that opiate-induced adaptations occur at the level of individual neuronal cells. A classic series of early studies conducted using cultured neuroblastoma cell lines proposed the existence of single-cell correlates of both physiological tolerance and dependence to opiate drugs (Sharma et al., 1977). Opiateinduced reduction in the functional activity of opioid receptors detected in acutely prepared tissue explants was associated with the expression of physiological tolerance (Chavkin and Goldstein, 1982). Evidence for such ‘‘cell-autonomous’’ components of opiate adaptation was a major advance that encouraged the use of in vitro models to elucidate specific mechanisms of opiate adaptation. However, it has been appreciated for many years that opiate-induced adaptations occurring in intact animal models, as in clinical observations of humans, are highly complex (Way et al., 1969). Indeed, opiate adaptations occurring in
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vivo have been convincingly shown to include modulation of a number of neural circuits that are only indirectly linked to the endogenous opioid system, and even in opioid receptor-expressing neurons, have been shown to involve regulation of numerous other cellular components in addition to the receptors themselves (Nestler, 2001; Vanderah et al., 2001; Williams et al., 2001). While it seems likely that regulatory processes affecting opioid receptors themselves play a fundamental role in physiological adaptation of the intact nervous system to opiates, clearly the complex physiology of opiate adaptation occurring in vivo cannot be understood solely at this level. With these concepts in mind, our laboratory is pursuing several goals toward understanding opiate adaptations. First, we seek to elucidate opioid receptor regulation in biochemical detail, and to link this information to mechanistic cell biology. These studies have involved extensive use of in vitro model systems, which are experimentally favorable but whose relevance to receptor regulation occurring in native neurons has not been established. Second, we seek to investigate the degree to which mechanisms of receptor regulation elucidated using non-neural model systems occur in physiologically relevant neurons and intact neural tissue, and to search for additional features of opioid receptor regulation in these preparations not anticipated from study of non-neural systems. Third, we seek to define how specific mechanisms of opioid receptor regulation contribute to adaptation of the intact nervous system to opiates. This goal involves a number of approaches, including the development of novel methods to manipulate specific regulatory mechanisms in vivo. The present article will briefly summarize progress that has been made so far, focusing on a set of membrane trafficking mechanisms that regulate the number and functional activity of opioid receptors accessible to native ligands and drugs in target neurons.
2. Regulated endocytosis of opioid receptors It is increasingly apparent that opiate drugs can modulate the membrane trafficking of opioid receptors between distinct membrane domains (Tsao and von Zastrow, 2001). A highly conserved mechanism of regulated endocytosis has been defined, based initially on studies of muscarinic and adrenergic receptors, and shown more recently to be relevant to certain other GPCRs including opioid receptors. Opioid receptors activated by naturally occurring opioid peptides and certain alkaloid agonists concentrate in clathrin-coated pits, which subsequently undergo dynamin-dependent fission from the plasma membrane and then fuse with early endosomes (Chu et al., 1997). This process is regulated by two key biochemical events: phosphoryla-
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tion of receptors by G protein-coupled receptor kinases (GRKs) and association of receptors with nonvisual (beta-) arrestins (Whistler and von Zastrow, 1998; Zhang et al., 1998; Zhang et al., 1999). Both of these events disrupt receptor signaling by inhibiting receptor interaction with heterotrimeric G proteins. In addition, these events promote endocytosis of receptors by promoting their concentration in clathrin-coated pits. These findings are similar to those obtained in earlier studies of the beta-2 adrenergic receptor, which established that nonvisual arrestins function as regulated endocytic adaptor proteins by binding both to receptors and the clathrin coat structure (Ferguson et al., 1996; Goodman et al., 1996). 3. Differences in endocytic regulatory effects between opiate drugs and peptides Early studies of radioligand binding to intact neuroblastoma cells suggested that distinct agonists differ substantially in their ability to induce endocytosis of opioid receptors (von Zastrow et al., 1993). This idea has been confirmed in studies using heterologous expression of cloned opioid receptors, where opioid peptides such as enkephalins promote internalization of a major fraction of mu and delta opioid receptors within several minutes but morphine fails to cause detectable internalization of receptors after acute (Keith et al., 1996) or chronic (Arden et al., 1995) application. Morphine was observed to promote regulatory phosphorylation and desensitization of opioid receptors to a much smaller extent than opioid peptides and certain other alkaloid agonists (Arden et al., 1995; Yu et al., 1997; Zhang et al., 1998). Overexpression of GRK2 renders morphine capable of promoting rapid phosphorylation and endocytosis of opioid receptors in transfected cells (Zhang et al., 1998). Similarly, overexpression of arrestin 2 or 3 (beta-arrestin-1 or 2), in the absence of GRK overexpression, also promotes endocytosis of morphine-activated receptors (Whistler and von Zastrow, 1998). These results suggest that morphine-activated receptors are not optimal substrates, when compared with receptors activated by opioid peptide ligands, either for GRK-mediated phosphorylation or for receptor interaction with arrestins. While the existence of pronounced ‘‘agonist-selective’’ differences in the regulatory effects of various opiate drugs is now well accepted, the molecular pharmacology underlying these differences remains controversial. Many opiate analgesics, including morphine, are partial agonists that have lower intrinsic efficacy than opioid peptides, as estimated by assay of receptor-mediated signaling via heterotrimeric G proteins. Agonist-specific differences in desensitization of cloned mu opioid receptors expressed in Xenopus oocytes correlated with differences in relative agonist
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efficacy, consistent with a simple two-state model of opioid receptor activation and arguing against the existence of significant functional differences between opiate agonists (Kovoor et al., 1998). Similar studies conducted in transfected mammalian cells supported the importance of agonist efficacy but also suggested that certain opiates, such as morphine, induce regulatory phosphorylation (Yu et al., 1997) and endocytosis (Whistler et al., 1999) of mu opioid receptors to a significantly smaller degree than would be predicted based on a strict correlation with relative agonist efficacy. Furthermore, a mutation of the cytoplasmic tail of the mu opioid receptor, which alters the rank order with which various opiates promote regulatory endocytosis of receptors, does so without detectably changing the order of relative agonist efficacy (Whistler et al., 1999). Such complex pharmacological behavior is probably not unique to opiates, as indicated by studies of certain other GPCRs such as serotonin receptors (Willins et al., 1998). One hypothesis is that distinct ligands induce or stabilize functionally distinct conformations of the receptor protein, in contrast to the single agonistinduced conformation of receptors proposed by a simple two-state model (Kenakin, 2004). Biophysical studies of the beta-2 adrenergic receptor support the existence of agonist-specific receptor conformations (Seifert et al., 2001; Swaminath et al., 2004). However, it is not yet formally known whether such agonistselective conformations exist for opioid receptors, and if so, whether (or how) they contribute to differences in the regulatory effects of opiate drugs.
ubiquitin, a mechanism first discovered in studies of GPCR trafficking to the yeast vacuole and subsequently found to be applicable to a variety of membrane proteins (Hicke, 2001). Ubiquitylation of opioid receptors is known to promote proteasome-dependent degradation of receptors from the biosynthetic pathway (Petaja-Repo et al., 2000) and perhaps also from the plasma membrane (Chaturvedi et al., 2001). However, ubiquitylation of delta opioid receptors does not appear to be required for their endocytic trafficking to lysosomes (Tanowitz and von Zastrow, 2002). Furthermore, lysosomal trafficking of opioid receptors can be modulated by non-covalent protein interactions with the cytoplasmic tail (Whistler et al., 2002). There is also evidence that non-covalent interactions with the cytoplasmic tail of opioid receptors inhibit lysosomal sorting of internalized receptors (Tanowitz and von Zastrow, 2003). Nevertheless, recent studies suggest that some endosome-associated proteins that mediate lysosomal sorting of ubiquitinated membrane proteins, including Hrs and Vps4, are also required for lysosomal sorting of opioid receptors (Hislop et al., 2004). While much remains to be learned about this important sorting mechanism, we currently believe that opioid receptor sorting involves conserved ‘‘core’’ endosomal sorting machinery together with additional specialized protein interactions, which may confer additional specificity and plasticity on opioid receptor sorting in complex mammalian cells.
5. Effects of opiate drugs on opioid receptor membrane trafficking in cultured neurons 4. Molecular sorting of opioid receptors between functionally distinct membrane pathways after endocytosis It turns out that endocytosis of GPCRs can have various functional consequences, which are dictated in large part by the specific ‘‘downstream’’ membrane pathway followed by internalized receptors (Tsao et al., 2001). Recycling of internalized opioid receptors to the plasma membrane is associated with dephosphorylation of receptors and functional recovery of signaling activity (Koch et al., 1998), similar to the extensively studied role of the recycling pathway in mediating ‘‘resensitization’’ of beta-2 adrenergic receptor signaling (Lefkowitz et al., 1998). In contrast, sorting of internalized opioid receptors to lysosomes leads to essentially the opposite consequence, proteolytic ‘‘down-regulation’’ of receptors and attenuated signal transduction (Law et al., 1984; Tsao and von Zastrow, 2000). The molecular basis of this endocytic sorting decision is presently an area of active investigation. Certain GPCRs are sorted to lysosomes by a mechanism involving covalent modification of receptors with
A number of studies have investigated the effects of opiate drugs on the localization of endogenously expressed opioid receptors in neural tissue. Overall, there is reasonably close agreement between systems. Opiate drugs that induce rapid endocytosis of opioid receptors in transfected cell models also appear to do so in native tissues. While morphine does not produce detectable internalization of the predominant splice variant of mu opioid receptor (MOR1) in several populations of native neurons (Abbadie and Pasternak, 2001; Keith et al., 1998; Sternini et al., 1996), recent studies suggest that there may be additional complexity in certain neurons or neuronal membrane domains. The endocytic effects of methadone (internalizing) and morphine (non-internalizing) observed in non-neural cells (Keith et al., 1998; Whistler et al., 1999) correlates with receptor localization observed in cell bodies of nucleus accumbens neurons. However, in dendrites of the same neurons, morphine-induced internalization of both virally transduced MOR1 and endogenously expressed mu opioid receptors (Haberstock-Debic et al., 2003). This result suggests that morphine may indeed
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induce rapid internalization of opioid receptors in certain populations of CNS neurons, and there may be additional differences in the regulatory effects of opiate drugs occurring in different membrane domains (e.g., axons vs. dendrites) of the same neurons (HaberstockDebic et al., 2003). 6. Agonist-selective effects on opioid receptor regulation in Locus Coeruleus neurons An extensively studied set of opiate-responsive neurons are present in the Locus Coeruleus, where agonist ligands inhibit firing via mu opioid receptor-mediated activation of potassium channels (Williams et al., 2001). The ability of a series of opiates to produce homologous desensitization of opiate responses, a phenomenon thought to represent regulation of the opioid receptor itself, correlated closely with differences in the ability of opiates to promote rapid endocytosis of receptors in non-neural cells but not with differences in relative agonist efficacy (Alvarez et al., 2002). A heterologous component of opiate desensitization has also been observed in this tissue, which is thought to reflect regulation of G protein coupling to potassium channels; the ability of opiates to induce this component of desensitization correlates directly with relative agonist efficacy (Blanchet and Luscher, 2002). Together these observations support the existence of multiple mechanisms mediating opiate adaptations in native neurons that differ in regulation, either by proposed agonistselective receptor conformational states (Alvarez et al., 2002) or by the overall strength of opioid signaling to downstream effectors (Blanchet and Luscher, 2002). 7. Role of opioid receptor endocytosis in physiological adaptation of the intact nervous system The importance of opioid receptor endocytosis to chronic opiate adaptation of the intact nervous system is not yet understood. Efforts to address this question have been made by several groups using assays of opiate-induced antinociception in rodent models. Disruption of the gene encoding beta-arrestin-2 (arrestin 3) in mice strongly impaired agonist-induced desensitization of mu opioid receptors, measured biochemically by G protein activation in brain membranes. These animals exhibited increased sensitivity to the anti-nociceptive effects of morphine (Bohn et al., 1999), as well as decreased development of anti-nociceptive tolerance to this opiate (Bohn et al., 2000). However, morphine tolerance was not completely blocked in these animals and evidence suggesting a distinct protein kinase Cmediated mechanism of tolerance was observed (Bohn et al., 2002). Nevertheless, this series of studies pro-
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vides strong support for the idea that arrestin-mediated desensitization (and perhaps also endocytosis) of opioid receptors can occur in CNS neurons in vivo, and contribute both to producing changes in baseline opiate sensitivity in drug-naive animals as well as opiate tolerance in the setting of chronic drug administration. Another study utilized the tendency of opioid receptors to exist in oligomeric complexes (Devi, 2001) as a means to pharmacologically enhance morphineinduced internalization of receptors in neurons of rat spinal neurons (He et al., 2002). Intrathecally administered opioid peptide, shown previously to produce antinociception in direct proportion to internalization of mu opioid receptors in spinal neurons (Trafton et al., 2000), potentiated the ability of morphine to produce rapid internalization of spinal opioid receptors when administered at low, sub-analgesic doses. This manipulation, which essentially converted morphine from a non-internalizing to an internalizing ligand, decreased the development of anti-nociceptive tolerance to morphine. Thus, it was proposed from these results that regulated endocytosis of opioid receptors may inhibit— rather than promote—the development of anti-nociceptive tolerance to opiates in vivo (He et al., 2002). These distinct genetic and pharmacological manipulations of opioid receptor regulation support different overall hypotheses regarding the precise role of rapid opioid receptor regulatory mechanisms in modulating antinociceptive tolerance in chronically treated animals, which remain to be resolved. Furthermore, it remains to be determined what role opioid receptor regulatory mechanisms play in additional neurobehavioral features of opiate adaptation observed in intact animals (Kieffer and Evans, 2002). Nevertheless, both approaches agree in supporting the existence of important functional consequence(s) of drug-mediated regulation of opioid receptors themselves on at least one major effect of chronic opiate exposure (anti-nociceptive tolerance) observed in vivo. 8. Conclusions and future directions The hypothesis that physiological mechanisms underlying opiate drug adaptation in vivo can be studied at a cell biological level, proposed over 20 years ago, has motivated a number of laboratories to investigate opioid receptor regulatory mechanisms in biochemical detail using simplified in vitro systems. These efforts, greatly facilitated by the molecular cloning of opioid receptor genes and methodological developments in modern cell biology, have led to rapid advance toward the first goal set forth in the introduction to this article, that of understanding of specific receptor regulatory mechanisms. In particular, much has been learned about a conserved mechanism mediating regulated endocytosis of opioid receptors. An inter-
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esting observation stemming from these studies is the appreciation of substantial differences in the regulatory effects of certain opiate drugs relative to opioid peptides, which have exciting—albeit still largely unexplored—implications for molecular pharmacology and therapeutics. The appreciation of regulated endocytosis induced by opiates led to the recognition that opioid receptors are also regulated at other stages of membrane trafficking. The mechanism by which opioid receptors are ‘‘sorted’’ between divergent recycling and degradative membrane pathways after endocytosis is of particular interest because of the distinct and functionally important effects of receptor trafficking via these pathways on cell signaling and opiate responsiveness after long-term exposure to drugs. Future studies in this area may yield additional insights relevant to opiate adaptations occurring in vivo, and could identify new targets for therapeutic manipulation. An example of this type of development is the identification of an unanticipated mechanism by which opioid receptor transport in the late biosynthetic pathway is regulated both by neurotrophins and electrical activity, suggesting a novel means by which opiate responses in certain target neurons could be rapidly enhanced by local signals (Kim and von Zastrow, 2003). Studies pursuing the second goal, that of correlating regulatory mechanisms elucidated in non-neural cell models with those occurring in physiologically relevant neurons, have yielded some surprising results. While the basic machinery mediating regulated endocytosis of opioid receptors appears to be conserved in neurons and can regulate natively expressed receptors in vivo, the effects of specific opiate drugs on this mechanism can differ in distinct populations of neurons and may also differ in different membrane domains of the same neuron. Overall, our present observations support the general utility of in vitro model systems but also emphasize the importance of studying opiate effects in a native cellular context. Further studies of toward this goal will yield additional insight to the opioid regulation occurring in vivo, and might help explain differences in adaptive changes in distinct neural circuits noted previously in opiate-dependent animals. It is anticipated that progress in these studies will be facilitated by further developments in gene transfer and livetissue imaging methodologies, by which it may become possible to monitor regulation of both opioid receptors and specific ‘‘downstream’’ signaling components in the same CNS neurons and in real time. Progress toward the third goal, that of linking specific opioid regulatory mechanisms to physiological adaptation observed in while-animal physiology and behavior, is experimentally the most challenging and remains the least well understood. Nevertheless, gene knockout methods provide a powerful approach toward this goal and have already yielded intriguing
results linking opioid receptor regulation to a form of physiological tolerance to morphine. A different approach to this question, using the principle of receptor oligomerization to modulate opiate-induced endocytosis, demonstrates the feasibility of devising new pharmacological strategies using in vitro models and applying them to study receptor regulation in vivo. While much controversy remains in this area, and the experimental challenges of these approaches should not be underestimated, in my opinion there is considerable reason for excitement and optimism. In addition to rapid advance in many other experimental approaches for studying mechanistic cell biology at the whole-animal level, I believe that studies using ‘‘oligomerization pharmacology’’ illustrate how information derived from mechanistic cell biology can be rapidly adapted to in vivo application. It is anticipated that further developments of this type, in addition to helping advance our understanding of opiate physiology, may facilitate more rapid development toward novel mechanismbased pharmacotherapy of opiate-induced clinical syndromes.
Acknowledgements I apologize for citing only a subset of studies contributing to the stated conclusions. The present article is intended to provide one (personal) perspective on the field, which is admittedly biased by studies carried out in my laboratory. I wish to acknowledge important contributions made by present and former members of my laboratory, without whom I would have a lot less fun and little original information to discuss. I also thank many colleagues and collaborators elsewhere for their generous and insightful contributions, particularly Chris Evans, Brian Kobilka and John Williams. Studies in my laboratory have been supported by the National Institute on Drug Abuse and the National Institutes of Health.
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A cell biologist’s perspective on physiological adaptation to opiate drugs Mark von Zastrow Departments of Psychiatry and Pharmacology, University of California, San Francisco, Room N212E Genentech Hall, 600 16th Street, San Francisco, CA 94143-2140, USA Received 20 April 2004; accepted 14 May 2004
Abstract Opiate drugs such as morphine and heroin are among the most effective analgesics known but are also highly addictive. The clinical utility of opiates is limited by adaptive changes in the nervous system occurring after prolonged or repeated drug administration. These adaptations are believed to play an important role in the development of physiological tolerance and dependence to opiates, and to contribute to additional changes underlying the complex neurobehavioral syndrome of drug addiction. All of these adaptive changes are initiated by the binding of opiate drugs to a subfamily of G protein-coupled receptors that are also activated by endogenously produced opioid neuropeptides. It is increasingly evident that opiate-induced adaptations occur at multiple levels in the nervous system, beginning with regulation of opioid receptors themselves and extending to a complex network of direct and indirect modifications of ‘‘downstream’’ signaling machinery. Efforts in my laboratory are directed at understanding the biochemical and cell biological basis of opiate adaptations. So far, we have focused primarily on adaptations occurring at the level of opioid receptors themselves. These studies have contributed to defining a set of membrane trafficking mechanisms by which the number and functional activity of opioid receptors are controlled. The role of these mechanisms in affecting adaptation of ‘‘downstream’’ neurobiological substrates, and in mediating opiate-induced changes in whole-animal physiology and behavior, are exciting questions that are only beginning to be explored. # 2004 Elsevier Ltd. All rights reserved. Keywords: Opiate; Opioid; Receptor; Addiction; Mechanism; Tolerance; Dependence; Plasticity
1. Introduction Opiate drugs mediate their physiological effects by binding to a subset of G protein-coupled receptors that are also activated by endogenously produced opioid neuropeptides. Three pharmacologically distinct types of mammalian opioid receptor (mu, delta and kappa) are encoded by separate structural genes (Zaki et al., 1996). Elegant studies using receptor-knockout mice, together with an extensive set of pharmacological studies, support the hypothesis that mu opioid receptors are of primary importance for mediating analgesic and addictive effects of clinically important opiate drugs (Kieffer, 1999). Early studies conducted using native tissue explants and neuroblastoma-derived cell cultures
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established evidence that opiate-induced adaptations occur at the level of individual neuronal cells. A classic series of early studies conducted using cultured neuroblastoma cell lines proposed the existence of single-cell correlates of both physiological tolerance and dependence to opiate drugs (Sharma et al., 1977). Opiateinduced reduction in the functional activity of opioid receptors detected in acutely prepared tissue explants was associated with the expression of physiological tolerance (Chavkin and Goldstein, 1982). Evidence for such ‘‘cell-autonomous’’ components of opiate adaptation was a major advance that encouraged the use of in vitro models to elucidate specific mechanisms of opiate adaptation. However, it has been appreciated for many years that opiate-induced adaptations occurring in intact animal models, as in clinical observations of humans, are highly complex (Way et al., 1969). Indeed, opiate adaptations occurring in
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vivo have been convincingly shown to include modulation of a number of neural circuits that are only indirectly linked to the endogenous opioid system, and even in opioid receptor-expressing neurons, have been shown to involve regulation of numerous other cellular components in addition to the receptors themselves (Nestler, 2001; Vanderah et al., 2001; Williams et al., 2001). While it seems likely that regulatory processes affecting opioid receptors themselves play a fundamental role in physiological adaptation of the intact nervous system to opiates, clearly the complex physiology of opiate adaptation occurring in vivo cannot be understood solely at this level. With these concepts in mind, our laboratory is pursuing several goals toward understanding opiate adaptations. First, we seek to elucidate opioid receptor regulation in biochemical detail, and to link this information to mechanistic cell biology. These studies have involved extensive use of in vitro model systems, which are experimentally favorable but whose relevance to receptor regulation occurring in native neurons has not been established. Second, we seek to investigate the degree to which mechanisms of receptor regulation elucidated using non-neural model systems occur in physiologically relevant neurons and intact neural tissue, and to search for additional features of opioid receptor regulation in these preparations not anticipated from study of non-neural systems. Third, we seek to define how specific mechanisms of opioid receptor regulation contribute to adaptation of the intact nervous system to opiates. This goal involves a number of approaches, including the development of novel methods to manipulate specific regulatory mechanisms in vivo. The present article will briefly summarize progress that has been made so far, focusing on a set of membrane trafficking mechanisms that regulate the number and functional activity of opioid receptors accessible to native ligands and drugs in target neurons.
2. Regulated endocytosis of opioid receptors It is increasingly apparent that opiate drugs can modulate the membrane trafficking of opioid receptors between distinct membrane domains (Tsao and von Zastrow, 2001). A highly conserved mechanism of regulated endocytosis has been defined, based initially on studies of muscarinic and adrenergic receptors, and shown more recently to be relevant to certain other GPCRs including opioid receptors. Opioid receptors activated by naturally occurring opioid peptides and certain alkaloid agonists concentrate in clathrin-coated pits, which subsequently undergo dynamin-dependent fission from the plasma membrane and then fuse with early endosomes (Chu et al., 1997). This process is regulated by two key biochemical events: phosphoryla-
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tion of receptors by G protein-coupled receptor kinases (GRKs) and association of receptors with nonvisual (beta-) arrestins (Whistler and von Zastrow, 1998; Zhang et al., 1998; Zhang et al., 1999). Both of these events disrupt receptor signaling by inhibiting receptor interaction with heterotrimeric G proteins. In addition, these events promote endocytosis of receptors by promoting their concentration in clathrin-coated pits. These findings are similar to those obtained in earlier studies of the beta-2 adrenergic receptor, which established that nonvisual arrestins function as regulated endocytic adaptor proteins by binding both to receptors and the clathrin coat structure (Ferguson et al., 1996; Goodman et al., 1996). 3. Differences in endocytic regulatory effects between opiate drugs and peptides Early studies of radioligand binding to intact neuroblastoma cells suggested that distinct agonists differ substantially in their ability to induce endocytosis of opioid receptors (von Zastrow et al., 1993). This idea has been confirmed in studies using heterologous expression of cloned opioid receptors, where opioid peptides such as enkephalins promote internalization of a major fraction of mu and delta opioid receptors within several minutes but morphine fails to cause detectable internalization of receptors after acute (Keith et al., 1996) or chronic (Arden et al., 1995) application. Morphine was observed to promote regulatory phosphorylation and desensitization of opioid receptors to a much smaller extent than opioid peptides and certain other alkaloid agonists (Arden et al., 1995; Yu et al., 1997; Zhang et al., 1998). Overexpression of GRK2 renders morphine capable of promoting rapid phosphorylation and endocytosis of opioid receptors in transfected cells (Zhang et al., 1998). Similarly, overexpression of arrestin 2 or 3 (beta-arrestin-1 or 2), in the absence of GRK overexpression, also promotes endocytosis of morphine-activated receptors (Whistler and von Zastrow, 1998). These results suggest that morphine-activated receptors are not optimal substrates, when compared with receptors activated by opioid peptide ligands, either for GRK-mediated phosphorylation or for receptor interaction with arrestins. While the existence of pronounced ‘‘agonist-selective’’ differences in the regulatory effects of various opiate drugs is now well accepted, the molecular pharmacology underlying these differences remains controversial. Many opiate analgesics, including morphine, are partial agonists that have lower intrinsic efficacy than opioid peptides, as estimated by assay of receptor-mediated signaling via heterotrimeric G proteins. Agonist-specific differences in desensitization of cloned mu opioid receptors expressed in Xenopus oocytes correlated with differences in relative agonist
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efficacy, consistent with a simple two-state model of opioid receptor activation and arguing against the existence of significant functional differences between opiate agonists (Kovoor et al., 1998). Similar studies conducted in transfected mammalian cells supported the importance of agonist efficacy but also suggested that certain opiates, such as morphine, induce regulatory phosphorylation (Yu et al., 1997) and endocytosis (Whistler et al., 1999) of mu opioid receptors to a significantly smaller degree than would be predicted based on a strict correlation with relative agonist efficacy. Furthermore, a mutation of the cytoplasmic tail of the mu opioid receptor, which alters the rank order with which various opiates promote regulatory endocytosis of receptors, does so without detectably changing the order of relative agonist efficacy (Whistler et al., 1999). Such complex pharmacological behavior is probably not unique to opiates, as indicated by studies of certain other GPCRs such as serotonin receptors (Willins et al., 1998). One hypothesis is that distinct ligands induce or stabilize functionally distinct conformations of the receptor protein, in contrast to the single agonistinduced conformation of receptors proposed by a simple two-state model (Kenakin, 2004). Biophysical studies of the beta-2 adrenergic receptor support the existence of agonist-specific receptor conformations (Seifert et al., 2001; Swaminath et al., 2004). However, it is not yet formally known whether such agonistselective conformations exist for opioid receptors, and if so, whether (or how) they contribute to differences in the regulatory effects of opiate drugs.
ubiquitin, a mechanism first discovered in studies of GPCR trafficking to the yeast vacuole and subsequently found to be applicable to a variety of membrane proteins (Hicke, 2001). Ubiquitylation of opioid receptors is known to promote proteasome-dependent degradation of receptors from the biosynthetic pathway (Petaja-Repo et al., 2000) and perhaps also from the plasma membrane (Chaturvedi et al., 2001). However, ubiquitylation of delta opioid receptors does not appear to be required for their endocytic trafficking to lysosomes (Tanowitz and von Zastrow, 2002). Furthermore, lysosomal trafficking of opioid receptors can be modulated by non-covalent protein interactions with the cytoplasmic tail (Whistler et al., 2002). There is also evidence that non-covalent interactions with the cytoplasmic tail of opioid receptors inhibit lysosomal sorting of internalized receptors (Tanowitz and von Zastrow, 2003). Nevertheless, recent studies suggest that some endosome-associated proteins that mediate lysosomal sorting of ubiquitinated membrane proteins, including Hrs and Vps4, are also required for lysosomal sorting of opioid receptors (Hislop et al., 2004). While much remains to be learned about this important sorting mechanism, we currently believe that opioid receptor sorting involves conserved ‘‘core’’ endosomal sorting machinery together with additional specialized protein interactions, which may confer additional specificity and plasticity on opioid receptor sorting in complex mammalian cells.
5. Effects of opiate drugs on opioid receptor membrane trafficking in cultured neurons 4. Molecular sorting of opioid receptors between functionally distinct membrane pathways after endocytosis It turns out that endocytosis of GPCRs can have various functional consequences, which are dictated in large part by the specific ‘‘downstream’’ membrane pathway followed by internalized receptors (Tsao et al., 2001). Recycling of internalized opioid receptors to the plasma membrane is associated with dephosphorylation of receptors and functional recovery of signaling activity (Koch et al., 1998), similar to the extensively studied role of the recycling pathway in mediating ‘‘resensitization’’ of beta-2 adrenergic receptor signaling (Lefkowitz et al., 1998). In contrast, sorting of internalized opioid receptors to lysosomes leads to essentially the opposite consequence, proteolytic ‘‘down-regulation’’ of receptors and attenuated signal transduction (Law et al., 1984; Tsao and von Zastrow, 2000). The molecular basis of this endocytic sorting decision is presently an area of active investigation. Certain GPCRs are sorted to lysosomes by a mechanism involving covalent modification of receptors with
A number of studies have investigated the effects of opiate drugs on the localization of endogenously expressed opioid receptors in neural tissue. Overall, there is reasonably close agreement between systems. Opiate drugs that induce rapid endocytosis of opioid receptors in transfected cell models also appear to do so in native tissues. While morphine does not produce detectable internalization of the predominant splice variant of mu opioid receptor (MOR1) in several populations of native neurons (Abbadie and Pasternak, 2001; Keith et al., 1998; Sternini et al., 1996), recent studies suggest that there may be additional complexity in certain neurons or neuronal membrane domains. The endocytic effects of methadone (internalizing) and morphine (non-internalizing) observed in non-neural cells (Keith et al., 1998; Whistler et al., 1999) correlates with receptor localization observed in cell bodies of nucleus accumbens neurons. However, in dendrites of the same neurons, morphine-induced internalization of both virally transduced MOR1 and endogenously expressed mu opioid receptors (Haberstock-Debic et al., 2003). This result suggests that morphine may indeed
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induce rapid internalization of opioid receptors in certain populations of CNS neurons, and there may be additional differences in the regulatory effects of opiate drugs occurring in different membrane domains (e.g., axons vs. dendrites) of the same neurons (HaberstockDebic et al., 2003). 6. Agonist-selective effects on opioid receptor regulation in Locus Coeruleus neurons An extensively studied set of opiate-responsive neurons are present in the Locus Coeruleus, where agonist ligands inhibit firing via mu opioid receptor-mediated activation of potassium channels (Williams et al., 2001). The ability of a series of opiates to produce homologous desensitization of opiate responses, a phenomenon thought to represent regulation of the opioid receptor itself, correlated closely with differences in the ability of opiates to promote rapid endocytosis of receptors in non-neural cells but not with differences in relative agonist efficacy (Alvarez et al., 2002). A heterologous component of opiate desensitization has also been observed in this tissue, which is thought to reflect regulation of G protein coupling to potassium channels; the ability of opiates to induce this component of desensitization correlates directly with relative agonist efficacy (Blanchet and Luscher, 2002). Together these observations support the existence of multiple mechanisms mediating opiate adaptations in native neurons that differ in regulation, either by proposed agonistselective receptor conformational states (Alvarez et al., 2002) or by the overall strength of opioid signaling to downstream effectors (Blanchet and Luscher, 2002). 7. Role of opioid receptor endocytosis in physiological adaptation of the intact nervous system The importance of opioid receptor endocytosis to chronic opiate adaptation of the intact nervous system is not yet understood. Efforts to address this question have been made by several groups using assays of opiate-induced antinociception in rodent models. Disruption of the gene encoding beta-arrestin-2 (arrestin 3) in mice strongly impaired agonist-induced desensitization of mu opioid receptors, measured biochemically by G protein activation in brain membranes. These animals exhibited increased sensitivity to the anti-nociceptive effects of morphine (Bohn et al., 1999), as well as decreased development of anti-nociceptive tolerance to this opiate (Bohn et al., 2000). However, morphine tolerance was not completely blocked in these animals and evidence suggesting a distinct protein kinase Cmediated mechanism of tolerance was observed (Bohn et al., 2002). Nevertheless, this series of studies pro-
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vides strong support for the idea that arrestin-mediated desensitization (and perhaps also endocytosis) of opioid receptors can occur in CNS neurons in vivo, and contribute both to producing changes in baseline opiate sensitivity in drug-naive animals as well as opiate tolerance in the setting of chronic drug administration. Another study utilized the tendency of opioid receptors to exist in oligomeric complexes (Devi, 2001) as a means to pharmacologically enhance morphineinduced internalization of receptors in neurons of rat spinal neurons (He et al., 2002). Intrathecally administered opioid peptide, shown previously to produce antinociception in direct proportion to internalization of mu opioid receptors in spinal neurons (Trafton et al., 2000), potentiated the ability of morphine to produce rapid internalization of spinal opioid receptors when administered at low, sub-analgesic doses. This manipulation, which essentially converted morphine from a non-internalizing to an internalizing ligand, decreased the development of anti-nociceptive tolerance to morphine. Thus, it was proposed from these results that regulated endocytosis of opioid receptors may inhibit— rather than promote—the development of anti-nociceptive tolerance to opiates in vivo (He et al., 2002). These distinct genetic and pharmacological manipulations of opioid receptor regulation support different overall hypotheses regarding the precise role of rapid opioid receptor regulatory mechanisms in modulating antinociceptive tolerance in chronically treated animals, which remain to be resolved. Furthermore, it remains to be determined what role opioid receptor regulatory mechanisms play in additional neurobehavioral features of opiate adaptation observed in intact animals (Kieffer and Evans, 2002). Nevertheless, both approaches agree in supporting the existence of important functional consequence(s) of drug-mediated regulation of opioid receptors themselves on at least one major effect of chronic opiate exposure (anti-nociceptive tolerance) observed in vivo. 8. Conclusions and future directions The hypothesis that physiological mechanisms underlying opiate drug adaptation in vivo can be studied at a cell biological level, proposed over 20 years ago, has motivated a number of laboratories to investigate opioid receptor regulatory mechanisms in biochemical detail using simplified in vitro systems. These efforts, greatly facilitated by the molecular cloning of opioid receptor genes and methodological developments in modern cell biology, have led to rapid advance toward the first goal set forth in the introduction to this article, that of understanding of specific receptor regulatory mechanisms. In particular, much has been learned about a conserved mechanism mediating regulated endocytosis of opioid receptors. An inter-
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esting observation stemming from these studies is the appreciation of substantial differences in the regulatory effects of certain opiate drugs relative to opioid peptides, which have exciting—albeit still largely unexplored—implications for molecular pharmacology and therapeutics. The appreciation of regulated endocytosis induced by opiates led to the recognition that opioid receptors are also regulated at other stages of membrane trafficking. The mechanism by which opioid receptors are ‘‘sorted’’ between divergent recycling and degradative membrane pathways after endocytosis is of particular interest because of the distinct and functionally important effects of receptor trafficking via these pathways on cell signaling and opiate responsiveness after long-term exposure to drugs. Future studies in this area may yield additional insights relevant to opiate adaptations occurring in vivo, and could identify new targets for therapeutic manipulation. An example of this type of development is the identification of an unanticipated mechanism by which opioid receptor transport in the late biosynthetic pathway is regulated both by neurotrophins and electrical activity, suggesting a novel means by which opiate responses in certain target neurons could be rapidly enhanced by local signals (Kim and von Zastrow, 2003). Studies pursuing the second goal, that of correlating regulatory mechanisms elucidated in non-neural cell models with those occurring in physiologically relevant neurons, have yielded some surprising results. While the basic machinery mediating regulated endocytosis of opioid receptors appears to be conserved in neurons and can regulate natively expressed receptors in vivo, the effects of specific opiate drugs on this mechanism can differ in distinct populations of neurons and may also differ in different membrane domains of the same neuron. Overall, our present observations support the general utility of in vitro model systems but also emphasize the importance of studying opiate effects in a native cellular context. Further studies of toward this goal will yield additional insight to the opioid regulation occurring in vivo, and might help explain differences in adaptive changes in distinct neural circuits noted previously in opiate-dependent animals. It is anticipated that progress in these studies will be facilitated by further developments in gene transfer and livetissue imaging methodologies, by which it may become possible to monitor regulation of both opioid receptors and specific ‘‘downstream’’ signaling components in the same CNS neurons and in real time. Progress toward the third goal, that of linking specific opioid regulatory mechanisms to physiological adaptation observed in while-animal physiology and behavior, is experimentally the most challenging and remains the least well understood. Nevertheless, gene knockout methods provide a powerful approach toward this goal and have already yielded intriguing
results linking opioid receptor regulation to a form of physiological tolerance to morphine. A different approach to this question, using the principle of receptor oligomerization to modulate opiate-induced endocytosis, demonstrates the feasibility of devising new pharmacological strategies using in vitro models and applying them to study receptor regulation in vivo. While much controversy remains in this area, and the experimental challenges of these approaches should not be underestimated, in my opinion there is considerable reason for excitement and optimism. In addition to rapid advance in many other experimental approaches for studying mechanistic cell biology at the whole-animal level, I believe that studies using ‘‘oligomerization pharmacology’’ illustrate how information derived from mechanistic cell biology can be rapidly adapted to in vivo application. It is anticipated that further developments of this type, in addition to helping advance our understanding of opiate physiology, may facilitate more rapid development toward novel mechanismbased pharmacotherapy of opiate-induced clinical syndromes.
Acknowledgements I apologize for citing only a subset of studies contributing to the stated conclusions. The present article is intended to provide one (personal) perspective on the field, which is admittedly biased by studies carried out in my laboratory. I wish to acknowledge important contributions made by present and former members of my laboratory, without whom I would have a lot less fun and little original information to discuss. I also thank many colleagues and collaborators elsewhere for their generous and insightful contributions, particularly Chris Evans, Brian Kobilka and John Williams. Studies in my laboratory have been supported by the National Institute on Drug Abuse and the National Institutes of Health.
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