Neuroscience and Biobehavioral Reviews 33 (2009) 11921197

Contents lists available at ScienceDirect

Neuroscience and Biobehavioral Reviews

journal homepage: www.elsevier.com/locate/neubiorev

Review

Receptor trafcking induced by m-opioid-receptor phosphorylation

Yan Zhang *, Wei Xiong, Xiaojing Lin, Xiang Ma, Long-Chuan Yu *
Laboratory of Neurobiology and State Key Laboratory of Biomembrane and Membrane Biotechnology, College of Life Sciences, Peking University, Beijing 100871, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Opiates m-Opioid receptor Phosphorylation Trafcking Tolerance Dependence G-protein coupled receptor kinase

Opiates, including morphine, are widely used drugs for antinociception in clinics. Prolonged treatments of opioids induce both tolerance and dependence, which are the major side effects of opioid therapy. One of the mechanisms for the development of tolerance and dependence is implicated to be opioid-receptor trafcking. Here we review the current understandings of opioid-receptor phosphorylation, endocytosis and desensitization after repeated agonist treatments. Also, the role of G-protein coupled receptor kinases in opioid-receptor phosphorylation is discussed. How to associate these observations to physiological and behavioral changes in animal models and clinics is still under investigation. 2009 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MOR internalization in opioid tolerance and dependence . Receptor trafcking induced by MOR phosphorylation . . . 3.1. Phosphorylation of MORs. . . . . . . . . . . . . . . . . . . . . 3.2. Acute agonist treatments . . . . . . . . . . . . . . . . . . . . . 3.3. Chronic agonist treatments . . . . . . . . . . . . . . . . . . . 3.4. Role of GRKs in MOR internalization. . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1192 1193 1193 1193 1194 1195 1195 1195 1196 1196

4.

1. Introduction Opioid dependence is the inability to stop drug application physically and mentally (von Zastrow et al., 2003). Opioid tolerance refers to a loss of effects after repeated use of drugs (Bailey and Connor, 2005). Although increasing data suggest that opioid dependence and tolerance may be mediated by separable pathways (Bailey and Connor, 2005), there is evidence strongly supporting that the trafcking of one of major opioid receptors,

m-opioid receptors (MORs), is involved in both processes (Bailey

and Connor, 2005). MORs belong to G-protein coupled receptor (GPCR) family. In general, agonist-exposure-induced receptor internalization is an important way to modulate GPCR function. Internalization of MORs has been indicated to play a critical role in opioid tolerance and dependence (von Zastrow et al., 2003). After acute or chronic exposure of agonists, MOR internalization reduces the number of membrane-located receptors, and therefore decreases the response of MOR to morphine, which is thought to be a mechanism of opioid tolerance and dependence (von Zastrow et al., 2003). There are many cellular events mediating MOR internalization. The involvement of receptor phosphorylation is demonstrated in regulating MOR internalization (Johnson et al., 2005). Many GPCRs can be modulated at serine, tyrosine or threonine sites by both basal and ligand-induced phosphorylation. GPCR kinases (GRKs) and second messenger dependent protein kinases are commonly

* Corresponding authors. Tel.: +86 10 62754880; fax: +86 10 62751526. E-mail addresses: yanzhang@pku.edu.cn (Y. Zhang), yulc@pku.edu.cn (L.-C. Yu). Abbreviations: DAMGO, [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin; GPCR, G-protein coupled receptor; GRK, G-protein coupled receptor kinase; MOR, m-opioid receptor; PKA, protein kinase A; PKC, protein kinase C; Ser, serine; Thr, threonine. 0149-7634/$ see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.neubiorev.2009.03.007

Y. Zhang et al. / Neuroscience and Biobehavioral Reviews 33 (2009) 11921197

1193

Fig. 1. MOR trafcking mediated by MOR phosphorylation. After binding with agonists, MOR is phosphorylated which triggers the activation of arrestin and clathrin-dependent endocytosis of MOR. Clathrin-dependent endocytosis then removes receptor from the cellular membrane into cytosol and forms endosome containing the internalized receptors. The reduction of number of membranebounded MOR is thought to be related to opioid tolerance and dependence. It is hypothesized that MOR internalization blocks signaling and prevents the development of tolerance and dependence. According to this idea, since the inability of morphine to induce MOR internalization, morphine-activated MORs continue to signal on the cell surface and induce the cellular adaptive response contributing to the development of tolerance and dependence. After endocytosed, MOR can be either degraded or recycled back to the membrane.

involved in the ligand-activated phosphorylation (Ferguson, 2001). General functional implications of phosphorylation include altering the types of receptor-binding G-proteins, activating or deactivating receptor interaction sites, creating adaptor or uncoupling-protein-binding sites and regulating receptor internalization and trafcking (Daaka et al., 1997; Maudsley et al., 2005). A common pathway for GPCR internalization is initiated by multiple phosphorylation of receptor, followed by activation of arrestin, which docks receptor to scaffold protein clathrin (Ferguson, 2001). Clathrin-dependent endocytosis then removes receptor from the cellular membrane into cytosol and forms endosome containing the internalized receptors, which nally get degraded or recycled (Ferguson, 2001). In summary, phosphorylation at the certain sites of MORs labels the receptors for internalization. Internalization reduces the number of membrane-bounded MORs and alters opioid tolerance and dependence (Fig. 1). The detailed mechanisms and the role of receptor phosphorylation in MOR trafcking are discussed in this review. 2. MOR internalization in opioid tolerance and dependence Continued exposure to morphine produces tolerance to the acute effects of this drug and may lead to physical and psychological dependence (von Zastrow et al., 2003). The role of opioid-receptor trafcking in the induction of tolerance and dependence has been investigated in many studies, which has been reviewed intensively (Bailey and Connor, 2005). One hypothesis regarding the role of MOR trafcking in modulating receptor signaling is that MOR internalization blocks signaling and prevents the development of tolerance and dependence (Finn and Whistler, 2001; Bushell et al., 2002). The investigators nd that although morphine is not sufcient to promote MOR internalization, introduction of MOR agonist [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DMAGO) or MOR mutations to facilitate MOR endocytosis reduces tolerance and dependence to morphine in either cellular level (Finn and Whistler, 2001) or behavioral level (Bushell et al., 2002). According to this idea, since the inability of morphine to

induce MOR internalization, morphine-activated MORs continue to signal on the cell surface and induce the cellular adaptive response, which contributes to the development of tolerance and dependence (Finn and Whistler, 2001; Bushell et al., 2002). The other hypothesis suggests that MOR internalization does not block signaling but maintains signaling by inducing recycle of uncoupled receptors to the membrane. The tolerance to morphine occurs because of the inability of morphine to recruiting receptor trafcking (Koch et al., 1998, 2001, 2005; Zhang et al., 1998). Both hypotheses base on the idea that morphine-activated MORs continue to stay on the cell surface and play normal functions. However, this theory cannot explain the role of other agonistinduced MOR internalization in the development of tolerance and dependence. The second problem with this idea is that in contrast to MOR-transfected cell lines, new proof indicates that morphine may induce signicant receptor desensitization or internalization in neurons (Haberstock-Debic et al., 2003, 2005; Dang and Williams, 2005). In addition, more studies should focus on the role of MOR internalization in vivo after long-time treatment with opioid, especially with morphine. Evidence shows that long-term treatment of mice with some opioid-receptor agonists, such as etorphine or sufentanil, downregulates the expression level of MORs (Tao et al., 1987; Diaz et al., 1995; Yoburn et al., 2004). However, chronic morphine-treated animals do not display the signicant change in the amounts of MORs (Werling et al., 1989; Bhargava and Gulati, 1990; Yoburn et al., 2004). It is possible that the internalization causes the loss of intracellular density of MORs, which may be degraded in lysosomes during each round of trafcking (Tanowitz and von Zastrow, 2003), although the details about the process of MOR degradation remain unknown. One study nds that cross-tolerance to antinociception induced by intrathecally administered DAMGO in morphine-tolerant rats can be produced without changing MOR internalization in the lamina II neurons of rat spinal cord (Trafton and Basbaum, 2004). Another in vivo study nds that chronic morphine-treatment leads to a decreased presence of MOR at the cell surface in the rostral ventrolateral medulla of morphine-treated rats, suggesting that chronic morphine application produces MOR internalization, without apparent alterations in receptor synthesis (Drake et al., 2005). The difference between in vivo and in vitro studies on agonist-dependent MOR internalization suggests that there may be huge difference between distinct types of cells, brain areas, and drug treatments in animals. These differences should be considered in the design of experiments when studying the role of MOR internalization in opioid tolerance and dependence. 3. Receptor trafcking induced by MOR phosphorylation Phosphorylation of GPCRs is a major rst step in a series of events that modulates receptor functions. The most commonly hypothesized consequences of MOR phosphorylation are the acute desensitization, receptor trafcking and chronic implication of receptor activity. Recently, it has been suggested that agonistinduced MOR phosphorylation enhances constitutive receptor activity so that MORs can continually function after agonist withdrawal (Sadee et al., 2005), although the phosphorylation residues and kinases are not determined. 3.1. Phosphorylation of MORs In MORs, there are approximately 20 serine, tyrosine and threonine sites that are accessible to protein kinases and can be potentially phosphorylated. All 3 intracellular loops and carboxyl tail contain these potential phosphorylation sites (Chavkin et al., 2001). However, not much data indicating which of these sites are

1194

Y. Zhang et al. / Neuroscience and Biobehavioral Reviews 33 (2009) 11921197

actually phosphorylated. Up to date, most of the studies of MOR phosphorylation sites have been done by mutating particular potential phosphorylation sites and observing the effects on general phosphorylation (Johnson et al., 2005). The shortcoming of this method is that it is difcult to distinguish a certain site is the actual phosphorylation site or is the binding site for phosphorylation regulation or other adaptor proteins. Therefore, in the literature, such shortcoming introduces many discrepancies in determining MOR phosphorylation sites (Johnson et al., 2005). MORs can be constitutively phosphorylated in unstimulated cells determined by the results of 32P incorporation experiments (Arden et al., 1995; Wang et al., 1996; Zhang et al., 1996, 1998; Yu et al., 1997; El Kouhen et al., 1999; Whistler et al., 1999; Deng et al., 2000, 2001; Koch et al., 2001; Schulz et al., 2004). Which protein kinases are responsible for MOR basal phosphorylation is not clear. There is evidence that protein kinase inhibitor H7 can prevent MOR basal phosphorylation (Wang et al., 1996). Activators of calcium dependent protein kinase and the catalytic subunit of protein kinase A (PKA) can increase the basal phosphorylation of MORs (Zhang et al., 1996; Chakrabarti et al., 1998; Deng et al., 2000). The functional signicance of basal phosphorylation is still unknown. It is suggested that phosphorylation of certain serine (Ser) or threonine (Thr) sites in the c-terminal of MOR can prevent receptor trafcking (El Kouhen et al., 1999). In addition, basal phosphorylation at tyrosine residues in the rst and second intracellular loops of MOR might modulate signaling efcacy (McLaughlin and Chavkin, 2001). Phosphorylation of MORs has been demonstrated in many studies (Arden et al., 1995; Zhang et al., 1996; El Kouhen et al., 1999). After agonist application, MORs undergo a rapid phosphorylation (Zhang et al., 1996, 1998; Whistler et al., 1999; Deng et al., 2000, 2001; Schulz et al., 2004). Rat MORs expressed in HEK293 cells are phosphorylated in the last two transmembrane domains and c-terminal tail (El Kouhen et al., 1999). Among many potential phosphorylation sites, Ser375 has been studied more intensively and is the only site with denitive conclusion of phosphorylation after agonist treatment demonstrated by specic antibody to phosphorylated Ser375 (Schulz et al., 2004). Agonists can also induce phosphorylation at Ser375 site in transfected HEK293 and embryonic cortical neurons (Schulz et al., 2004). Different agonist may induce variable levels of phosphorylation of MORs. This may be caused by phosphorylation of different residues (Johnson et al., 2005). For example, morphine produced more increase in Ser375 phosphorylation than other opioidreceptor agonist, such as DAMGO, sufentanil or etorphine (Arden et al., 1995; Zhang et al., 1996; Yu et al., 1997; Deng et al., 2001; Schulz et al., 2004). But mutation of Ser375 abolishes morphineinduced receptor phosphorylation while only inhibits 50% of DAMGO-induced phosphorylation (Schulz et al., 2004), suggesting the existence of additional phosphorylation sites for DAMGO. However, the results from the studies focusing on morphineinduced MOR phosphorylation are not consistent, which may depend on different cell lines and the expression level of specic kinases in these cells. The investigators nd that morphine can induce MOR phosphorylation in CHO cells (Yu et al., 1997), but not HEK293 cells (Zhang et al., 1998). However, over-expression of GRK2 in HEK293 cells results in the morphine-induced MOR phosphorylation (Zhang et al., 1998). In addition, the investigators have also observed promotion of morphine-induced phosphorylation of MORs by PKA (Chakrabarti et al., 1998). The mostly studied MOR agonist, morphine, induces less signicant phosphorylation than other agonists suggesting that different agonists might induce phosphorylation at different sites (Schulz et al., 2004). For example, morphine induces an increase of phosphorylation at Ser375 which is about 35% of that induced by DAMGO, sufentanil or etorphine (Schulz et al., 2004). However, there are data indicating

morphine does not induce phosphorylation of MORs (Zhang et al., 1998; Whistler et al., 1999). Subsidizing Ser375 with Ala almost completely prevents morphine-induced phosphorylation of MORs, but only reduces DAMGO-induced phosphorylation by 50% (Schulz et al., 2004), which suggests that DAMGO triggers different phosphorylation sites or pathways than morphine. The sites responsive to DAMGO have not been clearly demonstrated. In the morphine tolerance rats, basal incorporation of 32P by MOR is increased and DAMGO-induced phosphorylation of MOR is signicantly enhanced in the thalamic slices (Deng et al., 2001). Phosphorylation of human MORs transfected into CHO cells is recovered in 30 min (Yu et al., 1997). Dephosphorylation of rat MORs expressed in HEK293 cells happens 60 min after DAMGO withdrawal while morphine-induced phosphorylation does not reverse 6 h after morphine removal (Schulz et al., 2004). In addition, in Xemopus oocytes, dephosphorylation is shown to regulate MOR agonist efcacy (McLaughlin and Chavkin, 2001). In general, the data of MOR dephosphorylation are very limited and proteins and phosphotases involved in this process are not clearly identied. 3.2. Acute agonist treatments MORs internalization into clathrin-dependent endocytic pathways following agonist binding has been observed in MORtransfected mammalian cells, cultured neurons or in vivo (Sternini et al., 1996; Trafton et al., 2000; Bushell et al., 2002; Lee et al., 2002). MOR endocytosis in cell lines is attenuated by dominantnegative mutants of GRKs, arrestin or dynamin, and further promoted by overexpression of either GRK2 or arrestin (Whistler and von Zastrow, 1998; Zhang et al., 1998; Celver et al., 2004). The process usually happens with rapid internalization of MORs in 5 min after agonist treatment and then enters a dynamic steady state within 30 min (Keith et al., 1998; Trapaidze et al., 2000; Borgland et al., 2003). Distinct agonists, such as morphine and enkephalins, differ substantially in their ability to modulate MOR trafcking (Von Zastrow et al., 1993; Arden et al., 1995; Keith et al., 1996). The agonists which can promote obvious MOR internalization are DAMGO, etorphine, b-endorphin, fentanyl and methadone, while the other agonists that have lower ability to induce MOR internalization are morphine, pentazocine and buprenorphine (Arden et al., 1995; Keith et al., 1996, 1998; Bushell et al., 2002; Borgland et al., 2003; Celver et al., 2004). Internalization of MORs after acute agonist exposure is reported in vivo (Sternini et al., 1996; Trafton et al., 2000; He et al., 2002) and in cultured neurons (Lee et al., 2002). In cultured striatal neurons, morphine induces the similar degree of internalization of MOR as DAMGO (Haberstock-Debic et al., 2005). However, controversial data show that morphine does not induce MOR internalization in hippocampal neurons (Bushell et al., 2002), in the dendrites of cultured nucleus accumbens neurons (Haberstock-Debic et al., 2003) and in non-neuronal cells (Haberstock-Debic et al., 2005). One possible explanation is that in the cells lacking morphineinduced MOR internalization, the local concentrations of GRK and/ or arrestin are not high enough to trigger endocytosis and trafcking (Haberstock-Debic et al., 2005), although the direct evidence for this explanation is still elusive. The functional signicances of MOR internalization have been associated with desensitization and tolerance, while there are also results supporting that the analgesic doses of DAMGO can still induce MOR internalization (Trafton et al., 2000). The general assumption linking MOR phosphorylation and MOR internalization is that agonist application triggers receptor phosphorylation and recruitment of arrestin, which further activates clathrin and dynamin pathway to turn membrane-located MORs into endosome. However, the denite evidence showing this process is limited.

Y. Zhang et al. / Neuroscience and Biobehavioral Reviews 33 (2009) 11921197

1195

Kenski et al. (2005) have shown that inhibition of activity of overexpressed GRK2 prevents the initiation of morphine-induced MOR internalization, but not the maintenance of thus internalization, suggesting that recycled receptors may structurally differ from the membrane-located receptors (Kenski et al., 2005). Many studies show that morphine fails to induce MOR internalization in transfected mammalian cell lines (Von Zastrow et al., 1993; Arden et al., 1995; Keith et al., 1996, 1998; Borgland et al., 2003; Celver et al., 2004), however, several other studies get the opposite results in cultured striatal neurons (Haberstock-Debic et al., 2005). Interestingly, morphines ability to produce MOR internalization varies in different areas of brain. In the hippocampal neurons, MORs do not internalize in response to morphine (Bushell et al., 2002). In the nucleus accumbens neurons, however, morphine produces MOR internalization in the dendrites of neurons (Haberstock-Debic et al., 2003). The difference between non-neuronal cells and neurons may be caused by different expression levels of related kinases, arrestins or other molecules which have not been identied in MOR trafcking. After internalization, MORs have two possible distinct fates: up to 80% of internalized receptors will be recycled from endosomes to the plasma membrane, whereas the other receptors will trafc to lysosomes or proteasomes for degradation. The degradation will continue after long-term DAMGO treatment and induce signicant downregulation of MORs (Polakiewicz et al., 1998; Pak et al., 1999). 3.3. Chronic agonist treatments Chronic applications of etorphine and sufentanil induce signicant downregulation of MORs (Tao et al., 1987; Diaz et al., 1995; Yoburn et al., 2004). One explanation is part of the receptors get degraded during recycling and trafcking. In HEK293 cells, there are about 20% MORs degraded in lysosomes in each round of trafcking (Tanowitz and von Zastrow, 2003). In chronic morphine-treated rats, the relative amount of intracellular MORs increases versus membrane-located MORs (Drake et al., 2005). In DAMGO-treated rats, DAMGO-induced antinociception is greatly reduced while MOR internalization is almost unaffected, suggesting that chronic morphine-treatment may have less effects on receptor trafcking (Trafton and Basbaum, 2004). How MOR phosphorylation acts in chronic agonist-treatment-induced receptor trafcking and which protein kinases may involve in this process are still to be established. Phosphorylation of certain Ser and Thr residues within the carboxyl tail can regulate MOR internalization (El Kouhen et al., 2001). However, some opposite results show that the complete mutation of all Ser/Thr residues within the third intracellular loop and C-terminus of MORs does not block the agonist-induced receptor desensitization (Capeyrou et al., 1997). Mutation of Ser363 results in slower internalization when compared with wild-type MOR, but the level of receptor being internalized is similar to that of wild-type MOR after 4 h of etorphine treatment (Qiu et al., 2003). Some evidence suggests the involvement of GRKs in MOR trafcking, though the details of kinases involved in MOR internalization are not clearly known. Morphine induces MOR internalization in cells overexpressing GRK2 (Kenski et al., 2005). This effect can be blocked by using the substituted nucleotide analog 1-naphthyl-PP1 to inhibit the engineered version of GRK2 (Kenski et al., 2005). The desensitization of fentanyl- and morphine-induced population spike facilitation in the hippocampus is signicantly inhibited in GRK3 knockout mice, suggesting the role of GRK3 in modulating the MOR signaling (Terman et al., 2004). Other kinases, such as protein kinase C (PKC), can also promote the rapid MOR desensitization in neurons (Bailey et al., 2004).

3.4. Role of GRKs in MOR internalization The protein kinases involved in agonist-induced phosphorylation are still elusive. The role of GRKs in MOR phosphorylation has been studied (Wang and Wang, 2006). GRKs are a kind of Thr/Ser kinases which are consisted of seven subtypes and classied in three subfamilies (GRK1/7, GRK2/3 and GRK4/5/6) (Wang and Wang, 2006). GRK1/7 and GRK4 are specially expressed in the visual system and testes respectively and other four GRKs are expressed throughout the body and account for the regulation of most of the GPCRs (Wang and Wang, 2006). Large body of evidence supports the potentiating effects of GRK2 and GRK3 on MOR phosphorylation in response to agonist exposure (Zhang et al., 1998; Deng et al., 2000; Schulz et al., 2004). Chronic morphine treatment results in the upregulation of GRK2 in the locus coeruleus and the prefrontal cortex of rats (Wang and Wang, 2006). The levels of GRK2/6 are signicantly decreased in the prefrontal cortex of opioid addicts but upregulated in the cortex of chronic naloxone- or naltrexone-treated rats (Wang and Wang, 2006). Morphine induces similar analgesia and tolerance in the wild-type and GRK3 knockout mice but the tolerance to antinociceptive effects of fentanyl, which had more efciency to activate MOR, was reduced in the GRK3 knockout mice (Terman et al., 2004). In vitro GRK2 can phosphorylate Ser375 but not Thr370 residue of MOR. Overexpression of GRK2 enhances morphineinduced phosphorylation but has no effect on DAMGO-induced phosphorylation, implicating that different subtypes of GRKs are responsible for phosphorylation of specic agonist-receptor complex which may result from the afnity between the GRKs and the complex (Wang and Wang, 2006). However, the relationship between GRKs and MOR phosphorylation in vivo has not been denitely proved. Other studies have suggested the role of CaM kinase II in MOR phosphorylation. The mutation results show that CaM kinase II can phosphorylate the MORs via Ser261 and Ser266 in the third intracellular loop (Koch et al., 1997). The role of MOR trafcking in opioid tolerance and dependence is also investigated in GRK3 or arrestin3 knockout mice. The tolerance to continuous treatment of fentanyl is inhibited in GRK3 knockout mice, that display similar antinociceptive effects on fentanyl and morphine as wild-type mice (Terman et al., 2004). However, chronic morphine-induced tolerance does not change in GRK3 knockout mice (Gurevich and Gurevich, 2004; Terman et al., 2004). Arrestin3 knockout animals display decreased development of tolerance to morphine antinociception (Bohn et al., 1999, 2002). Similar to GRK3 knockout mice, however, the investigators fail to observe the naloxone-precipitated withdrawal in the arrestin3 knockout mice (Bohn et al., 2000). Since GRKs and arrestins are associated with receptor phosphorylation and internalization (Ferguson, 2001), the above studies on GRK3 or arrestin3 knockout mice indicate the role of MOR phosphorylation or internalization in the development of opioid tolerance and dependence. 4. Conclusions Phosphorylation is probably one of the most common protein posttranslational modications. It regulates huge variety of cellular processes and activities. Opioid-receptor phosphorylation is involved in receptor trafcking, and therefore, plays an important role in receptor desensitization and opioid tolerance and dependence (Fig. 1). There is limited knowledge about how opioid-receptor phosphorylation is regulated and how phosphorylation, in turn, modies the downstream molecular events. Fortunately, the recent advance of high through-put technology makes it possible of screening differential phosphorylation proles with various treatments in vitro, in vivo and in clinical samples. However, how to

1196

Y. Zhang et al. / Neuroscience and Biobehavioral Reviews 33 (2009) 11921197 carboxyl tail differentially regulates mu-opioid receptor internalization. Journal of Biological Chemistry 276, 1277412780. Ferguson, S.S.G., 2001. Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacological Reviews 53, 124. Finn, A.K., Whistler, J.L., 2001. Endocytosis of the mu opioid receptor reduces tolerance and a cellular hallmark of opiate withdrawal. Neuron 32, 829839. Gurevich, V.V., Gurevich, E.V., 2004. The molecular acrobatics of arrestin activation. Trends in Pharmacological Sciences 25, 105111. Haberstock-Debic, H., Kim, K.A., Yu, Y.J., von Zastrow, M., 2005. Morphine promotes rapid, arrestin-dependent endocytosis of mu-opioid receptors in striatal neurons. Journal of Neuroscience 25, 78477857. Haberstock-Debic, H., Wein, M., Barrot, M., Colago, E.E.O., Rahman, Z., Neve, R.L., Pickel, V.M., Nestler, E.J., von Zastrow, M., Svingos, A.L., 2003. Morphine acutely regulates opioid receptor trafcking selectively in dendrites of nucleus accumbens neurons. Journal of Neuroscience 23, 43244332. He, L., Fong, J., von Zastrow, M., Whistler, J.L., 2002. Regulation of opioid receptor trafcking and morphine tolerance by receptor oligornerization. Cell 108, 271 282. Johnson, E.E., Christie, M.J., Connor, M., 2005. The role of opioid receptor phosphorylation and trafcking in adaptations to persistent opioid treatment. Neurosignals 14, 290302. Keith, D.E., Murray, S.R., Zaki, P.A., Chu, P.C., Lissin, D.V., Kang, L., Evans, C.J., von Zastrow, M., 1996. Morphine activates opioid receptors without causing their rapid internalization. Journal of Biological Chemistry 271, 1902119024. Keith, D.E., Anton, B., Murray, S.R., Zaki, P.A., Chu, P.C., Lissin, D.V., Monteillet-Agius, G., Stewart, P.L., Evans, C.J., von Zastrow, M., 1998. Mu-opioid receptor internalization: opiate drugs have differential effects on a conserved endocytic mechanism in vitro and in the mammalian brain. Molecular Pharmacology 53, 377384. Kenski, D.M., Zhang, C., von Zastrow, M., Shokat, K.M., 2005. Chemical genetic engineering of G protein-coupled receptor kinase 2. Journal of Biological Chemistry 280, 3505135061. Koch, T., Kroslak, T., Mayer, P., Raulf, E., Hollt, V., 1997. Site mutation in the rat muopioid receptor demonstrates the involvement of calcium/calmodulin-dependent protein kinase II in agonist-mediated desensitization. Journal of Neurochemistry 69, 17671770. Koch, T., Schulz, S., Schroder, H., Wolf, R., Raulf, E., Hollt, V., 1998. Carboxyl-terminal splicing of the rat mu opioid receptor modulates agonist-mediated internalization and receptor resensitization. Journal of Biological Chemistry 273, 13652 13657. Koch, T., Schulz, S., Pfeiffer, M., Klutzny, M., Schroder, H., Kahl, E., Hollt, V., 2001. Cterminal splice variants of the mouse mu-opioid receptor differ in morphineinduced internalization and receptor resensitization. Journal of Biological Chemistry 276, 3140831414. Koch, T., Widera, A., Bartzsch, K., Schulz, S., Brandenburg, L.O., Wundrack, N., Beyer, A., Grecksch, G., Hollt, V., 2005. Receptor endocytosis counteracts the development of opioid tolerance. Molecular Pharmacology 67, 280287. Lee, M.C., Cahill, C.M., Vincent, J.P., Beaudet, A., 2002. Internalization and trafcking of opioid receptor ligands in rat cortical neurons. Synapse 43, 102111. Maudsley, S., Martin, B., Luttrell, L.M., 2005. The origins of diversity and specicity in G protein-coupled receptor signaling. Journal of Pharmacology and Experimental Therapeutics 314, 485494. McLaughlin, J.P., Chavkin, C., 2001. Tyrosine phosphorylation of the mu-opioid receptor regulates agonist intrinsic efcacy. Molecular Pharmacology 59, 1360 1368. Pak, Y., ODowd, B.F., Wang, J.B., George, S.R., 1999. Agonist-induced, G proteindependent and -independent down-regulation of the mu opioid receptor. The receptor is a direct substrate for protein-tyrosine kinase. Journal of Biological Chemistry 274, 2761027616. Polakiewicz, R.D., Schieferl, S.M., Dorner, L.F., Kansra, V., Comb, M.J., 1998. A mitogen-activated protein kinase pathway is required for mu-opioid receptor desensitization. Journal of Biological Chemistry 273, 1240212406. Qiu, Y., Law, P.Y., Loh, H.H., 2003. Mu-opioid receptor desensitization: role of receptor phosphorylation, internalization, and representation. Journal of Biological Chemistry 278, 3673336739. Sadee, W., Wang, D.X., Bilsky, E.J., 2005. Basal opioid receptor activity, neutral antagonists, and therapeutic opportunities. Life Sciences 76, 14271437. Schulz, S., Mayer, D., Pfeiffer, M., Stumm, R., Koch, T., Hollt, V., 2004. Morphine induces terminal mu-opioid receptor desensitization by sustained phosphorylation of serine-375. EMBO Journal 23, 32823289. Sternini, C., Spann, M., Anton, B., Keith Jr., D.E., Bunnett, N.W., von Zastrow, M., Evans, C., Brecha, N.C., 1996. Agonist-selective endocytosis of mu opioid receptor by neurons in vivo. Proceedings of the National Academy of Sciences of the United States of America 93, 92419246. Tanowitz, M., von Zastrow, M., 2003. A novel endocytic recycling signal that distinguishes the membrane trafcking of naturally occurring opioid receptors. Journal of Biological Chemistry 278, 4597845986. Tao, P.L., Law, P.Y., Loh, H.H., 1987. Decrease in delta and mu opioid receptor binding capacity in rat brain after chronic etorphine treatment. Journal of Pharmacology and Experimental Therapeutics 240, 809816. Terman, G.W., Jin, W., Cheong, Y.P., Lowe, J., Caron, M.G., Lefkowitz, R.J., Chavkin, C., 2004. G-protein receptor kinase 3 (GRK3) inuences opioid analgesic tolerance but not opioid withdrawal. British Journal of Pharmacology 141, 5564. Trafton, J.A., Basbaum, A., 2004. [D-Ala2,N-mephe4,Gly-ol5]enkephalin-induced internalization of the mu opioid receptor in the spinal cord of morphine tolerant rats. Neuroscience 125, 541543.

interpret the data generated by these studies and how to associate them with potential therapies or prevention of opioid addiction and side effects are still challenging. Acknowledgements This work was supported by the National Program of Basic Research sponsored by the Ministry of Science and Technology of China (2006CB500706, 2009CB941301), National Science Foundation of China (NSFC) General Research Grant (30670658, 30370455 and 30470542), Peking University President Research Grant and Ministry of Education Recruiting Research Grant. References
Arden, J.R., Segredo, F., Wang, Z.J., Lameh, J., Sadee, W., 1995. Phosphorylation and agonist-specic intracellular trafcking of an epitope-tagged mu-opioid receptor expressed in HEH-293 cells. Journal of Neurochemistry 65, 1636 1645. Bailey, C.P., Connor, M., 2005. Opioids: cellular mechanisms of tolerance and physical dependence. Current Opinion in Pharmacology 5, 6068. Bailey, C.P., Kelly, E., Henderson, G., 2004. Protein kinase C activation enhances morphine-induced rapid desensitization of mu-opioid receptors in mature rat locus coeruleus neurons. Molecular Pharmacology 66, 15921598. Bhargava, H.N., Gulati, A., 1990. Down-regulation of brain and spinal cord muopiate receptors in morphine tolerant-dependent rats. European Journal of Pharmacology 190, 305311. Bohn, L.M., Lefkowitz, R.J., Caron, M.G., 2002. Differential mechanisms of morphine antinociceptive tolerance revealed in beta arrestin-2 knock-out mice. Journal of Neuroscience 22, 1049410500. Bohn, L.M., Gainetdinov, R.R., Lin, F.T., Lefkowitz, R.J., Caron, M.G., 2000. Mu-opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence. Nature 408, 720723. Bohn, L.M., Lefkowitz, R.J., Gainetdinov, R.R., Peppel, K., Caron, M.G., Lin, F.T., 1999. Enhanced morphine analgesia in mice lacking beta-arrestin 2. Science 286, 24952498. Borgland, S.L., Connor, M., Osborne, P.B., Furness, J.B., Christie, M.J., 2003. Opioid agonists have different efcacy proles for G protein activation, rapid desensitization, and endocytosis of mu-opioid receptors. Journal of Biological Chemistry 278, 1877618784. Bushell, T., Endoh, T., Simen, A.A., Ren, D., Bindokas, V.P., Miller, R.J., 2002. Molecular components of tolerance to opiates in single hippocampal neurons. Molecular Pharmacology 61, 5564. Capeyrou, R., Riond, J., Corbani, M., Lepage, J.F., Bertin, B., Emorine, L.J., 1997. Agonist-induced signaling and trafcking of the mu-opioid receptor: role of serine and threonine residues in the third cytoplasmic loop and C-terminal domain. FEBS Letter 415, 200205. Celver, J., Xu, M., Jin, W., Lowe, J., Chavkin, C., 2004. Distinct domains of the muopioid receptor control uncoupling and internalization. Molecular Pharmacology 65, 528537. Chakrabarti, S., Law, P.Y., Loh, H.H., 1998. Distinct differences between morphineand [D-Ala(2),N-MePhe(4),Gly-ol(5)]-enkephalin-mu-opioid receptor complexes demonstrated by cyclic AMP-dependent protein kinase phosphorylation. Journal of Neurochemistry 71, 231239. Chavkin, C., McLaughlin, J.P., Celver, J.P., 2001. Regulation of opioid receptor function by chronic agonist exposure: constitutive activity and desensitization. Molecular Pharmacology 60, 2025. Daaka, Y., Luttrell, L.M., Lefkowitz, R.J., 1997. Switching of the coupling of the beta(2)-adrenergic receptor to different G proteins by protein kinase A. Nature 390, 8891. Dang, V.C., Williams, J.T., 2005. Morphine-induced mu-opioid receptor desensitization. Molecular Pharmacology 68, 11271132. Deng, H.B., Yu, Y.K., Wang, H.Y., Guang, W., Wang, J.B., 2001. Agonist-induced mu opioid receptor phosphorylation and functional desensitization in rat thalamus. Brain Research 898, 204214. Deng, H.B., Yu, Y., Pak, Y., ODowd, B.F., George, S.R., Surratt, C.K., Uhl, G.R., Wang, J.B., 2000. Role for the C-terminus in agonist-induced mu opioid receptor phosphorylation and desensitization. Biochemistry 39, 54925499. Diaz, A., Ruiz, F., Florez, J., Hurle, M.A., Pazos, A., 1995. Mu-opioid receptor regulation during opioid tolerance and supersensitivity in rat central-nervous-system. Journal of Pharmacology and Experimental Therapeutics 274, 15451551. Drake, C.T., Aicher, S.A., Montalmant, F.L., Milner, T.A., 2005. Redistribution of muopioid receptors in C1 adrenergic neurons following chronic administration of morphine. Experimental Neurology 196, 365372. El Kouhen, R., Kouhen, O.M., Law, P.Y., Loh, H.H., 1999. The absence of a direct correlation between the loss of [D-Ala(2),MePhe(4),Gly(5)-ol]enkephalin inhibition of adenylyl cyclase activity and agonist-induced mu-opioid receptor phosphorylation. Journal of Biological Chemistry 274, 92079215. El Kouhen, R., Burd, A.L., Erickson-Herbrandson, L.J., Chang, C.Y., Law, P.Y., Loh, H.H., 2001. Phosphorylation of Ser363, Thr370, and Ser375 residues within the

Y. Zhang et al. / Neuroscience and Biobehavioral Reviews 33 (2009) 11921197 Trafton, J.A., Abbadie, C., Marek, K., Basbaum, A.I., 2000. Postsynaptic signaling via the mu-opioid receptor: responses of dorsal horn neurons to exogenous opioids and noxious stimulation. Journal of Neuroscience 20, 85788584. Trapaidze, N., Gomes, I., Cvejic, S., Bansinath, M., Devi, L.A., 2000. Opioid receptor endocytosis and activation of MAP kinase pathway. Brain Research Molecular Brain Research 76, 220228. Von Zastrow, M., Keith Jr., D.E., Evans, C.J., 1993. Agonist-induced state of the deltaopioid receptor that discriminates between opioid peptides and opiate alkaloids. Molecular Pharmacology 44, 166172. von Zastrow, M., Svingos, A., Haberstock-Debic, H., Evans, C., 2003. Regulated endocytosis of opioid receptors: cellular mechanisms and proposed roles in physiological adaptation to opiate drugs. Current Opinion in Neurobiology 13, 348353. Wang, Z., Arden, J., Sadee, W., 1996. Basal phosphorylation of mu opioid receptor is agonist modulated and Ca2+-dependent. FEBS Letter 387, 5357. Wang, Z.J., Wang, L.X., 2006. Phosphorylation: a molecular switch in opioid tolerance. Life Science 79, 16811691. Werling, L.L., McMahon, P.N., Cox, B.M., 1989. Selective changes in mu opioid receptor properties induced by chronic morphine exposure. Proceedings of the National Academy of Sciences of the United States of America 86, 63936397.

1197

Whistler, J.L., von Zastrow, M., 1998. Morphine-activated opioid receptors elude desensitization by beta-arrestin. Proceedings of the National Academy of Sciences of the United States of America 95, 99149919. Whistler, J.L., Chuang, H.H., Chu, P., Jan, L.Y., von Zastrow, M., 1999. Functional dissociation of mu opioid receptor signaling and endocytosis: implications for the biology of opiate tolerance and addiction. Neuron 23, 737746. Yoburn, B.C., Purohit, V., Patel, K., Zhang, Q., 2004. Opioid agonist and antagonist treatment differentially regulates immunoreactive mu-opioid receptors and dynamin-2 in vivo. European Journal of Pharmacology 498, 8796. Yu, Y., Zhang, L., Yin, X., Sun, H., Uhl, G.R., Wang, J.B., 1997. Mu opioid receptor phosphorylation, desensitization, and ligand efcacy. Journal of Biological Chemistry 272, 2886928874. Zhang, J., Ferguson, S.S.G., Barak, L.S., Bodduluri, S.R., Laporte, S.A., Law, P.Y., Caron, M.G., 1998. Role for G protein-coupled receptor kinase in agonist-specic regulation of mu-opioid receptor responsiveness. Proceedings of the National Academy of Sciences of the United States of America 95, 71577162. Zhang, L., Yu, Y.K., Mackin, S., Weight, F.F., Uhl, G.R., Wang, J.B., 1996. Differential mu opiate receptor phosphorylation and desensitization induced by agonists and phorbol esters. Journal of Biological Chemistry 271, 1144911454.