Tracking the opioid receptors on the way of desensitization

Cellular Signalling 18 (2006) 1815–1833www.elsevier.com/locate/cellsig

Review

Tracking the opioid receptors on the way of desensitization

Nicolas Marie a, Benjamin Aguila b, Stéphane Allouche b,⁎

a Neuropsychopharmacologie des addictions, CNRS 7157, INSERM U705, Université Paris V, Franceb Laboratoire de Biologie cellulaire et moléculaire de la signalisation, UPRES-EA 3919, Université de Caen, France

Received 24 February 2006; accepted 21 March 2006Available online 15 April 2006

Abstract

Opioid receptors belong to the super family of G-protein coupled receptors (GPCRs) and are the targets of numerous opioid analgesicdrugs. Prolonged use of these drugs results in a reduction of their effectiveness in pain relief also called tolerance, a phenomenon wellknown by physicians. Opioid receptor desensitization is thought to play a major role in tolerance and a lot of work has been dedicated toelucidate the molecular basis of desensitization. As described for most of GPCRs, opioid receptor desensitization involves theirphosphorylation by kinases and their uncoupling from G-proteins realized by arrestins. More recently, opioid receptor trafficking was shownto contribute to desensitization. In this review, our knowledge on the molecular mechanisms of desensitization and recent progress on therole of opioid receptor internalization, recycling or degradation in desensitization will be reported. A better understanding of these regulatorymechanisms would be helpful to develop new analgesic drugs or new strategies for pain treatment by limiting opioid receptor desensitizationand tolerance.© 2006 Elsevier Inc. All rights reserved.

Keywords: Opioid receptors; Desensitization; G-protein coupled receptors; Internalization; Phosphorylation; β-arrestins

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18162. Opioid receptor desensitization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1816

2.1. Characterization of opioid receptor desensitization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18172.1.1. Opioid receptor desensitization on adenylyl cyclase pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18172.1.2. Opioid receptor desensitization on other pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1818

2.2. Quantitative modification of opioid receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18192.3. Phosphorylation of opioid receptors as a mechanism for desensitization . . . . . . . . . . . . . . . . . . . . . . . . . . . 1820

2.3.1. Phosphorylation of opioid receptors by GRKs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18202.3.2. Phosphorylation and desensitization of opioid receptors by other kinases . . . . . . . . . . . . . . . . . . . . . . 18212.3.3. Identification of phosphorylation sites on opioid receptors involved in desensitization . . . . . . . . . . . . . . . 1822

2.4. Regulation of opioid receptors activity by β-arrestins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18232.4.1. Interactions between opioid receptors and β-arrestins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18242.4.2. Role of β-arrestins in opioid receptors uncoupling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18252.4.3. Role of β-arrestins in opioid receptor desensitization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1825

3. Internalization of opioid receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18253.1. Role of receptor phosphorylation in internalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18263.2. Role of β-arrestins in opioid receptors internalization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1827

⁎ Corresponding author. Laboratoire de Biochimie, Centre Hospitalier et Universitaire, Avenue côte de nacre, 14033 Caen cedex, France. Tel.: +33 231064560; fax:+33 231064985.

E-mail address: [email protected] (S. Allouche).

0898-6568/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.cellsig.2006.03.015

mailto:[email protected]

http://dx.doi.org/10.1016/j.cellsig.2006.03.015

1816 N. Marie et al. / Cellular Signalling 18 (2006) 1815–1833

3.3. Post-endocytic fate of internalized receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18283.4. Which mechanisms dictate the post-endocytic fate of receptors? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1828

3.4.1. Receptors phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18283.4.2. β-Arrestins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18293.4.3. Accessory proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1829

3.5. Functional consequences of receptor endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18304. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1830Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1830References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1831

1. Introduction

Opioids are the most potent analgesic drugs used not only forpain relief but also for treatment of diarrhoea or cough.However, their prolonged administration for chronic painproduces tolerance to the analgesic effects requiring escalatingdoses that are associated with side effects such as respiratorydepression, and thus limiting their therapeutic potential. A lot ofwork has been dedicated to identify molecular mechanisms oftolerance and now it is well admitted that opioid receptordesensitization, that is defined as a decrease of receptorsignalling after sustained agonist activation, is closelyconnected to this phenomenon. As a huge amount of workhas been accumulated on desensitization of opioid receptors andtheir molecular mechanisms for more than 20 years, we attemptto review all these data stressing on conflicting results betweenstudies; we discuss about different models and experimentalconditions (opioid agonists, periods of pretreatment, etc.) thatare used trying to find explanations for these discrepancies. Wealso focus on recent data revealing the role of receptortrafficking in desensitization and tolerance. We summarizeddata in this large field that were published before November2005.

Knock-out of opioid receptor genes by homologousrecombination in mice showed that among the different typesof opioid receptors (μ, δ and κ), only μ receptors are involved inanalgesia and tolerance induced by morphine [1]. However,other data obtained by the same laboratory in a further studyrevealed a decrease of analgesia induced by δ-selective agonistsin mice lacking μ-opioid receptors. The authors proposed acooperativity between μ and δ receptors resulting rather frominterconnections of neurons expressing these opioid receptorsthan a physical interaction between these opioid receptors [2].However, a physical interaction between μ and δ receptorseffectively occurs as revealed by formation of μ–δ heterodimerswhose pharmacological properties would differ from monomersand would be preferentially activated by deltorphin II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2) [3]. Such a regulation of δ-opioid receptors-mediated antinociception by μ receptors wasrecently documented both in rats and mice by Morinville et al.[4]. Hence, sustained activation of μ receptors by variousagonists including morphine for 48 h enhanced δ receptorimmunoreactivity at the plasma membrane of neurons in thespinal cord which was correlated with an increase ofantinociception promoted by δ selective agonists. Obviously,not only μ- but also δ-opioid receptors are regulated by

morphine and they both participate in the development oftolerance.

Tolerance is highly complex and includes modifications ofopioidergic but also of many other neurotransmitter pathways.Indeed, blockade of N-methyl-D-aspartate (NMDA) [5],cholecystokinin (CCK) [6,7] or calcitonin gene-relatedpeptide-receptors [8] by antagonists was shown to reducemorphine-induced tolerance. So, it was speculated thatprolonged administration of opioid drugs would promotealterations of neuronal networks resulting from an exacerba-tion of excitatory pathways and a reduction of neuronalinhibition by opioids. Cellular modifications of opioidergicneurons in various cerebral regions, including thalamus andperiaqueductal gray matter, have also been observed in chronicmorphine-treated animals and are characterized by a desensi-tization of opioid receptors [9]. Actually, the experimental datasupport both mechanisms (i.e. exacerbation of pronociceptivepathways and opioid receptor desensitization) in opioidtolerance development but only molecular mechanisms ofopioid receptor desensitization will be discussed in the presentreview.

2. Opioid receptor desensitization

Historically, first studies of opioid receptor desensitizationwere conducted in vivo, but data interpretation was subjected toquestioning for two major reasons:

– The three different classes of opioid receptors μ, δ and κexpressed in the central nervous system (CNS), aredifferentially regulated as described thereafter in thisreview.

– Even when using “selective” ligands, it is difficult tospecifically activate one class of receptor in vivo since theselectivity is obtained at low concentrations and generallyhigh doses are used in most of the studies.

So, many laboratories moved from animal experimentalmodels to cell lines endogenously expressing opioid receptorsto study biochemical processes of desensitization. For instance,we mention the hybridoma NG108-15 only expressing murineδ-opioid receptors [10], the two human neuroblastoma SK–N–SH [11] and SH-SY5Y [12] expressing both μ- and δ-opioidreceptors and the neuroblastoma cell line SK–N–BE expressingan homogenous population of human δ-opioid receptors [13].Since the cloning of the first opioid receptor in 1992 [14,15],

1817N. Marie et al. / Cellular Signalling 18 (2006) 1815–1833

those cellular models have been largely supplanted byheterologous expression systems whose major advantage is toeasily express a desired receptor type. Indeed, most of thestudies related to the regulation of opioid receptors are actuallyconducted on non-neuronal transfected systems as COS-7(green monkey kidney) cells, Chinese Hamster Ovary cells(CHO) or Human Embryonic Kidney (HEK293) cells in whichopioid receptors are generally over-expressed. These cellularmodels are widely used for studies of most of the GPCRs butextrapolation of data obtained in such systems to neurons couldbe sometimes hazardous as recently shown by Haberstock-Debic et al. who observed that morphine is able to promote μreceptor internalization in cultured striatal neurons while whenexpressed in HEK293, these opioid receptors remain localizedat the plasma membrane even after prolonged periods ofmorphine exposure [16]. Western blot analysis of neurons andnon-neuronal cells (HEK293) showed different protein expres-sion levels of opioid receptor regulators (G protein receptorkinases (GRKs) or arrestins). These differences would provideputative explanations for such discrepancies between studiesand would indicate the limitations of heterologous systems aspertinent models for studying opioid receptor regulation [16].Regardless of their expression levels, it is also possible that inneuronal cells compartmentalization of opioid receptors andtheir regulators in highly organized structures would allowefficient and rapid interaction between these proteins while suchstructures in heterologous systems would not exist. It is alsoworthy to note that evident disparities also exist betweenHEK293 and CHO cells when opioid receptor internalization isstudied. When a truncated mutant of δ-opioid receptor isexpressed in CHO cells, no obvious sequestration is observedupon etorphine exposure whereas expression of this mutant inHEK293 cells allows its internalization (discussed later inSection 3.1) [17].

2.1. Characterization of opioid receptor desensitization

Desensitization of μ-, δ- and κ-opioid receptors has beenessentially studied on the inhibition of adenylyl cyclase forpractical reasons; quantification of cAMP intracellular levelsis a more popular method than measurement of ionic currentsby electrophysiology. Most of the studies about desensitiza-tion are conducted on second messengers, which makesdifficult to distinguish the involvement of uncoupling,internalization and post-endocytic trafficking in this complexphenomenon. However, pharmacological agents such assucrose or monensin allow to study desensitization indepen-dently from internalization and post-endocytic trafficking,respectively [18,19].

Two kinds of desensitization can be described but withdistinct mechanisms underlying each process ([20]):

– The homologous desensitization results from inactivation ofprolonged agonist-activated receptors

– The heterologous desensitization concerns both agonist-activated and non-activated receptors sharing the samesignalling pathways.

While opioid receptor desensitization has been the object ofintensive investigations and was thought to be the basicmechanism protecting cells against an excessive stimulation,experimental data from the literature tend to reveal a morecomplex process than expected. Indeed, molecular mechan-isms of tolerance to morphine were proposed to result fromthe absence of μ receptor desensitization [21]. These authorsproposed that an excess of transduction from these opioidreceptors would activate certain pathways triggering tolerance.However, two recent studies seem to challenge the basis ofthis theory since morphine can also induce desensitization[22,23].

2.1.1. Opioid receptor desensitization on adenylyl cyclasepathway

To illustrate this complexity, we selected arbitrarily somerepresentative studies that are summarized in Table 1 andshow that in vitro opioid receptor desensitization is observedrapidly as soon as 5 to 15 min of agonist pretreatment [24,25]contrasting with longer exposure required to induce tolerancein vivo [9]. As expected intuitively, the level of opioidreceptor desensitization is time- and agonist concentration-dependent. The more receptors are activated by agonist, themore they desensitize [26,27]. This could explain the greatdisparity of the observations between various studies aboutdesensitization of opioid receptors since experimental condi-tions are rarely identical from one study to another. Thestudies investigating the nature of the desensitization, showedthat it was either homologous [28] or heterologous [9,25,26]and dependent on the cellular model, suggesting the existenceof distinct mechanisms contributing to this reduction ofsignalling. The homologous desensitization involves phos-phorylation of the agonist activated-receptor by the GRKfamily. Once phosphorylated, the receptor is uncoupled fromits G proteins and then internalized. In the case ofheterologous desensitization, agonist-activated and non-acti-vated receptors are phosphorylated by second messengers-dependent kinases; this covalent modification would impairsignal transduction from receptors. Receptors bind endogenousopioid peptides, natural products such as morphine andsynthetic ligands. But depending on the agonist used, itappears that the opioid receptors are differentially desensitizedas demonstrated by works from Bot et al. [29] and from ourlaboratory [18,24,30–32]. In early studies, we proposed thathuman δ-opioid receptor desensitization was faster and deeperupon peptides than alkaloid agonists exposure [30] but using alarger sample of drugs, we noticed that this property wasrather dependent on the δ agonist's selectivity [18,31,32]. InHEK293 transfected cells, Blake et al. [33] observed thatmorphine failed to induce μ-opioid receptor desensitizationwhile other agonists such as methadone and buprenorphinedid. They suggested a possible link between the ability ofdrugs to induce desensitization and their reduced abuseliability. However, recent studies showed that morphine wasable to promote μ receptor desensitization. When expressed inHEK293 transfected cells, μ receptors were shown todesensitize as soon as 30 min after activation by morphine

Table 1Desensitization of opioid receptors on the inhibition of adenylyl cyclase

Receptor Models Agonist Pretreatment Desensitization References

μ, δ Periaqueductalgrey matter

Morphine 6 days Heterologous desensitization between opioid (μ, δ)metabotropic glutamate and 5-HT1A receptors

[9]

SH-SY5Y PLO17, DPDPE 24 h Dose-dependent and homologous desensitization betweenopioid and α2-adrenergic receptors

[28]

μ 7315 c Morphine 100 μM 1 h→48 h Desensitization by ∼ 80–90% after 9-h pretreatment [61]HEK293 Etorphine, methadone,

buprenorphine, morphinelevorphanol, DAMGO

3 h Desensitization (increase of IC50 and reduction of maximalinhibition) after etorphine, methadone, buprenorphinetreatment. No desensitization after exposure to morphine,levorphanol or DAMGO

[33]

Fentanyl, sulfentanil,lofentanil

More important desensitization after sulfentanil or lofentanilexposure compared to fentanyl

[70]

DAMGO 30 min→4 h More important desensitization of MOR1 compared to MOR1Bafter 30-min pretreatment but similar after 4-h agonist exposure

[19]

δ NG 108-15 Etorphine 24 h Total desensitization at 1 μM etorphine. Heterologousdesensitization between opioid, muscarinic and α2-adrenergicreceptors

[26]

CHO DADLE, 10 pM→0.1 μM At low concentrations, more important desensitization in clonesexpressing low levels of opioid receptors. Similardesensitization when DADLE = 100 nM

[27]

SNC-80, 100 nM Partial desensitization with an increase of IC50 and reduction ofmaximal inhibition

[65]

SK–N–BE Etorphine 15 min→2 h Heterologous desensitization between opioid, dopaminergicand α2-adrenergic receptors

[25]

HEK 293 DPDPE, DSLET,DADLE, morphine

3 h Desensitization after DPDPE, DSLET, DADLE, exposure(decrease of maximal inhibition) and sensitization (decrease ofIC50 and increase of maximal inhibition) after morphinetreatment

[29]

κ R1.1 U50,488 15 min, 24 h and 48 h No desensitization with no change either in IC50 or maximalinhibition

[35]

HEK 293 U50,488, Dynorphine A,levorphanol, etorphine

3 h Pretreatment with U50,488 or Dynorphine A decreases theirinhibitory action (desensitization). Exposure either tolevorphanol or etorphine increases the inhibitory action ofU50,488

[34]

CHO U50,488, 100 nM 1 h and 24 h Desensitization with an increase of IC50 but withoutmodification of maximal inhibition

[75]


on the inhibition of adenylyl cyclase [23]. In contrast,exposure to some opioid agonists was demonstrated topromote rather a sensitization than desensitization. This isthe case of the κ-opioid receptor that desensitized uponU50,488 (trans-3,4-dichloro-N-methyl-N[2-(1-pyrrolinyl)-cyclohexyl]-benzeneacetamide) pretreatment but after etor-phine exposure, a greater potency of U50,488 to inhibit cAMPaccumulation was observed [34]. These latter data obtained inHEK293 cells are in contradiction with the absence ofdesensitization observed in the thymoma cell line R1.1naturally expressing the mouse κ receptors [35]. Differencesbetween the two cellular models could probably account forsuch discrepancies but this observation illustrates alsodivergent data concerning opioid receptor desensitization.One explanation for these contrasting results is the differentopioid receptor levels between such cell lines; the κ-opioidreceptors are expressed at relative high level in HEK293 cells(1.44 pmol/mg of protein) by comparison with the R1.1 cellline (55 fmol/mg of protein) [36]. Indeed, by using CHO celllines expressing different levels of receptors either by clonalselection or via a hormone-inducible expression vector, Law etal. demonstrated that opioid receptor desensitization wasdirectly related to their expression level [27,37]. The more

receptors are expressed, the more the desensitization is low.According to this rule, a higher desensitization of opioidreceptors was expected in the thymoma cell line compared tothe transfected HEK293 cells suggesting the plethora ofparameters influencing desensitization. Indeed, striking differ-ences between rodent and human κ receptor desensitizationwere also reported [38,39] While upon U50,488 exposurehuman κ-opioid receptors were shown to desensitize, rat κ-opioid receptors failed. This difference is attributable to theSer358 localized in the carboxyl-terminal domain of the humanκ receptor that undergoes a phosphorylation by GRKs (seebelow). This underlines the risk of extrapolation of dataobtained from rodents to humans. In spite of elucidatingparameters directly influencing desensitization, it is notpossible to reconcile all data of the literature. Opioid receptorsregulate other effectors than adenylyl cyclase and desensiti-zation was also studied on such pathways.

2.1.2. Opioid receptor desensitization on other pathwaysIn the hybridoma cell line NG108-15, activation of

δ receptors triggers intracellular calcium rise, a response thatundergoes a fast desensitization after 30–90 s [40]. Such kineticof desensitization has also been observed for the activation of


phospholipase C by the μ-opioid receptors in the SH-SY5Ycells [41]. In the neuroblastoma ND8-47, longer agonistexposure (24 h) is required to observe a decrease of the δreceptors-mediated intracellular calcium rise contrasting withdata obtained by Song and Chueh [40]. However, thisdesensitization is dose-dependent and can be induced onlyafter 1 h but in the presence of a high concentration of 1 μM [D-Ser2-Leu5-Thr6]enkephalin (DSLET) [42]. In the NG108-15cell line, the inhibition of voltage-dependent calcium channelsby δ-opioid receptors is decreased after a prolonged treatment(10–30 min) by [D-Ala2-D-Leu5]enkephalin (DADLE) [43]. Inanother model, the dorsal root ganglion neurons, the loss ofinhibition of calcium currents observed after 10 min pretreat-ment by [D-Ala2-MePhe4-Gly5-ol]enkephalin (DAMGO) israther attributed to the inactivation of ionic channels than thereceptor desensitization. However, when agonist treatment isprolonged until 24 h, the reduction of the response on thecalcium channels is due to desensitized μ receptors. Moreover,this desensitization is homologous as no modification of theresponse to γ-amino butyric acid (GABA) B receptors wasevidenced on calcium currents after DAMGO pretreatment [44].In the locus coeruleus neurons, activation of μ receptors eitherby DAMGO or Met-enkephalin produces opening of potassiumchannels which rapidly diminishes after 5 min. The authorsdemonstrated rather an alteration at the receptor level than at thechannel itself [45]. More recently and using a similar model,Blanchet and Lüscher [46] have an opposite conclusion: the lossof potassium current induced by a sustained activation of μreceptors directly results from the inactivation of the channels.This has also been demonstrated when μ-opioid receptors andpotassium channels are co-expressed in the Xenopus oocytes[47]. In locus coeruleus neurons, morphine was shown to causea desensitization on outward potassium currents but to a lowerextent and to a slower rate compared to Met-enkephalin (Tyr-Gly-Gly-Phe-Met) [22]. The differential ability of these twoagonists to desensitize opioid receptors would be due to thedifferent intrinsic activity of morphine (partial μ agonist) andMet-enkephalin (full μ agonist) or to the more robust ability ofMet-enkephalin to induce μ receptor phosphorylation [48].Subsequently, it is easier for the highly phosphorylated-μreceptor to recruit β-arrestins which in turn decrease receptorsignalling [49]. This morphine-induced desensitization wasabolished by a prior brief exposure of Met-enkephalin, whichby itself would uncouple receptor. So, a further application ofmorphine after Met-enkephalin exposure leads to a weakoutward potassium currents activation, then in this condition, μreceptor desensitization would be difficult to observe. That isprobably why in previous works, the authors failed to observe adecrease of potassium current after morphine exposure since abrief challenge with Met-enkephalin was used to determine amaximum response [50]. This is in contrast with previousresults obtained by Kovoor et al. who observed no obviousrapid μ receptor desensitization on potassium channels uponmorphine treatment even without any pretreatment with a fullagonist [51].

The opioid receptors also modulate the mitogen-activatedprotein kinase (MAPK) pathway [52–54]. In CHO cells,

Polakiewicz et al. observed no heterologous desensitizationbetween μ- and lysophosphatidic acid-receptors on theextracellular-regulated kinases 1 and 2 (ERK1/2) pathway[54]. This desensitization is dose-dependent, detected as soon as5 min and the response was lost over 2-h pretreatment withDAMGO. Identical results were reported by Schulz et al. inHEK293 cells transfected with μ-opioid receptors [23]. Moreinterestingly, interconnection between activation of the ERK1/2protein kinases and the inhibition of adenylyl cyclase is thoughtto occur since the blockade of the MAPK pathway by the 2′-amino-3′methoxyflavone (PD98059) prevents μ receptor de-sensitization on the cAMP pathway even after 2 h of 100-nMDAMGO exposure.

All those data indicate that regulation of opioid receptorsignalling is highly complex, depending of numerous para-meters such as the chemical nature of agonists, their concentra-tions, the time of pretreatment, the opioid receptor levels, thecellular model, and affects either the receptor itself or theeffector (i.e. the potassium channels) depending on theconsidered pathway. Desensitization of opioid receptors,whose molecular mechanisms are discussed later, is due to arapid uncoupling between receptors and G proteins. Decrease ofopioid receptors from cell surface has also been proposed as anexplanation for the reduction of signalling after agonistpretreatment (i.e. desensitization).

2.2. Quantitative modification of opioid receptors

Disappearance of total (cell surface+ intracellular) opioidreceptors is referred as down-regulation and is generallymeasured by using lipophilic radioligands in whole cell or incrude membrane fraction and by Western blot using antibodiesdirected against either the native or tagged receptors. From invivo studies, there is no clear relationship between agonisttreatments and the opioid receptor levels. Indeed, in tolerantmice, rats or rabbits, morphine exposure produces either animportant decrease [55] or no significant variation of opioidreceptor number [56,57]. Reduction of μ receptors in the CNSwas also reported after a prolonged treatment by high doses ofbuprenorphine in rats [58] and by etorphine in mouse spinalcord but not upon morphine exposure [59]. However, when theloss of opioid binding sites was detected, it could not beattributed to the decrease of mRNA level encoding for the μreceptors [60]. From these results, it is difficult to assume thattolerance is due to a quantitative loss of opioid receptors. Whatabout relationship between opioid receptor number anddesensitization in vitro?

In numerous studies, even a brief activation (few hours) ofμ-, δ- and κ-opioid receptors leads to a reduction of theirnumber both in cellular models endogenously expressing thesereceptors and in transfected cells [19,26,28,29,33–35,61–71].δ receptor down-regulation is achieved in lysosomes as shownby the use of chloroquine that impairs lysosomal enzymeactivity [18,72,73] but also by the ubiquitin/proteasomepathway [71].

In transfected cells such as COS-7 [74], CHO [75] or babyhamster kidney cells [64], short-term activation (1 h) of κ- and


μ-opioid receptors was demonstrated to promote desensitizationwithout affecting receptor density suggesting that bothprocesses are independent as previously demonstrated for δ-receptors in NG108-15 cells [26]. In contrast, other studiesreported a close correlation between loss of opioid receptors andtheir desensitization as demonstrated for μ receptors expressedin HEK293 [19] and SH-SY5Y cells [28], as well as forδ receptors expressed in SH-SY5Y cells [28], CHO cells [65]and SK–N–BE cell line [18,31]. In other reports, desensitiza-tion was null or weak compared to the robust reduction ofopioid binding sites [66,76] as illustrated with the κ receptorsexpressed in the R1.1 thymoma cell line that are down-regulatedbut not desensitized upon U50,488 exposure [35]. This suggeststhat, when active receptors are down-regulated, spare receptorswould be recruited and in these conditions only a modestdesensitization could be observed. Conversely, it is possible toobserve a strong desensitization associated with a weakreduction of opioid binding sites demonstrating the absence ofsuch spare receptors in certain experimental models [29,61,68].Moreover, opioid agonists have obviously not the same abilityto promote desensitization and down-regulation whatever thetype of opioid receptors [18,29–34,69,70]. This is also true forregulation of mRNA levels of opioid receptors. Whenmessengers of μ receptors are measured by quantitative-competitive RT-PCR in SH-SY5Y cells, morphine treatmentwas able to reduce their levels but not endomorphin-1 and -2[77]. However, it appears that loss of opioid binding sites is notcorrelated with the decrease of mRNA levels encoding foropioid receptors [63,67].

2.3. Phosphorylation of opioid receptors as a mechanism fordesensitization

Since reduction of opioid receptor numbers, which isinconstant, cannot alone explain the decrease of opioid receptoractivity upon chronic stimulation, other pathways wereexplored and particularly the role of receptor phosphorylationas demonstrated for the β2-adrenergic receptor (see for review[78]).

The first direct demonstrations of opioid receptor phosphor-ylation were reported in 1995 by using metabolic labelling with[32P]orthophosphate. Pei et al. [79] demonstrated a rapid and atime-dependent phosphorylation of δ receptor expressed inHEK293 cells upon [D-Pen2-D-Pen5]enkephalin (DPDPE)exposure. Both at the same time and in the same transfectedcells, Arden et al. reported that μ receptor was phosphorylatedin the basal state (in the absence of agonist) and after 15 min ofstimulation with a high concentration of morphine (10 μM), thelevel of receptor phosphorylation was increased [80]. In acellular model endogenously expressing human δ receptors, weobserved a correlation between phosphorylation and desensiti-zation upon etorphine exposure [81]. On hippocampal slicesfrom guinea pig, the κ-opioid receptor is also phosphorylatedunder the basal condition and its phosphorylation stateincreased by 40% following U50,488 exposure (2 μM for75 min). Parallel to this phosphorylation process, the κreceptors are desensitized as measured by electrophysiology on

the response evoked by perforant path stimulation [82].Moreover, these opioid receptors remained phosphorylated inU50,488-tolerant animals [82]. Similar results were publishedby Deng et al. [83] for the μ receptors in striatum and thalamusfrom rat brain. In thalamus, DAMGO and morphine (1 μM forboth) were shown to enhance μ receptor phosphorylation butto a lesser extent in the presence of morphine as compared toDAMGO. The time-course and dose–response experimentsdemonstrated that opioid receptor phosphorylation wasdetected as soon as 5 min, maximal at 10 μM DAMGO andcorrelated with their desensitization when measured on theinhibition of adenylyl cyclase. The DAMGO-induced increaseof μ receptor phosphorylation was greater in morphine-tolerantrats than naive animals suggesting that opioid receptorphosphorylation is probably a crucial event in the developmentof tolerance.

In order to establish a direct link between desensitizationand receptor phosphorylation, Yu et al. [48] comparedvarious opioid agonists in promoting phosphorylation anddesensitization of human μ-opioid receptors expressed inCHO cells (phosphorylation and functional experiments) andin Xenopus oocytes (functional experiments). This studyclearly established that the ability of an agonist to promote μreceptor phosphorylation was correlated with its ability toinduce desensitization [48]. While in this latter study theauthors demonstrated that morphine was able to promote μreceptor phosphorylation, Zhang et al. [49] observed that thisalkaloid ligand failed to induce incorporation of [32P]orthophosphate in μ receptors expressed in HEK293 cells.Such discrepancies could be related to the different cellularmodels used and consequently to their content in proteinsinvolved in such processes (GRKs or other kinases). Thishighlights the difficulty to find an experimental model closeto physiology.

In spite of their complexity, all these data suggest that opioidreceptor phosphorylation plays an important role in theirregulation. Thus, it is crucial to identify which kinase(s) is(are) involved in opioid receptor phosphorylation anddesensitization?

2.3.1. Phosphorylation of opioid receptors by GRKsThe first study demonstrating the role of GRKs in opioid

receptor phosphorylation was published by Pei et al. [79]. Whenexpressed in HEK293 cells, the GRK2 (β-Adrenergic ReceptorKinase 1 or βARK1) and GRK5 were shown to increasephosphorylation level of δ receptors upon DPDPE treatment[79]. However, in functional experiments only the role of GRK2was explored. Hence, over-expression of GRK2 enhanceddesensitization on the inhibition of cAMP pathway whereas theover-expression of a dominant negative mutant of this kinase(GRK2-K220R) totally blocked DPDPE-induced opioid recep-tor desensitization.

GRKs belong to the Ser/Thr kinases family that phosphor-ylates GPCRs when activated by their agonist and arecomposed by 7 different members [84]. While GRK1, 4 and7 have a restricted expression pattern, the other isoforms arewidely expressed in different tissues. These kinases display a


highly conserved structure with variable regions at the amino-and carboxyl-terminal tail; this latter domain by interacting withdifferent cellular components is responsible for the subcellulardistribution of GRKs and modulates their activity. For instance,GRK2 and 3, that are cytosolic proteins, undergo a translocationto the plasma membrane by interacting with free Gβγ dimers viatheir pleckstrin homology (PH) domain localized at the tail. Inthese conditions, the GRKs are close enough to the agonist-activated receptors to promote their phosphorylation.

Involvement of GRKs in opioid receptor regulation wasdemonstrated in various models such as HEK293 cells, inwhich over-expression of GRK2 potentiates the ability ofmorphine to induce phosphorylation of μ receptor and reducessignificantly the inhibition on the adenylyl cyclase [49]. Thiskinase participates directly to desensitization of the μ-opioidreceptors in neurons of the nucleus raphe magnus [85] and theκ receptors expressed in COS-7 cells [74]. By over-expressinga carboxyl-terminal region of GRK2 (from Gly495 to Leu689)known to interact with free Gβγ dimers and consequentlyimpairing recruitment of endogenous GRK2 to plasmamembrane, Wang [86] observed a major decrease of rat μreceptor desensitization expressed in HEK293 cells. Involve-ment of other GRKs in regulation of opioid receptors was alsodemonstrated; GRK3 expression in Xenopus oocytes wasshown to mediate μ receptor desensitization [87]. In NG108-15 cells, GRK6 over-expression was shown to reduceinhibition of cAMP formation after pretreatment with a highconcentration of DPDPE (10 μM) while GRK2 or its dominantnegative mutant were devoid of any effect on desensitizationof δ receptors endogenously expressed in this neuroblasto-ma×glioma hybrid cells [88]. In the human neuroblastomaSK–N–BE cells, heparin, an inhibitor of GRKs, was shown todecrease effectively etorphine-induced δ receptor phosphory-lation and desensitization [81]. Desensitization of these opioidreceptors observed on voltage-dependent calcium channelswas also reduced by pretreatment with heparin suggesting therole of one or more GRKs in these processes [43]. Differencesin the ability of opioid receptors to mobilize GRK2 and 3 wereobserved by Schulz et al. [89]. To visualize GRKs and opioidreceptors, these authors generate opioid receptors fused toenhanced green fluorescent protein (EGFP) and GRKs fused toDsRed (a red fluorescent protein) that were expressed either inHEK293 cells or the neuroblastoma cell line NG108-15. Whileδ receptor activation by deltorphin II allows a rapidtranslocation of either GRK2 or 3 to the plasma membraneafter 1 min, μ receptors failed to trigger GRKs recruitmentupon sufentanil, etorphine, DAMGO or endomorphin 1exposure. These results illustrate the different capacity ofopioid receptors to mobilize GRKs. However, GRKs are notthe only kinases known to mediate opioid receptor phosphor-ylation and desensitization.

2.3.2. Phosphorylation and desensitization of opioid receptorsby other kinases

2.3.2.1. PKA. PKA is a Ser/Thr kinase activated by cAMP. Itis a heterotetramer in its inactive form, composed of two

regulatory and two catalytic subunits. Molecular cloningrevealed the presence of four different regulatory subunits(RIα, RIβ, RIIα, RIIβ) and four different catalytic subunits(Cα, Cβ, Cγ, PrKX) [90,91]. Binding of cAMP to regulatorysubunits leads to release of the catalytic subunits that couldphosphorylate their substrates [90].

Role of PKA in desensitization seems unlikely since opioidreceptors are known to inhibit adenylyl cyclase and thusdecrease cAMP level. However, chronic exposure to morphineresults in a compensatory up-regulation of adenylyl cyclaseactivity (or overshoot) and a subsequent increase of intracellularcAMP level that in turn can stimulate PKA activity [92]. In vitrostudies showed that, in the presence of catalytic subunit of PKA,morphine and levorphanol were able to stimulate μ receptorphosphorylation while other agonists as DAMGO or [D-Ala2-D-Leu5]enkephalin (DADLE) were not [93]. Even if this kinasephosphorylates μ receptors, PKA is not involved in desensiti-zation when examined on the cAMP pathway [93]. In thehuman neuroblastoma SH-SY5Y, Wang and Sadée [94] alsoconcluded that PKA was not involved in morphine-induced μreceptor desensitization while activation of this kinase byforskolin pretreatment effectively decreased the ability ofmorphine to inhibit cAMP accumulation. These data rule outthe role of cAMP and PKA in agonist-induced opioid receptordesensitization.

2.3.2.2. PKC. The term “PKC” defines a large family of Ser/Thr kinases composed of at least 12 members and is divided inthree subfamilies based on their amino-terminal ends condi-tioning their Ca2+ and diacylglycerol (DAG) dependency. Thethree subfamilies are: the classical isoforms (PKCα, PKCβI,PKCβII and PKCγ) that are regulated by both Ca2+ and DAG,the novel isoforms (PKCδ, PKCε, PKCθ and PKCη) thatrequire DAG only and the atypical isoforms (PKCξ, PKMξ, andPKCι/λ) that need neither Ca2+ nor DAG [95].

As described above, activation of opioid receptors couldincrease intracellular calcium and thus leading to PKCactivation. So, it is tempting to speculate on a role of PKC inopioid receptor desensitization and phosphorylation. Indeed, inNG108-15 cells, a prolonged DADLE treatment induceddesensitization of Ca2+ stores mobilization, which is inhibitedby two different PKC inhibitors (staurosporine and bisindo-lylmaleimide GF109203X) [96]. Using a different protocol toinduce desensitization (repetitive exposure to Leu-enkephalin(Tyr-Gly-Gly-Phe-Leu), Song and Chueh (1999) [40] demon-strated that this desensitization was blocked by staurosporine. Inthe same cell line, promoting calcium entry by activatingionotropic glutamate receptor (AMPA/kainate or NMDAreceptor) reduced activation of G proteins by DPDPE orinhibitory effect on cAMP accumulation by this agonistwhereas ketamine and AMPA/kainate receptor antagonistreduced DPDPE-induced desensitization. The effect of gluta-mate receptor activation on δ receptor activity is reduced whenPKC are inhibited [97,98]. Moreover, NMDA receptoractivation induced δ receptor phosphorylation but the authorsdid not show PKC involvement in this process. Mestek et al.[99] showed that activation of PKC by phorbol esters


potentiates desensitization of μ receptors on K+ channels uponDAMGO exposure. Again, it is worthy to note that theseauthors did not show that this increase in desensitization wasrelated to a stimulation of receptor phosphorylation. All thesedata suggest a role for PKC in opioid receptor desensitization,but even if PKC activation by phorbol esters promotesphosphorylation of δ- [79] and μ-opioid receptors [100,101],these kinases are involved neither in this phosphorylation[79,100,101] nor in receptor desensitization upon agonistexposure as shown by using selective inhibitors or PKC-depleted cells [43,100]. These data strongly argues for a lack offunction of PKCs, at least in desensitization.

Recently, by an electrophysiology study of K+ currents onbrain slices preparations Bailey et al. [102] evidenced that directactivation of PKC by a phorbol ester or by stimulation of M3muscarinic receptors causes a rapid μ receptor desensitizationafter morphine exposure while the decrease of the response afterMet-enkephalin treatment was shown to be insensitive to PKCinhibitor (chelerythrine). This effect of PKC activation is ratherdue to a modification of opioid receptors than K+ channels butthese authors did not explore phosphorylation state of thereceptors and its is not possible to rule out that this PKCinhibitor modify activity of other proteins involved in thetransduction pathway (G proteins, GRKs, arrestins). Indeed, inrabbit smooth muscle cells, PKCα and ε were shown tophosphorylate Gαi1 and Gαi2 after activation of CCK receptor,and this effect mediate heterologous desensitization of δ-opioidreceptor by CCK receptors [103]. In the human neuroblastomaBE(2)-C, heterologous desensitization of μ-opioid receptors byORL1 receptors involved PKC [104] and subsequent decipher-ing of mechanism showed that this heterologous desensitizationwas caused by an increase of GRK2 activity mediated by PKCactivation and subsequent enhancement of the ability forDAMGO to induced μ receptor phosphorylation after nocicep-tin pretreatment [105]. Taken altogether, data converged to arole for PKC in heterologous desensitization of opioid receptor.Nevertheless, particular attention is needed when interpretingresults using chemical kinase inhibitors, as the lack of effect ofone compound does not systematically rule out all the kinasefamily. For instance, whereas heterologous desensitization ofchemokine receptor by opioid receptor is not inhibited byGo6976 (a PKC inhibitor used in many studies), it is involvedPKC anyhow but a Ca2+-insensitive isoform [106].

2.3.2.3. CaM kinase II, MAPK. Functional analysis of anallelic variant of human μ receptor (S268P), whose consensussequence of phosphorylation RXXS by CaM kinase II is altered,suggest that this kinase is involved in phosphorylation anddesensitization of μ receptors. However, until now no proof of μreceptor phosphorylation by CaM kinase II was brought [107].

First reports demonstrating mitogen-activated protein (MAP)kinase stimulation, and more precisely ERK1/2 by opioids werepublished in 1996 using CHO cells and Rat-1 fibroblasts [108–110]. Later, experimental data were accumulated to demonstratethe involvement of this Ser/Thr kinases family in μ- and δ-opioid receptor regulation [54,111–113]. Indeed, inhibitors ofERK1/2 pathway were shown to block μ receptor desensitiza-

tion induced by DAMGO exposure and examined on theinhibition of adenylyl cyclase [54,111]. In addition, whereas aconcomitant decrease of opioid receptor phosphorylation wasalso observed in the presence of PD98059, a potent MAPKkinase inhibitor, phosphorylation studies revealed that μreceptors failed to incorporate (32P) in the presence of purifiedMAP kinases demonstrating that ERK1/2 were not directlyinvolved but rather regulate other kinases that in turnphosphorylate the receptor [111]. Indeed, in SH-SY5Y cells, apretreatment with PD98059 blocked DAMGO-induced homol-ogous and heterologous desensitization with ORL1 receptorsprobably by inhibiting GRK2 up-regulation [113] confirmingthe indirect action of MAPK on opioid receptor desensitization.

2.3.3. Identification of phosphorylation sites on opioidreceptors involved in desensitization

Identification of phosphorylation sites in the intracellularregions including the three intracellular loops and thecarboxyl-terminal tail of opioid receptors was investigatedby using site-directed mutagenesis, truncation of receptors andthe different μ receptor variants. All these approaches alloweda critical examination of relationship between receptorphosphorylation and desensitization. However, in thesestudies, it is hard to ensure that the lack of phosphorylationin one receptor mutant is due either to the absence of a Ser,Thr or Tyr or to a modification of the receptor structureavoiding kinase accessibility.

Koch et al. showed that the presence of Thr394, a putativephosphorylation site located in the carboxyl-terminal region ofthe rat μ receptor (MOR1), which is not found in the MOR1Bvariant, confers a greater sensitivity for desensitization uponshort exposure to DAMGO (between 1 and 4 h) [19]. However,in this study, there is no direct evidence that in the presence ofDAMGO the phosphorylation level of MOR1B is reducedcompared to the MOR1. By substituting this Thr394 to Ala,Deng et al. [114] established the link between the phosphor-ylation of this residue and the receptor desensitization. Indeed,desensitization of the mutant receptor induced by DAMGO wastotally abolished and the phosphorylation state greatly reducedcompared to the wild-type receptor. While Thr394 is required forμ receptor desensitization, other residues located in this domainwere also shown to participate in this process. For instance,substitution of Thr383 into Ala was demonstrated to reducedesensitization by 30%. Other residues at positions 388, 391and 393 (Glu) would modulate the accessibility of kinases toThr394 since substitution of these amino acids into Gln totallyabolished μ receptor desensitization [115]. Using carboxyl-terminal tail truncated mutants of the μ receptor, Wang [86]showed that the desensitization, measured on adenylyl cyclaseinhibition, was dependent on the presence of a Ser/Thr cluster(Thr354, Ser355, Ser356, Thr357), which could be recognized andphosphorylated by the GRK2. The replacement of these aminoacids by alanine revealed the importance of the Ser355 andThr357 as the double mutant receptor displayed a reducedDAMGO-induced desensitization and phosphorylation [116]. Astudy conducted on μ receptor splice variants differing by thepresence of putative phosphorylation sites in the carboxyl-


terminal tail did not reveal any difference in the desensitizationkinetics following DAMGO exposure. Interestingly, the fasterdesensitization of the variants MOR1 and MOR1C followingmorphine treatment, compared to MOR1D and MOR1E, wasassociated with a weaker phosphorylation level [117]. Thiscontrasts with previous works demonstrating a close correlationbetween phosphorylation and desensitization [48,81]. Morerecently, Schulz et al. [23] identified the Ser375 in the carboxyl-terminal tail of the μ receptor, as necessary for bothdesensitization and phosphorylation upon morphine treatment.The importance of this μ receptor domain in desensitization andphosphorylation was questioned by a work of Celver et al.[118]. Indeed, when expressed in Xenopus oocytes, μ-opioidreceptor desensitization, observed on the K+ currents, doesinvolve neither the carboxyl-terminal tail nor the Ser261, 266, 268

in the third intracellular loop nor the Thr97, 101, 103 in the firstintracellular loop but the Thr180 in the second intracellular loop.It is noteworthy that these results were obtained with an over-expression of GRK3 and β-arrestin 2.

Regarding the δ-opioid receptor, two independent groupsdemonstrated that the Thr358 and Ser363 in the carboxyl-terminal tail were phosphorylated upon DPDPE treatment.This latter residue seems crucial as its substitution by an Alaabolished the δ receptor phosphorylation following DPDPE[37,119] and also deltorphin II exposure [120]. More recently,by using a specific antibody directed against the Ser363-phosphorylated δ-opioid receptor (corresponding to the Ser375

of the μ receptor), Navratilova et al. showed that morphinetreatment (30 μM for 30 min) also promotes receptorphosphorylation at this position but to a lesser extent thandeltorphin II (100 nM for 30 min) [121]. When the T358Amutant is activated by DPDPE it is still possible to observe aphosphorylation of δ receptor but to a lesser extent comparedto wild type [37]. Those authors concluded that the GRK2-mediated δ-opioid receptor phosphorylation would proceed ina hierarchical manner with the phosphorylation of Ser363 asthe first step followed by the Thr358. A third phosphorylationsite (Ser344) was identified by Pei et al. as the exclusive targetof phorbol 12-myristate 13-acetate-activated PKC but notGRK2 since the S344G receptor mutant is still phosphory-lated in the presence of DPDPE [119,122]. Concerning therelationship between phosphorylation and desensitization, thedata are less clear. Indeed, the δ receptor mutant S363A,showing no phosphorylation after deltorphin II exposure, isstill able to desensitize in the presence of this agonist[37,120]. This suggests that changes in the receptorconformation induced by agonist binding would allowrecruitment of proteins involved in desensitization (i.e.arrestins) but receptor phosphorylation would just favourthese interactions.

Less data are available for the kappa receptor, but it seemsthat the Ser358 (for the human receptor and corresponding toAsn of the rat receptor) and Ser369 (for the rodent receptor andcorresponding to Tyr for the human receptor) residues located inthe carboxyl-terminal tail are of particular importance for boththe desensitization and phosphorylation. So, when the Ser358 issubstituted to Ala in the human kappa receptor, U50,488 is

unable to induce phosphorylation and shows a reduced ability topromote desensitization of this mutant receptor [123]. In the ratkappa receptor expressed in Xenopus oocytes, the Ser369 isphosphorylated in the presence of U50,488 and its replacementby an Ala impairs the ability of this agonist to desensitize thereceptor [124].

2.4. Regulation of opioid receptors activity by β-arrestins

Opioid receptor phosphorylation cannot solely explain theirdesensitization as illustrated by studies of Law's laboratory. Forinstance, there is no relationship between levels of μ or δreceptor phosphorylation and their desensitization. While amaximal receptor phosphorylation is observed after 5–10 min,no decrease of their activity can be measured on the inhibitionof adenylyl cyclase [37,101]. However, when examining otherpathways (ionic currents) by electrophysiological tools, opioidreceptor phosphorylation and desensitization are concomitantwith time scale of seconds to minutes [22,51]. So, whenconsidering ionic channels, that are the primary regulationtargets of opioid receptors in neurons, a close correlationbetween such processes could be found while this is not truewhen examining other effectors such as adenylyl cyclase,whose involvement in analgesia is discussed. Among otherpartners of GRKs that participate in inactivation of phosphor-ylated GPCRs are arrestins originally discovered as arrestingagent of rhodopsin transduction [125]. The arrestin familyincludes 4 members which are regrouped into 2 classes: (1) thevisual and the cone arrestins mainly expressed in the retina, (2)the β-arrestins: β-arrestin 1 (or arrestin 2) and β-arrestin 2 (orarrestin 3) ubiquitously expressed but with a predominantlocation in the CNS (for review see [126]). Therefore thissecond class of arrestins regulates most of GPCRs includingopioid receptors. A model of GPCRs signalling regulation wasproposed from Lefkowitz's works based on the β2-adrenergicreceptor that was further extended to the large family of 7transmembrane domains receptors and in which β-arrestins playa central role. In this model, these proteins are involved in the 3following events:

1. homologous desensitization results from the binding of β-arrestins to the agonist-activated and GRK-phosphorylatedreceptor which physically uncouple receptor from its cognateG-proteins.

2. internalization: receptor-bound β-arrestins also act as anadapter protein recruiting components of the endocyticmachinery including AP-2 complex and clathrin.

3. post-endocytic trafficking: co-internalization of receptor andβ-arrestins targets this complex to degradation in lysosomes.

In this paragraph, we debate on the role of β-arrestins inopioid receptors uncoupling and desensitization whereas wewill discuss data supporting involvement of β-arrestins ininternalization and post-endocytic trafficking of opioid recep-tors in a next section. We decided to choose objectively the mostpertinent data on the multiple role of β-arrestins in opioidreceptor regulation which are summarized in Table 2.

Table 2Role of β-arrestins in opioid receptor regulation

Process studied Receptor Models Agonist Effect References

Uncoupling μ, δ, κ HEK293 DPDPE and U69,593 Selective uncoupling between β-arrestin 1 and δ/κ-receptors [133]μ HEK293 Morphine and DAMGO Over-expression of β-arrestin forces morphine-activated β

receptor uncoupling[134]

β-Arrestin 2-KO mice Morphine No μ receptor uncoupling is observed in absence of β-arrestin 2expression

[136]

Desensitization μ, δ Xenopus oocyte DAMGO and DPDPE Co-expression of both GRK3 and β-arrestin 2 induces a fasterdesensitization of δ-opioid receptors than μ receptors

[87,138]

HEK293 DAMGO and DPDPE [101]μ HEK293 Morphine Over-expression of β-arrestin alone makes possible μ receptor

desensitization[134]

κ Xenopus oocyte U69,593 and U50,488 Desensitization of κ receptor by GRK3 or GRK5 and β-arrestin2 co-expression

[132]

Sequestration μ HEK293 DAMGO Over-expression of β-arrestin 1 enhances μ receptorinternalization

[49]

HEK293 Etorphine β-Arrestin 1, V53D and S412D mutants reduce μ receptorinternalization

[162]

HEK293 Morphine Over-expression of β-arrestin allows μ receptor endocytosis [134]AtT20 DAMGO and morphine β-Arrestin 1 319–418 mutant reduces μ receptor endocytosis [130]Rat striatal neurons DAMGO and morphine [16]HEK293 Morphine β-Arrestin 1 V53D mutant inhibits the rescuing effect of GRK2

on morphine-mediated μ receptor internalization[49]

μ 363D Neuro2A DAMGO Internalization without plasma membrane translocation ofGFP-tagged β-arrestin 2

[131]

δ HEK293 DPDPE While over-expression of β-arrestin 1 enhances δ receptorendocytosis the β-arrestin 1 V53D mutant totally blocks thisprocess

[122]

HEK293 DPDPE Phosphorylated-δ receptor endocytosis required β-arrestin 1and 2 whereas unphosphorylated δ receptor endocytosisrequired solely β-arrestin 2

[156]

κ CHO U50,488 Over-expression of β-arrestin 1 319–418 mutant impairs κreceptor endocytosis

[143]

Sorting μ HEK293 Not specified μ-Opioid receptor internalization is followed by its recycling:μ receptor belongs to the GPCRs class A

[168]

HEK293 DAMGO μ-Opioid receptors by interacting with NK1-β-arrestin2 complexes switch from recycling to degradation pathway

[161]

μ 363 D Neuro2A DAMGO The μ 363D receptor mutant which does not promote β-arrestin2 plasma membrane recruitment is unable to recycle after itsinternalization

[131]


2.4.1. Interactions between opioid receptors and β-arrestinsBy using surface plasmon resonance with synthetic peptides

derived from opioid receptors, Cen et al. [127] showed that boththe third intracellular loop and the carboxyl-terminal tail of the δreceptors interact equally with β-arrestins 1 and 2. Only arestricted domain of κ receptors corresponding to the carboxyl-terminal tail was shown to bind β-arrestins. Regarding the μreceptors, no interaction either with β-arrestin 1 or 2 wasevidenced [127]. In contrast by real-time microscopy, etorphine-activated μ-opioid receptors were shown to recruit GFP-fusedβ-arrestins 1 and 2 when both receptors and β-arrestins are over-expressed in HEK293 cells [128]. Identification of amino acidsof δ-opioid receptors interacting with β-arrestins was addressedby site-directed mutagenesis and revealed that the last 5 Ser/Thrresidues localized in the carboxyl-terminal domain are essentialfor GRK3- and β-arrestin 2-dependent desensitization whenassessed on K+ channels [87]. By using GST pull-down assay,another group demonstrated that the third intracellular loop of δreceptors interacts with β-arrestin 1 or 2 independently of thecarboxyl terminal region [129].

As demonstrated for δ receptors, two domains of μreceptor were shown to interact with β-arrestin 2. Onelocated in the second intracellular loop (T180) and a secondin the carboxyl-terminal region. Substitution of Thr180 intoAla impairs DAMGO- and β-arrestin 2-dependent desensi-tization [118]. The second domain, which is involved in β-arrestin 2-dependent internalization [130], was revealed byQiu et al. [131] by using a truncated μ receptor afterSer363.

When the last four Ser/Thr of the rat κ-opioid receptorare deleted, the GRK3- and β-arrestin 2-dependentdesensitization is not observed any more upon U69,593exposure (N-methyl-N[7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-8-yl]-benzeneacetamide) whereas the three substitutionsinto Ala of putative phosphorylable amino acids in thethird intracellular loop was devoid of any effect [132].This is consistent with surface plasmon resonance dataobtained by Pei's laboratory [127] showing that only thecarboxyl-terminal domain of κ receptors is able to interactwith β-arrestins.


2.4.2. Role of β-arrestins in opioid receptors uncoupling

2.4.2.1. In vitro data. Participation of β-arrestins in opioidreceptors uncoupling was demonstrated by functional experi-ments in vitro when those proteins are over-expressed (Table 2).Co-transfection of opioid receptors with β-arrestin 1 revealedselective uncoupling of κ and δ receptors observed in [35S]GTPγS binding studies without any effect on the μ receptor.When the carboxyl-terminal tail of the μ receptor wasexchanged with the corresponding domain of the δ receptor,Cheng et al. [133] measured a reduced ability of DAMGO toinhibit cAMP accumulation confirming the importance of thisdomain for interaction with β-arrestin 1. In HEK293 expressingμ receptors, a differential uncoupling and desensitization wasreported by Whistler and von Zastrow [134] upon pretreatmentwith equal concentration of DAMGO and morphine. Indeed, assoon as 5-min DAMGO was shown to promote a totaluncoupling and desensitization while morphine did not.However, over-expression of a β-arrestin (non-specified iso-form) abolished this different behaviour of μ receptor since inthis situation, a 5-min morphine pretreatment caused asignificant uncoupling and desensitization. Thus, morphine-activated μ receptors have a poor ability to recruit and activateendogenous β-arrestins, but enhanced β-arrestin levels cancompensate these weak interactions.

2.4.2.2. In vivo data. β-Arrestins involvement in opioidreceptors uncoupling was also studied in vivo, in mice lackingeither β-arrestin 1 or β-arrestin 2 (Table 2). Invalidation of theβ-arrestin 2 gene allows a stronger G-proteins activation byDAMGO-activated μ receptors compared to wild-type micesuggesting that this protein regulates the basal state of thereceptor [135]. Furthermore, in mice lacking β-arrestin 2neither sign of tolerance nor uncoupling of μ receptors areobserved in chronic morphine-treated animals [136]. However,no significant difference of morphine analgesia was detectedbetween wild-type and β-arrestin 1 KO mice [128] confirmingthe in vitro data that previously showed a selective interactionbetween μ-opioid receptors and β-arrestin 2. In this paper, usingβ-arrestin 2 KO mice, the authors failed to observe anypotentiation of analgesia when using other agonists such asetorphine, fentanyl and methadone [128]. This data suggeststhat μ-opioid receptors activated by either morphine orDAMGO interact selectively with β-arrestin 2, whereasregulation of those receptors stimulated by others agonistsinvolves both β-arrestins 1 and 2. β-Arrestin 2 seems to be anegative regulator of morphine-activated μ receptor signallingon analgesia, but would be directly responsible for side effectssuch as when physical dependence [136], constipation andrespiratory suppression [137] since in β-arrestin 2 KO micethese latter effects were decreased.

2.4.3. Role of β-arrestins in opioid receptor desensitizationThe main strategy to demonstrate the role of β-arrestins in

opioid receptor desensitization was to over-express theseproteins in either HEK293 or Xenopus oocytes. As suggestedby the model of Lefkowitz, β-arrestins were shown to

potentiate opioid receptor desensitization but only when oneGRK isoform was also over-expressed. Introduction of cRNAencoding either for GRK3 or β-arrestin 2 alone was devoid ofany effect on μ and δ receptor desensitization in Xenopusoocytes. However, co-expression of these proteins promotes astrong desensitization measured on K+ channels [87]. Usingthe same strategy and the same cellular model, Appleyard et al.also confirmed that κ-opioid receptor desensitization requiresboth one GRK isoform (3 or 5) and β-arrestin 2 [132]. Adifferential GRK3- and β-arrestin 2-dependent desensitizationbetween μ and δ receptors were reported by Lowe et al. [138]in Xenopus oocytes on the K+ channels activation but also byEl Kouhen et al. [101] in HEK293 cells on the inhibition ofadenylyl cyclase. μ receptors were shown to desensitize at aslower rate and to a lesser extent than the δ receptors. Thisdifference is probably due to a differential activation of β-arrestin 2 by these opioid receptors since the same level ofdesensitization is observed when this protein is replaced by aconstitutive active mutant β-arrestin 1-R169E [138]. Celver etal. [118] identified the Thr180 in the rat μ receptor as a criticalamino acid responsible for desensitization while the putativephosphorylation sites of δ receptor that promote desensitiza-tion are multiple and rather localized in the carboxyl-terminaldomain [37,119]. According to the model of visual arrestinactivation by rhodopsin [139], β-arrestin 2 activation would beproportional to the number of phosphate attached to thereceptor. So, the difference of the phosphorylation state ofopioid receptors would explain the differential β-arrestin 2activation observed between μ and δ. In contrast with the datacited above, Whistler and von Zastrow [134] demonstrated thatμ opioid receptor desensitization did not require over-expression of both GRK and β-arrestin. Indeed, a strongover-expression of β-arrestin (non-specified isoform), by morethan 30-fold than the endogenous level, was sufficient toinduce a total μ receptor desensitization after morphinepretreatment [134]. Such an over-expression of β-arrestinwould force interactions with poorly phosphorylated μreceptor. These experimental conditions are far from physio-logical models and these results should be interpreted withcaution. Since β-arrestins were described as proteins involvedboth in receptor uncoupling and internalization, and themajority of these desensitization studies were realized on aneffector response, it is hard to estimate the part of β-arrestins indesensitization. This will be discussed in the next section.

3. Internalization of opioid receptors

Internalization, sequestration or endocytosis of GPCRs is acommon way to regulate their activity by removing activereceptors from the cell surface into intracellular compartmentsand has been widely studied. Development of new tools such asepitope-tagged receptors, GFP-receptor fusion proteins orfluorescent ligands affords visualization of such processes foropioid receptors.

GPCRs internalization is mediated by the clathrin-coatedpits, caveolae and uncoated vesicles (for review see [141]) butthe clathrin pathway is the most-well described: following their


activation by agonists, receptors are phosphorylated by GRKsand then interact with the β-arrestins. Those oligomersaccumulated into pits at the plasma membrane and β-arrestindirectly or via AP-2 (adapter protein) complex recruits theclathrin at the cytosolic side. Then, the dynamin, a large GTPaseprotein, removes GPCRs accumulated into clathrin-coated pitsfrom cell surface into intracellular compartments.

The first demonstration of opioid receptor internalizationwas done by Arden et al. in 1995. They visualizedinternalization of epitope-tagged μ-opioid receptors expressedin HEK293 cells, in the presence of DAMGO but not in thepresence of morphine, suggesting complex mechanisms inthis process (discussed later) [80]. Using the same strategy,endocytosis of δ- and κ-opioid receptors was evidenced bydifferent groups [142,143]. Besides these works in transfectedcells, many papers reported internalization of opioid receptorsin different tissues. For example, using specific antibodiesagainst the μ-opioid receptor, internalization was observed inmyenteric neurons in the presence of etorphine [144] and inrats spinal cord slices following DAMGO or etorphinetreatment [145]. More recently, Haberstock-Debic et al.(2005) [16] challenged the well-admitted idea that morphinewas unable to promote μ receptor sequestration [16]. Indeed,they observed in rat striatal neurons that both endogenousand transfected FLAG-tagged μ receptor were redistributedfrom cell surface to intracellular compartments uponmorphine and DAMGO exposure. This work emphasizesthe importance of cellular model used to study opioidreceptor internalization.

An alternative strategy to follow receptor internalization,originally developed by Vincent et al., consists of usingfluorescent ligands to track the receptor. Using deltorphin I(Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) or dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) labelled with fluorophores,they were able to visualize μ and δ opioid receptorredistribution to intracellular compartments in COS-7 cells[146]. Nevertheless, this method has the disadvantage to followexclusively the ligand independently of the receptor. Therefore,using this technique in primary cultures of cortical neurons, Leeet al. demonstrated that after internalization and receptor/ligandcomplex dissociation, the receptor was recycled back to theplasma membrane and the fluorescent ligand was transported tothe soma [147].

Internalization of μ and δ-opioid receptors is a fast clathrin-dependent process (t1/2<10 min) [148]. Regarding the κreceptor, data from the literature are more controversial sinceit was demonstrated that etorphine at a saturating concentrationof 5 μM [149] or U50,488, a κ agonist, at a low concentration[150] were unable to promote internalization of the human ormouse κ receptors, respectively, while Li et al. [143] observed aclathrin-dependent endocytosis of the human receptor afterU50,488 treatment.

Besides the proteins cited above, the role of dynamin inopioid receptor endocytosis was also evidenced. Indeed, over-expression of dynamin I or its dominant negative mutant(dynamin I-K44A/E) demonstrated the role of this protein in δ[17,149], μ [49,134] and κ [143] receptor endocytosis.

3.1. Role of receptor phosphorylation in internalization

Many evidences have been accumulated for a major role ofphosphorylation in opioid receptor endocytosis. By studyingsplice variants of the μ receptor, Koch et al. [19] demonstratedthat the MOR1B, lacking a putative phosphorylation site(Thr394), was internalized faster than the μ receptor (MOR1).They also provided evidence that the mutation of this residue toalanine accelerates the internalization of μ receptor suggestingthat phosphorylation of Thr394 would act as a brake for receptorendocytosis [151]. In contrast, for other μ receptor splicevariants, this group established a correlation between receptorphosphorylation and their endocytosis promoted by morphine[117]. As mentioned earlier, morphine is unable to induce μreceptor internalization in non-neuronal cells [80], but the over-expression of GRK2 could reverse this phenotype and thiseffect is correlated to the ability of the kinase to phosphorylatethe receptors [49,134]. Same results were obtained with thehuman κ receptor expressed in HEK293 cells in which over-expression of GRK2 or GRK3 was required to induce receptorinternalization following U50,488 treatment, with a morepronounced effect for GRK3 [150]. Using heparin, a GRKsinhibitor, we demonstrated the involvement of this kinasesfamily in etorphine-induced internalization of endogenouslyexpressed δ-opioid receptors in SK–N–BE cell line [152].These latter findings support the assumption that opioidreceptor phosphorylation is essential for their endocytosis.

Several studies using site-directed mutagenesis have pointout the importance of particular phosphorylated residues inopioid receptor endocytosis. Therefore, for the rat μ receptor,Ser375 phosphorylation stimulated the receptor internalization,whereas phosphorylation of Ser363 and Thr370 in the presence ofDAMGO inhibited sequestration [153]. Regarding the mouse δ-opioid receptor, it was demonstrated that the phosphorylation ofthe Ser344 and Ser363, respectively, by PKC activation anddeltorphin II, allowed receptor sequestration via clathrin-coatedpits [120,122]. The Ser369 substitution to alanine in thecarboxyl-terminal tail of the rat κ opioid receptor blocks itsU50,488-induced phosphorylation and internalization inHEK293 cells [124]. In this latter case, it is noteworthy thatthe agonist concentration was high (10 μM), because with alower U50,488 concentration (≤1 μM) no internalization of therat κ receptor is detected [143]. Ser/Thr residues are not the onlyputative phospho-acceptor sites involved in opioid receptorinternalization. Therefore, the mutation of Tyr318 into Phe in thecarboxyl-terminal tail of the murine δ receptor reduced theability of [D-Thr2-Leu5-Thr6]enkephalin (DTLET) to promotephosphorylation and internalization of this receptor [154]. Thus,it appears that internalization of opioid receptors involves theirphosphorylation but the exact location of phosphorylatedresidues is greatly dependent on experimental conditions.

As usual in biology, the picture is not so clear and someworks have questioned the role of phosphorylation in opioidreceptors endocytosis. A murine δ receptor truncated from theSer344 or DOR344T (missing putative phosphorylation sites atthe carboxyl-terminal tail and displaying no detectablephosphorylation) was still able to internalize in the presence


of DADLE or etorphine to a similar degree than the wild typereceptor [17]. However, this is only true when mutant opioidreceptors are expressed in HEK293 cells since when experi-ments were conducted in CHO cells, a strong inhibition ofendocytosis was observed indicating that the molecularmechanisms of receptor internalization are cell type-dependent.Later, the same group conducted studies on mutated δ-opioidreceptor (substitution of the last five Ser/Thr into Ala orDOR5A), and showed that this unphosphorylated mutant failsto internalize [155]. They concluded that internalizationrequires phosphorylation of amino acids in the carboxyl-terminal tail, and this domain would also contain other residueswhich act as inhibitors of sequestration. That is probably whythey observed an internalization of the DOR344T which isdevoid of this inhibitory region [17]. In contrast, mutant ofmouse δ-opioid receptor, in which the last three Ser/Thr (atpositions of 358, 361, 363) were substituted into Ala or m4/5/6,is unable to undergo phosphorylation upon DPDPE exposurebut still able to internalize [156]. Those authors checked thatthis lack of phosphorylation of the receptor mutant m4/5/6 is notdue to a decrease of GRK2 recruitment These discrepanciescould not be explained neither by the cellular model nor thespecies of the studied receptor, but we note that differentagonists were used and in the paper of Whistler two additionalresidues were substituted. More recently, Qiu et al. [131] foundthat the μ receptor truncated after its Ser363 underwentphosphorylation-independent internalization. Therefore, noreceptor phosphorylation was detected after 30-min etorphinetreatment whereas internalization occurred, but to a lesser extentcompared with wild-type receptor suggesting a non-essentialrole for receptor phosphorylation in this process.

Oligomerization is another parameter to take into account forthe opioid receptors internalization. When Cvejic and Devi[157] observed that a δ receptor deleted from its last 15 aminoacids was deficient in endocytosis and exits exclusively inmonomeric form, they suggested a role for receptor dimeriza-tion in endocytosis by a putative formation of homodimersinteracting by the carboxyl-terminal tail. Conversely, in cellswhere δ/κ receptors dimerization occurred, δ receptor inter-nalizes to lesser extent compared with cells only expressing δreceptor [158]. In this situation, heterodimer formation wouldinhibit δ receptor endocytosis. Interestingly, in cells co-expressing β2-adrenergic and δ-opioid receptors, etorphinewas able to drag β2-adrenergic receptor to the endocytosispathway and vice versa with the isoproterenol [159]. Thisprocess that could be defined as a heterologous internalizationwas also noticed with neurokinin type 1 (NK1) receptor/μreceptor dimers [160]. In some cases, the heterologousinternalization is observed in one way, as for μ/sst2A receptorsheterodimers; a selective somatostatin receptor agonist was ableto promote μ receptors endocytosis whereas DAMGO had noeffect on sst2A receptors sequestration [161]. In contrast, thisdragging phenomenon is not detected when β2-adrenergic and κ-opioid receptors are co-expressed suggesting that this is not auniversal mechanism for opioid receptors [159]. It is noteworthy,that so far, these studies did not bring direct evidence for aninternalization of these receptors in oligomeric form. Indeed, it is

possible that agonist-activated receptors would drag othersGPCRs without physical interactions during clathrin coated-pitsformation.

3.2. Role of β-arrestins in opioid receptors internalization

Few studies were conducted to determine the role of β-arrestins in opioid receptors internalization (Table 2). Demon-strations of β-arrestins-dependent opioid receptors internaliza-tion were obtained by over-expressing wild type β-arrestin 1 or2, dominant negative mutants (V53D, 319–418 correspondingto the last 100 amino acids of β-arrestin 1, and S412D),constitutive active arrestins (R169E) or by using siRNAdirected against the mRNA encoding for the β-arrestin isoformstudied. Regarding μ receptor, etorphine-induced receptorsequestration is increased by 30% when the β-arrestin 1 isover-expressed [49] and is significantly impaired when thedominant negative mutant β-arrestin 1 V53D is co-expressedin HEK293 cells [162]. Morphine fails to promote μ receptorinternalization in heterologous expression systems but it ispossible to overcome this by over-expressing exogenous β-arrestins (non-specified isoform) [134]. Co-expression of β-arrestin dominant-negative mutant (319–418), which trapsclathrin and prevents its further recruitment to plasmamembrane, and μ receptors either in atT20 cells [130] or inprimary cultured striatal neurons [16], decreases significantlyinternalization observed in confocal microscopy. GRK2 over-expression forces morphine-mediated μ receptors internaliza-tion which is impaired when the β-arrestin 1 dominant-negative mutant (V53D) is co-expressed with this kinase [49].Claing et al. [162] observed a partial but significantimpairment of μ receptor internalization when the β-arrestin1 mutant S412D, that mimics a constitutive inactive andphosphorylated form of arrestin, is expressed. All these datastrongly argue for a major role of β-arrestins in μ receptorsequestration while recently the study of Qiu et al. [131]showed that a μ receptor mutant truncated after Ser363 was ableto internalize without recruiting GFP-tagged β-arrestin 2.These authors suggest that both receptor phosphorylation andtheir interactions with β-arrestins are not absolutely essentialfor endocytosis. δ-opioid receptors were also demonstrated tointernalize in β-arrestins-dependent manner. Over-expressionof β-arrestin 1 enhances DPDPE-stimulated δ receptorsequestration by 1.5-fold while the expression of the β-arrestin1 dominant-negative mutant (V53D) totally blocks thisinternalization [122]. Interestingly, Zhang et al. [156] recentlydescribed two independent ways of δ receptor internalization,differentially requiring β-arrestins:

1 A phosphorylation-dependent pathway, which requires bothβ-arrestins 1 and 2, as demonstrated by using siRNA againstthese two proteins.

2 A phosphorylation-independent pathway, which exclusive-ly requires β-arrestin 2. Indeed, the unphosphorylated-δmutant receptor m4/5/6 is still able to interact with β-arrestins and is internalized in a GRK-2-independent- butβ-arrestin 2-dependent manner.


Concerning κ receptor, there only exist two studiesconducted by Li et al. [143,163] which showed that human κ-opioid receptors sequestration is strongly diminished by the319–418 mutant over-expression, when activated by U50, 488.These data support that κ receptors internalize through β-arrestins-dependent mechanisms as described for most ofGPCRs.

3.3. Post-endocytic fate of internalized receptors

Once internalized, receptors could be either recycled back toplasma membrane, trapped in endosomes, or targeted tolysosomes to be degraded (Fig. 1). In the case of the recycling,the receptor is dephosphorylated by intracellular phosphatasesallowing the return of functional receptor to the plasmamembrane and resensitization (see for review [78]).

Many parameters could influence the post-endocytictargeting of opioid receptors. The selectivity of an agonisttoward one type of opioid receptor is one of them. In ourlaboratory, we observed that δ selective agonists (DPDPE,deltorphin I and SNC-80 ((+)-4-[(alpha R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethyl-benzamide) targeted the receptor to degradative path-way, whereas non-selective opioid agonists (enkephalins andetorphine) preferentially induced a recycling of this receptor[18,31]. In this latter case, the recycling promotes resensitizationas evidenced by monensin which is described as an endosomalrecycling inhibitor. The cellular model could be also a criticalparameter. Therefore, in HEK293 cells expressing δ receptor, 1-

Oakley’s hypothesis applied to opioid receptors

1- GRK phosphorylation / βarrestin translocation

2- βarrestin dependent internalization

3- Strong interactions between βarrestin and opioidreceptor target this complex to the lysosome

3’- Poor interactions between βarrestin and opioidreceptor allow receptor dephosphorylation and recycling

Class AClass B

PPP

P PP

2

33’

βarr

1

βarr

βarr

βarr

Fig. 1. Role of β-arrestins in opioid recepto

h etorphine exposure leads to receptor degradation [72] whereasexpressed in SK–N–BE neuroblastoma cells, the same receptoris recycled to the plasma membrane with the same treatment[18]. The time treatment may influence the fate of sequestratedreceptors. Whereas 1h etorphine exposure leads to δ receptorrecycling, this latter process is no longer observed after 4 h,suggesting a receptor degradation [152]. Such a sorting towardsdegradation was also described for δ receptors expressed inneuro2a cells but after 24 h of DADLE pretreatment [164]. It isnoteworthy that the continuous presence of the agonist does notseem to be necessary for receptor degradation as demonstratedby Tsao and von Zastrow [72].WhenHEK293 cells expressing δreceptor are challenged with etorphine for 30 min andsubsequently exposed to naloxone, receptor degradation is stillobserved.

3.4. Which mechanisms dictate the post-endocytic fate ofreceptors?

3.4.1. Receptors phosphorylationGiven the importance of the phosphorylation in receptor

internalization and the role of internalization in down-regulation, one could assume that opioid receptor phosphory-lation is involved in sorting of internalized receptors. Therefore,after 150 min of etorphine treatment, rat δ receptor down-regulation which requires their internalization, involves differ-ent kinases : GRKs, tyrosine kinases and MAPKs [73]. Whenexpressed in CHO cells, human κ-opioid receptors were unableto be sequestrated and consequently down-regulated upon

Prossnitz’s hypothesis applied to opioid receptors

1- No GRK phosphorylation / no βarrestin translocation

2- βarrestin independent internalization

3- Lack of βarrestin-receptor interactions leads thereceptor to the lysosomes

3’-βarrestin-internalized receptor interactions lead thereceptor to the recycling pathway

2

33’

1

βarr

βarr

rs trafficking: two opposite hypothesis.


etorphine exposure [143]. But, co-expression of GRK2 and β-arrestin-2 allowed this agonist to promote κ receptor down-regulation, suggesting a role for phosphorylation in thismechanism [143,163]. Other studies have questioned thephosphorylation as a key event in receptor targeting todegradative pathway. By examining different carboxyl-terminaltail mutants of mouse δ receptor, Whistler et al. [155]demonstrated that the phosphorylation was required forinitiating the receptor endocytosis, but not for the targeting tolysosomes and its subsequent degradation. More recently, it wasdemonstrated that U50,488 was able to promote internalizationand down-regulation of human κ receptors, but not of rat κreceptors. This discrepancy seems to be related to the presenceof Ser358 in the human receptor, which is substituted into Arg inthe rat receptor. When this Arg was replaced by Ser, the rat κreceptor mutant N358S was able to be phosphorylated andinternalized but not down-regulated following U50,488 treat-ment supporting a role for phosphorylation in sequestrationinitiation but not in receptor post-endocytic fate [39,165]. Takentogether, these data strongly suggest the requirement of otherfactors in opioid receptors intracellular trafficking.

3.4.2. β-Arrestinsβ-arrestins regulate GPCRs internalization by acting as

scaffold proteins but they were also reported to be involved inreceptors sorting. Indeed, according to the model proposed byOakley, β-arrestins target GPCRs to the degradation pathwaywhile Vines et al. showed that they rather allow receptorsrecycling [166] (Fig. 1). Oakley et al. [167–169] identified twoclasses of GPCRs (A and B) depending on interactions betweenreceptors and β-arrestins. Indeed, following β-arrestinstranslocation to the plasma membrane and receptors internal-ization, receptor-β-arrestin complexes can either dissociatethemselves and thus the receptor is recycled (class A) or stronginteractions between those partners prevent further dissociation,receptor dephosphorylation and recycling (class B). The abilityof β-arrestins to remain associated with the internalizedreceptors is mediated by clusters of Ser/Thr located in thecarboxyl-terminal tail of GPCRs [169]. Regarding μ-opioidreceptors, the same authors showed that they belong to the classA. However, it is possible to induce a switch of μ receptors fromclass A to class B when these opioid receptors are co-expressedwith NK1 receptors (class B) and consequently, resensitizationof μ receptors is severely delayed. Trans-phosphorylation andheterodimerization between those receptors would drag μreceptors to degradation and not to recycling [160]. Analternative approach to switch the μ receptor from the class Ato class B consists of exchanging its carboxyl-terminal tail withδ receptor [155]. Concerning the δ receptors, von Zastrow'slaboratory showed that etorphine exposure promotes transloca-tion of β-arrestin 2 to the plasma membrane and receptorinternalization followed by their degradation [72,155] suggest-ing that conversely to the μ receptor, the δ type belongs rather tothe class B receptors. A new concept was developed byProssnitz et al. [166,170] suggesting a new class of GPCRsinternalizing through a β-arrestins-independent mechanism.Indeed, using a mouse embryonic fibroblast (MEF) cells

generated from β-arrestins 1 and 2 double knock-out mouse,those authors observed an internalization of N-formyl peptidereceptor to the same extent as in the wild type MEF cellsdemonstrating that β-arrestins are not required for endocytosisof this receptor [166]. However, β-arrestins were shown tointervene but later in GPCRs trafficking by promoting theirrecycling. That is precisely what Qiu et al. [131] suggested forthe μ receptor and the β-arrestin 2. Truncation of μ receptorafter its Ser363 abolished GFP-tagged β-arrestin 2 translocationto the plasma membrane without affecting the level ofinternalization after 4-h etorphine exposure but impairs itsrecycling. This underlines the role of β-arrestins rather in thesorting of internalized receptors to the recycling pathway ratherthan in endocytosis.

3.4.3. Accessory proteinsOther studies have implicated particular amino acids

sequences in opioid receptors and the role of accessory proteinsin controlling the post-endocytic fate of receptors. At the end ofthe human β2-adrenergic receptor carboxyl-terminal tail, asequence was identified as the mediator of the receptorrecycling. This sequence, consisting of fours amino acids:Asp-Ser-Leu-Leu (DSLL), was shown to interact with a proteincalled: EBP50 (Ezrin-Radixin-Moesin (ERM) binding phos-phoprotein 50). This interaction, dependent on the Serphosphorylation by GRK5, allowed the receptor recycling byinteractions via actin cytoskeleton [171]. Interestingly, when thissequence was added to mouse δ receptor carboxyl-terminal tail,the resulted mutant receptor was re-routed to recycling pathwaywhereas the wild-type receptor was targeted to degradation,when expressed in HEK293 cells [172]. As mentioned earlier,mouse μ receptor is usually recycled following its sequestration,leading von Zastrow et al. to wander if the μ receptor possesses arecycling signal like the β2-adrenergic receptor. So, theyidentified the last 17 amino acids as a sufficient and necessaryrecycling signal. This sequence was distinct from the β2-adrenergic receptor's one, even if they were functionallyinterchangeable. The μ receptor recycling mechanism seems tobe different from that described for the β2-adrenergic receptor,as it was not dependent on the actin cytoskeleton and did notinvolve EBP50 [173]. This also indicates that μ receptors aremainly internalized and recycled while their sorting to thedegradative pathway requires additional factors or signals.Concerning the human κ-opioid receptor, it interacts withEBP50, when these two proteins are co-expressed in CHO cells.This interaction, which is enhanced following U50,488treatment, abolishes the agonist-induced receptor down-regula-tion by increasing the recycling rate. Interestingly, the bindingsequence to EBP50, located at the carboxyl-terminal tail of the κreceptor, is different from the motif described for the β2-adrenergic receptor indicating that interactions between GPCRsand EBP50 are achieved by different ways [123]. All together,these data have pointed out the crucial role of the opioid receptorcarboxyl-terminal tail not only in endocytosis but also in thereceptor targeting to the recycling pathway. Then, does thesignal leading receptors to degradation lie also in the carboxyl-terminal tail of the receptors?


As mouse δ receptor was preferentially degraded following3-h etorphine treatment while mouse μ receptor was mainlyrecycled to plasma membrane, Whistler et al. [174] sought toidentify the molecular basis of this difference. From a yeast two-hybrid screen, using δ receptor carboxyl-terminal tail as a bait,they identified a protein named GASP (G protein-coupledreceptor associated sorting protein). This latter was demon-strated to interact with δ- but not with μ-opioid receptors and totarget the sequestrated receptor to lysosomes. More recently,Wang et al. [175] noted that, when expressed in neuro2A cells, aμ receptor chimera containing the δ carboxyl-terminal tail didnot display the complete phenotype of the wild-type δ receptor.For instance, after etorphine treatment, the co-localization of thechimera with lysosomes was observed at a lower degreecompared to wild-type δ receptor. These authors then suggestedthe presence of additional signal in δ receptor required forreceptor lysosomal targeting and demonstrated the presence of adi-leucine motif in the third intracellular loop of the receptor.

3.5. Functional consequences of receptor endocytosis

As mentioned above, internalized receptors could be eithertargeted to degradative pathway or recycling. In the case ofrecycling, resensitization is preceded by receptor dephosphor-ylation, and in the case of lysosomal targeting of receptors, apotentiation of desensitization is observed.

Höllt's group did first studies on a relation between receptorpost-endocytic fate and its functional activity by studyingMOR1 and MOR1B (see Section 2.3.3). MOR1B was shown tobe more resistant to desensitization after DAMGO exposurecompared to MOR1 [76]. They demonstrated that this resistancewas due to the great ability of MOR1B to undergo endocytosisafter DAMGO treatment allowing recycling and resensitizationof the receptor. So, in this case internalization counteracteddesensitization by targeting receptor to recycling pathway [19].We observed the same phenomena with human δ receptor whenexposed to etorphine or enkephalins [18,31]. On the other hand,targeting receptor to degradation might contribute to desensi-tization. For instance, when μ receptor carboxyl-terminal endwas replaced by the δ receptor's one, morphine was able topromote internalization followed by degradation of thischimeric receptor and in this case a potentiation of thedesensitization was observed [21].

The molecular mechanisms underlying the receptor post-endocytic fate seem very complex and need further attentionregarding the last data provided by Whistler et al., concerning alink between opioid receptors post-internalization events andtolerance. Using μ receptor chimeras, which differed from thewild-type by their ability to internalize following morphineexposure, these authors demonstrated a reduced “cellulartolerance” in HEK293 cells expressing these mutants comparedto cells expressing wild-type receptors. The sorting of thesequestrated receptors is also important, as the cells expressingchimeras targeted to the recycling pathway after morphineexposure, displayed a lower “cellular tolerance” compared withcells expressing a chimera which is targeted to degradativepathway after the same agonist exposure [21]. One could regret

the term of “tolerance”, employed by the authors, in the case ofcellular experiments. Nevertheless, they confirmed theirhypothesis in a later study realized in vivo. Whereas chronicmorphine treatment induced tolerance to analgesia in rats, a co-administration of morphine and DAMGO, at a sub-analgesicand sub-sequestrating concentration, promoted μ receptorsinternalization and reduced morphine-induced tolerance toanalgesia [176]. These data suggest that the lack of internali-zation following morphine treatment would maintain acontinuous receptor signalling contributing to tolerance devel-opment. However, two recent studies realized in HEK293 cellsfailed to demonstrate that addition of DAMGO could enhancemorphine-activated μ receptor endocytosis [140,177]. More-over, in Koch's study, even if the authors found an inversedcorrelation between internalization and desensitization (andpossibly tolerance) in agreement with Whistler's group, theyattributed the reduction of opioid signalling after chronicmorphine exposure to receptor desensitization and not to cAMPsuperactivation as supposed by Finn and Whistler [21].

4. Conclusions

First, since cloning of opioid receptors, their regulation havebeen mainly investigated in heterologous expression systems.Consequently, extra-care should be taken regarding the interpre-tation of data as most of the models may be unsuitable. So, the useof neuronal cell lines endogenously expressing opioid receptorsmay be more appropriate by mimicking the cellular environmentin which these receptors are naturally found.

Second, opioid receptor phosphorylation seems to be a majorsignal triggering decrease of receptors signalling by recruitingother proteins such as β-arrestins. Even if this receptorphosphorylation is mainly achieved by GRKs, other kinases(PKC, CaM-kinases, tyr-kinases) also phosphorylate either thereceptor or downstream proteins in the transduction cascade.Consistent with the model proposed by Lefkowitz, receptorphosphorylation appears essential for desensitization while itsrole for opioid receptors internalization would be a facilitatoryfactor by enhancing this process.

Third, recent advances demonstrate inter-relationship be-tween desensitization, internalization and receptor sorting. Toobtain a weak desensitization, two conditions are required:internalization of opioid receptors and their recycling. In vivo,when these conditions are fulfilled, a reduction of morphinetolerance has been observed. While molecular mechanismsinvolved in opioid receptors internalization are now welldescribed, post-endocytic pathways and their associated proteinsneed to be clarified. Several candidates have been identified(GASP, EBP50, etc.) but probably other proteins will bediscovered in these complex mechanisms. These new insightsregarding opioid receptors trafficking will enable developmentof more effective new drugs with reduced addictive properties.

Acknowledgments

The authors thank Dr. Florence Noble (Neuropsychophar-macologie des addictions, UMR CNRS 7157, INSERM U705,


Université Paris V, France) for her advises and critical readingof the manuscript.

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