Morphine-like Opiates Selectively Antagonize Receptor-Arrestin Interactions
2010; Elsevier BV; Volume: 285; Issue: 17 Linguagem: Inglês
10.1074/jbc.m109.059410
ISSN1083-351X
AutoresPaola Molinari, Vanessa Vezzi, Maria Sbraccia, Cristina Grò, Daniela Riitano, Caterina Ambrosio, Ida Casella, Tommaso Costa,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoThe addictive potential of opioids may be related to their differential ability to induce G protein signaling and endocytosis. We compared the ability of 20 ligands (sampled from the main chemical classes of opioids) to promote the association of μ and δ receptors with G protein or β-arrestin 2. Receptor-arrestin binding was monitored by bioluminescence resonance energy transfer (BRET) in intact cells, where pertussis toxin experiments indicated that the interaction was minimally affected by receptor signaling. To assess receptor-G protein coupling without competition from arrestins, we employed a cell-free BRET assay using membranes isolated from cells expressing luminescent receptors and fluorescent Gβ1. In this system, the agonist-induced enhancement of BRET (indicating shortening of distance between the two proteins) was Gα-mediated (as shown by sensitivity to pertussis toxin and guanine nucleotides) and yielded data consistent with the known pharmacology of the ligands. We found marked differences of efficacy for G protein and arrestin, with a pattern suggesting more restrictive structural requirements for arrestin efficacy. The analysis of such differences identified a subset of structures showing a marked discrepancy between efficacies for G protein and arrestin. Addictive opiates like morphine and oxymorphone exhibited large differences both at δ and μ receptors. Thus, they were effective agonists for G protein coupling but acted as competitive enkephalins antagonists (δ) or partial agonists (μ) for arrestin. This arrestin-selective antagonism resulted in inhibition of short and long term events mediated by arrestin, such as rapid receptor internalization and down-regulation. The addictive potential of opioids may be related to their differential ability to induce G protein signaling and endocytosis. We compared the ability of 20 ligands (sampled from the main chemical classes of opioids) to promote the association of μ and δ receptors with G protein or β-arrestin 2. Receptor-arrestin binding was monitored by bioluminescence resonance energy transfer (BRET) in intact cells, where pertussis toxin experiments indicated that the interaction was minimally affected by receptor signaling. To assess receptor-G protein coupling without competition from arrestins, we employed a cell-free BRET assay using membranes isolated from cells expressing luminescent receptors and fluorescent Gβ1. In this system, the agonist-induced enhancement of BRET (indicating shortening of distance between the two proteins) was Gα-mediated (as shown by sensitivity to pertussis toxin and guanine nucleotides) and yielded data consistent with the known pharmacology of the ligands. We found marked differences of efficacy for G protein and arrestin, with a pattern suggesting more restrictive structural requirements for arrestin efficacy. The analysis of such differences identified a subset of structures showing a marked discrepancy between efficacies for G protein and arrestin. Addictive opiates like morphine and oxymorphone exhibited large differences both at δ and μ receptors. Thus, they were effective agonists for G protein coupling but acted as competitive enkephalins antagonists (δ) or partial agonists (μ) for arrestin. This arrestin-selective antagonism resulted in inhibition of short and long term events mediated by arrestin, such as rapid receptor internalization and down-regulation. IntroductionPhysiological agonists are usually equally efficient in promoting the interaction of receptors with G protein and arrestin, but manmade analogues can show divergent molecular efficacies for the two transducers (1Violin J.D. Lefkowitz R.J. Trends Pharmacol. Sci. 2007; 28: 416-422Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar, 2Masri B. Salahpour A. Didriksen M. Ghisi V. Beaulieu J.M. Gainetdinov R.R. Caron M.G. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13656-13661Crossref PubMed Scopus (264) Google Scholar). This phenomenon, often addressed with a pictorial terminology (3Urban J.D. Clarke W.P. von Zastrow M. Nichols D.E. Kobilka B. Weinstein H. Javitch J.A. Roth B.L. Christopoulos A. Sexton P.M. Miller K.J. Spedding M. Mailman R.B. J. Pharmacol. Exp. Ther. 2007; 320: 1-13Crossref PubMed Scopus (891) Google Scholar, 4Kenakin T. Mol. Pharmacol. 2007; 72: 1393-1401Crossref PubMed Scopus (238) Google Scholar, 5Kenakin T.P. Br. J. Pharmacol. 2008; 153: 432-438Crossref PubMed Scopus (36) Google Scholar), has attracted great interest and if better understood might lead to new types of drugs.The differential efficacy of opioids for G protein and arrestin interactions is also important in the mechanism of opiate addiction. As reported earlier, the addictive opiate morphine cannot induce and actually blocks desensitization and G protein uncoupling of δ-opioid receptors (DOPR) 2The abbreviations used are: DOPRδ-opioid receptorMOPRμ-opioid receptorRETresonance energy transferHEK293human embryonic kidney 293 cellsRGFPRenilla green fluorescent proteinRlucRenilla luciferaseGPCRG-protein-coupled receptorcpscounts/sβArr1 and βArr2β-arrestin 1 and 2bDOCbisdeoxy-coelenterazineDADLE[d-Ala2, d-Leu5]-enkephalinsEKCethylketocyclazocineGTPγSguanosine 5′-O-(thiotriphosphate)mtmembrane-targetedDmt2′,6′-dimethyltyrosineTic1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. in neuroblastoma or in transfected cells (6Vachon L. Costa T. Herz A. Biochem. Pharmacol. 1987; 36: 2889-2897Crossref PubMed Scopus (32) Google Scholar, 7Eisinger D.A. Ammer H. Schulz R. J. Neurosci. 2002; 22: 10192-10200Crossref PubMed Google Scholar). Subsequent work shows that morphine is a poor inducer of rapid arrestin-dependent endocytosis for both δ and μ (MOPR) receptors (8Arttamangkul S. Quillinan N. Low M.J. von Zastrow M. Pintar J. Williams J.T. Mol. Pharmacol. 2008; 74: 972-979Crossref PubMed Scopus (90) Google Scholar, 9Keith D.E. Anton B. Murray S.R. Zaki P.A. Chu P.C. Lissin D.V. Monteillet-Agius G. Stewart P.L. Evans C.J. von Zastrow M. Mol. Pharmacol. 1998; 53: 377-384Crossref PubMed Scopus (289) Google Scholar, 10Keith D.E. Murray S.R. Zaki P.A. Chu P.C. Lissin D.V. Kang L. Evans C.J. von Zastrow M. J. Biol. Chem. 1996; 271: 19021-19024Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar, 11Whistler J.L. von Zastrow M. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 9914-9919Crossref PubMed Scopus (314) Google Scholar), although one exception is in striatum neurons with high levels of G protein receptor kinases (12Haberstock-Debic H. Kim K.A. Yu Y.J. von Zastrow M. J. Neurosci. 2005; 25: 7847-7857Crossref PubMed Scopus (117) Google Scholar).Two theories predict a relation between lack of endocytosis and the addiction liability of opioids, but the proposed explanations are radically different. One sees rapid endocytosis as a means to quench receptor signaling. Thus, the abnormally sustained signaling pattern produced by a drug that cannot internalize the receptor would promote post-receptor compensatory mechanisms, which may be responsible for the process of tolerance and withdrawal in vivo (13Finn A.K. Whistler J.L. Neuron. 2001; 32: 829-839Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). The other regards endocytosis and the rapid recycling process that follows (more relevant in μ than in δ receptors (14Whistler J.L. Enquist J. Marley A. Fong J. Gladher F. Tsuruda P. Murray S.R. Von Zastrow M. Science. 2002; 297: 615-620Crossref PubMed Scopus (268) Google Scholar)) as a tool for receptor recovery (15Koch T. Widera A. Bartzsch K. Schulz S. Brandenburg L.O. Wundrack N. Beyer A. Grecksch G. Höllt V. Mol. Pharmacol. 2005; 67: 280-287Crossref PubMed Scopus (144) Google Scholar). Thus, a ligand promoting negligible endocytosis (e.g. morphine), even if interacts with arrestin weakly, would cause progressive accumulation of arrestin-bound desensitized receptors on prolonged exposure, as no significant receptor recovery would occur. That might cause tolerance and dependence in vivo (16Bohn L.M. Dykstra L.A. Lefkowitz R.J. Caron M.G. Barak L.S. Mol. Pharmacol. 2004; 66: 106-112Crossref PubMed Scopus (132) Google Scholar, 17Koch T. Höllt V. Pharmacol. Ther. 2008; 117: 199-206Crossref PubMed Scopus (147) Google Scholar).Recent knock-in mice models have established that the loss of signaling due to receptor endocytosis is related to tolerance in vivo. In a transgenic line expressing a fluorescent replacement of DOPR, the loss of analgesic effect upon repeated dosing of an agonist was clearly related to the ability of the agonist to promote receptor endocytosis (18Pradhan A.A. Becker J.A. Scherrer G. Tryoen-Toth P. Filliol D. Matifas A. Massotte D. Gavériaux-Ruff C. Kieffer B.L. PLoS One. 2009; 4: e5425Crossref PubMed Scopus (151) Google Scholar). Likewise, reduced tolerance to morphine was found in mice bearing a MOP/DOPR chimeric receptor that can be internalized by morphine (19Kim J.A. Bartlett S. He L. Nielsen C.K. Chang A.M. Kharazia V. Waldhoer M. Ou C.J. Taylor S. Ferwerda M. Cado D. Whistler J.L. Curr. Biol. 2008; 18: 129-135Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). However, because DOPR and the MOP/DOPR chimera undergo lysosomal down-regulation (20Marie N. Lecoq I. Jauzac P. Allouche S. J. Biol. Chem. 2003; 278: 22795-22804Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 21Tsao P.I. von Zastrow M. J. Biol. Chem. 2000; 275: 11130-11140Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) more than recycling (13Finn A.K. Whistler J.L. Neuron. 2001; 32: 829-839Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 14Whistler J.L. Enquist J. Marley A. Fong J. Gladher F. Tsuruda P. Murray S.R. Von Zastrow M. Science. 2002; 297: 615-620Crossref PubMed Scopus (268) Google Scholar), the data do not dismiss the suspicion that the outcome in vivo of endocytosis in MOPR might be different in DOPRs (18Pradhan A.A. Becker J.A. Scherrer G. Tryoen-Toth P. Filliol D. Matifas A. Massotte D. Gavériaux-Ruff C. Kieffer B.L. PLoS One. 2009; 4: e5425Crossref PubMed Scopus (151) Google Scholar).The role of arrestin in morphine antinociception and tolerance needs clarification. Evidence that β-arrestin 2 plays a role in vivo despite the weak interactions observed in vitro comes from knock-out animals. Targeted deletion of the β-arrestin 2 gene results in an enhanced analgesic effect (22Bohn L.M. Lefkowitz R.J. Gainetdinov R.R. Peppel K. Caron M.G. Lin F.T. Science. 1999; 286: 2495-2498Crossref PubMed Scopus (781) Google Scholar) and reduced tolerance to morphine but not other opioids (23Bohn L.M. Gainetdinov R.R. Lin F.T. Lefkowitz R.J. Caron M.G. Nature. 2000; 408: 720-723Crossref PubMed Scopus (703) Google Scholar). Similarly, delayed tolerance to morphine occurs in rats after antisense targeting of the β-arrestin 2 gene in the spinal cord (24Przewlocka B. Sieja A. Starowicz K. Maj M. Bilecki W. Przewlocki R. Neurosci. Lett. 2002; 325: 107-110Crossref PubMed Scopus (44) Google Scholar). To explain this "morphine paradox," it was proposed that the weak interaction that morphine-bound MOPR establishes with arrestin is the key factor, as it may lead to a progressive build-up of desensitized receptors that cannot be restored by endocytic recycling (16Bohn L.M. Dykstra L.A. Lefkowitz R.J. Caron M.G. Barak L.S. Mol. Pharmacol. 2004; 66: 106-112Crossref PubMed Scopus (132) Google Scholar).Given such background, we thought it useful to measure the differential efficacy for G protein and arrestin of μ and δ receptors, which are the main receptor subtypes involved in tolerance and addiction (25Kieffer B.L. Trends Pharmacol. Sci. 1999; 20: 19-26Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar). We monitored the direct binding interaction between receptors and the two transducers using resonance energy transfer (RET) techniques (26Molinari P. Casella I. Costa T. Biochem. J. 2008; 409: 251-261Crossref PubMed Scopus (50) Google Scholar) to obtain estimates of ligand efficacies unbiased by nonlinear amplification factors and cross-transducer antagonism that are inherent in indirect determinations from second messenger and protein kinase assays. We show that morphine-like ligands are mixed agonist-antagonists for the two transducers; i.e. they can activate G protein but block competitively arrestin.DISCUSSIONWe have used two assays based on RET technology to measure the efficacy of δ- and μ-opioids in driving the interaction of the receptor with β-arrestin 2 and G protein. Efficacy data were derived from direct measurements of protein-protein interactions, rather than indirectly from biochemical events resulting from those interactions (e.g. second messenger levels, ERK phosphorylation, or receptor internalization). Thus, they should closely reflect the intrinsic property of each ligand structure to stabilize a receptor conformation that can favor the interaction with either arrestin or G protein.The interaction receptor-arrestin was only marginally affected by treatment of cells with pertussis toxin, suggesting that the assessment of ligand efficacy for this interaction is not altered by the concurrent interaction of the receptor with G proteins and the consequent signaling. To achieve a similar situation for the determination of receptor-G protein interaction, we developed a RET assay of G protein coupling in membrane particles. This application was not described previously. Therefore, although not the focus of the study, some experiments were directed to better characterize the GPCR/Gβ response in membranes.Receptor-Gβ interactions have been described previously in cell suspensions using different bioluminescence RET acceptor/donors (34Galés C. Rebois R.V. Hogue M. Trieu P. Breit A. Hébert T.E. Bouvier M. Nat. Methods. 2005; 2: 177-184Crossref PubMed Scopus (319) Google Scholar) or in single cells by epifluorescence fluorescence RET microscopy (35Hein P. Frank M. Hoffmann C. Lohse M.J. Bünemann M. EMBO J. 2005; 24: 4106-4114Crossref PubMed Scopus (176) Google Scholar). The kinetics reported in those studies (subsecond halftimes) is faster than what we measure in membranes. However, considering the strong effect of nucleotides on the interaction plus the diversity in reaction environment and membrane integrity between the experimental systems, a difference in kinetics properties might be not surprising.The enhancement of RET in membranes was both receptor- and Gα-mediated, indicating that the change that brings the N terminus of Gβ closer to the receptor C terminus requires the α-subunit. This increased proximity might either result from the motion of one (or both) reacting partners inside the membrane layer to form a complex with the "dark" Gα subunit, or equally likely, it might result from intermolecular rearrangement among the three partners within a preassembled complex. Whatever the underlying mechanism, the rapid reversal by guanine nucleotide suggests that the driving force behind is release of bound GDP from the Gα subunit. Our working hypothesis is that the RET signal observed in membranes may gauge the power of activated receptor to induce the "empty" state of Gαβγ.Another question is the direction of the change in RET. Agonists do not increase but shorten the distance between receptor and β subunit. This is in contrast with one fluorescence RET study of Gα-Gβγ interactions indicating heterotrimer dissociation by agonists (36Gibson S.K. Gilman A.G. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 212-217Crossref PubMed Scopus (102) Google Scholar), but it does agree with many other RET investigations of receptor-G protein coupling (34Galés C. Rebois R.V. Hogue M. Trieu P. Breit A. Hébert T.E. Bouvier M. Nat. Methods. 2005; 2: 177-184Crossref PubMed Scopus (319) Google Scholar, 35Hein P. Frank M. Hoffmann C. Lohse M.J. Bünemann M. EMBO J. 2005; 24: 4106-4114Crossref PubMed Scopus (176) Google Scholar, 37Ayoub M.A. Maurel D. Binet V. Fink M. Prézeau L. Ansanay H. Pin J.P. Mol. Pharmacol. 2007; 71: 1329-1340Crossref PubMed Scopus (76) Google Scholar, 38Galés C. Van Durm J.J. Schaak S. Pontier S. Percherancier Y. Audet M. Paris H. Bouvier M. Nat. Struct. Mol. Biol. 2006; 13: 778-786Crossref PubMed Scopus (339) Google Scholar). Such findings may challenge the idea that Gβγ dissociation is the primary step of G protein activation and, together with other lines of evidence (39Klein S. Reuveni H. Levitzki A. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 3219-3223Crossref PubMed Scopus (79) Google Scholar), may suggest that in several GPCR/G protein systems activation would involve subunit rearrangements within a preformed complex without physical dissociation of the heterotrimer. Alternatively we may speculate that our results reflect a characteristic of reactions carried over in isolated membranes, where the absence of guanine nucleotides might favor the formation of a dead-end intermediate consisting of nucleotide-free Gα that may trap receptor and Gβγ in an undissociable complex.Regardless of the underlying molecular mechanism, the best evidence that what we measured in membranes is a faithful indicator of ligand ability to promote receptor-mediated G protein activation comes from pharmacological data. Relative potencies, efficacies, and μ/δ selectivity of the panel of opioids measured via RET in this study are in broad agreement with results from a variety of in vivo and in vitro pharmacological assays. We, thus, conclude that the membrane assay of receptor-G protein coupling described in this paper can provide estimates of ligand intrinsic activities that are neither biased by nonlinear amplification factors of the signaling network nor by competitive inhibition from arrestin (40Violin J.D. DiPilato L.M. Yildirim N. Elston T.C. Zhang J. Lefkowitz R.J. J. Biol. Chem. 2008; 283: 2949-2961Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar).Ligand efficacy for G protein and arrestin interactions were compared in both μ and δ receptors. Although not exhaustive, the list of studied ligands represents a fairly broad exploration of the conformational space that can fit δ/μ-opioid receptor binding sites. At least one member of the major chemical classes of opioid was in fact tested, including peptides, natural or semi-synthetic alkaloids, benzomorphans, 4-phenyl- and 4-anilidopiperidines, benzhydrylpiperazines, and Dmt-Tic "peptoids."The arrestin and G protein efficacies of ligands are markedly different both at DOPR and MOPR. However, they are not randomly independent. No ligand displayed greater efficacy for arrestin than for G protein, and partial agonism at G protein was invariably associated with lesser or no efficacy for arrestin. This pattern suggests that the structural requirements for an agonist to trigger the interaction of the receptor with arrestin are more stringent than those sufficient to initiate G protein coupling. All tested antagonists were inactive on arrestin, including two inverse agonists. This finding is in contrast with observations made on different GPCRs (41Azzi M. Charest P.G. Angers S. Rousseau G. Kohout T. Bouvier M. Piñeyro G. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 11406-11411Crossref PubMed Scopus (417) Google Scholar) and tells that a positive efficacy for β-arrestin 2 is not a general property of inverse agonism. We cannot obviously rule out that an opioid with greater efficacy for arrestin than for G protein may actually exist. A high-throughput screening search might be necessary to discover a structure with such property.The molecular mechanism underlying the differential agonism of ligands for arrestin and G protein deserves further study. Because two receptor domains (intracellular loops and the C terminus) are differentially involved in G protein and arrestin recognition (42Gurevich V.V. Gurevich E.V. Pharmacol. Ther. 2006; 110: 465-502Crossref PubMed Scopus (356) Google Scholar), the balance of molecular perturbations transmitted from the ligand binding pocket to the cytosolic area may be a key factor. Structural requirements needed to propagate a change to the somewhat segregated C terminus might be more stringent than those sufficient to affect loops that are contiguous to the transmembrane bundle. That may explain why full agonists for both interactions are more frequently encountered in analogues of the endogenous enkephalins (which was "fine-tuned" by evolution to reach both targets) than when manipulating the structure of opium-derived phenanthrene alkaloids or other synthetic templates.However, the existence of a subset of ligands with poor arrestin efficacy despite a close to full G protein efficacy suggests that in those structures molecular interactions that are optimal to allosterically induce the receptor interface for G protein may be detrimental to shape that for arrestin. Thus, there might be specific contacts with receptor residues that differentially channel conformational perturbations resulting in alternative three-dimensional configurations of the cytosolic area. If so, the shortening from N-allyl to the N-methyl group that converts naloxone (a dual G protein/arrestin antagonist) into oxymorphone (a mixed agonist/antagonist) is particularly interesting because it seems sufficient to restore full receptor efficacy for G protein but not for arrestin. Conversely, the double addition of one methyl and one carboxymethyl group to the piperidine ring of fentanyl (which makes lofentanyl) is sufficient to convert in DOPR an agonist/antagonist into a dual full agonist. More detailed structure-activity relationship analysis of the data might provide further insight into this issue.It is interesting that the discrepancy between G protein and arrestin efficacy of partial agonists described here for opioids is much stronger than what reported for the β2-adrenergic receptor (28Drake M.T. Violin J.D. Whalen E.J. Wisler J.W. Shenoy S.K. Lefkowitz R.J. J. Biol. Chem. 2008; 283: 5669-5676Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). This suggests that our observations may not be broadly applicable to other GPCR or ligand systems and that factors related to the cellular expression host may also play a key role. Particularly important in the case of arrestin-receptor interactions is the contribution of G protein receptor kinase-mediated receptor phosphorylation.Although studies with radiolabeled arrestin indicate that agonists can regulate arrestin binding regardless of phosphorylation (43Gurevich V.V. Dion S.B. Onorato J.J. Ptasienski J. Kim C.M. Sterne-Marr R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: 720-731Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar), recent findings on the CCR7 chemokine receptors show that the differential activation of G protein receptor kinase isotypes by distinct agonist can result in marked differences of arrestin recruitment (44Zidar D.A. Violin J.D. Whalen E.J. Lefkowitz R.J. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 9649-9654Crossref PubMed Scopus (221) Google Scholar). This implies the possibility that intrinsic differences in phosphorylation might play a key role in determining distinct relationships between transducers efficacy across different ligands and GPCR types.The biological consequence of receptor occupation by a ligand that has strong or full efficacy for G protein but weak or null efficacy for arrestin is agonism for the first interaction and competitive antagonism for the second. This defines a new type of transducer-selective antagonism, where the conflicting efficacies reflect the interaction of the same receptor with two distinct transduction partners. As shown here, oxymorphone and morphine are indeed virtually pure arrestin antagonists at DOPR and weak partial agonists with strong antagonistic properties at MOPR.Our data confirm the morphine paradox mentioned in the introduction; targeted deletion of the β-arrestin 2 gene affects the acute and chronic pharmacology of morphine more than other opiates (16Bohn L.M. Dykstra L.A. Lefkowitz R.J. Caron M.G. Barak L.S. Mol. Pharmacol. 2004; 66: 106-112Crossref PubMed Scopus (132) Google Scholar). Yet, as shown here, morphine and oxymorphone are among the agonists with the worst efficiency to promote the interaction of the receptor with β-arrestin 2. The additional point emerging from our study is that morphine-like alkaloids are mixed agonist/antagonists of receptor-transducer interactions. In fact, as shown here, they can activate G protein-mediated signaling but also protect the receptor from internalization and down-regulation induced by endogenous enkephalins. This explains the antagonistic effects reported earlier in neuroblastoma cells (6Vachon L. Costa T. Herz A. Biochem. Pharmacol. 1987; 36: 2889-2897Crossref PubMed Scopus (32) Google Scholar). Whether such dual property might be related to the ability of morphine to up-regulate DOPR trafficking, often observed in brain (45Hack S.P. Bagley E.E. Chieng B.C. Christie M.J. J. Neurosci. 2005; 25: 3192-3198Crossref PubMed Scopus (69) Google Scholar, 46Morinville A. Cahill C.M. Esdaile M.J. Aibak H. Collier B. Kieffer B.L. Beaudet A. J. Neurosci. 2003; 23: 4888-4898Crossref PubMed Google Scholar), or to the strong tolerance that such ligands exert in vivo remains to be established. Other ligands share similar although not identical profiles. Fentanyl at δ but not μ receptors is one example. Some benzomorphans and oripavines also have G protein agonism and arrestin antagonism, although they are weaker agonists for G proteins and have considerable activity for κ receptors.The mix of agonism and antagonism that each ligand can exhibit not only across different receptor subtypes but through the same subtype also across different transducers draws a very complex maze of potential signaling outputs. This makes it really difficult to predict how such molecular interactions may be linked to the acute and chronic drug responses occurring in vivo. As recently shown by groundbreaking work in the knock-in mouse carrying a fluorescent replacement of the δ receptor (18Pradhan A.A. Becker J.A. Scherrer G. Tryoen-Toth P. Filliol D. Matifas A. Massotte D. Gavériaux-Ruff C. Kieffer B.L. PLoS One. 2009; 4: e5425Crossref PubMed Scopus (151) Google Scholar, 47Scherrer G. Tryoen-Tóth P. Filliol D. Matifas A. Laustriat D. Cao Y.Q. Basbaum A.I. Dierich A. Vonesh J.L. Gavériaux-Ruff C. Kieffer B.L. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 9691-9696Crossref PubMed Scopus (187) Google Scholar), animal models in which molecular interactions are engineered for in vivo detection may be a solution to the problem and are likely to provide the kind of physiological and clinical relevant insight that progress in molecular knowledge alone cannot deliver. IntroductionPhysiological agonists are usually equally efficient in promoting the interaction of receptors with G protein and arrestin, but manmade analogues can show divergent molecular efficacies for the two transducers (1Violin J.D. Lefkowitz R.J. Trends Pharmacol. Sci. 2007; 28: 416-422Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar, 2Masri B. Salahpour A. Didriksen M. Ghisi V. Beaulieu J.M. Gainetdinov R.R. Caron M.G. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13656-13661Crossref PubMed Scopus (264) Google Scholar). This phenomenon, often addressed with a pictorial terminology (3Urban J.D. Clarke W.P. von Zastrow M. Nichols D.E. Kobilka B. Weinstein H. Javitch J.A. Roth B.L. Christopoulos A. Sexton P.M. Miller K.J. Spedding M. Mailman R.B. J. Pharmacol. Exp. Ther. 2007; 320: 1-13Crossref PubMed Scopus (891) Google Scholar, 4Kenakin T. Mol. Pharmacol. 2007; 72: 1393-1401Crossref PubMed Scopus (238) Google Scholar, 5Kenakin T.P. Br. J. Pharmacol. 2008; 153: 432-438Crossref PubMed Scopus (36) Google Scholar), has attracted great interest and if better understood might lead to new types of drugs.The differential efficacy of opioids for G protein and arrestin interactions is also important in the mechanism of opiate addiction. As reported earlier, the addictive opiate morphine cannot induce and actually blocks desensitization and G protein uncoupling of δ-opioid receptors (DOPR) 2The abbreviations used are: DOPRδ-opioid receptorMOPRμ-opioid receptorRETresonance energy transferHEK293human embryonic kidney 293 cellsRGFPRenilla green fluorescent proteinRlucRenilla luciferaseGPCRG-protein-coupled receptorcpscounts/sβArr1 and βArr2β-arrestin 1 and 2bDOCbisdeoxy-coelenterazineDADLE[d-Ala2, d-Leu5]-enkephalinsEKCethylketocyclazocineGTPγSguanosine 5′-O-(thiotriphosphate)mtmembrane-targetedDmt2′,6′-dimethyltyrosineTic1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. in neuroblastoma or in transfected cells (6Vachon L. Costa T. Herz A. Biochem. Pharmacol. 1987; 36: 2889-2897Crossref PubMed Scopus (32) Google Scholar, 7Eisinger D.A. Ammer H. Schulz R. J. Neurosci. 2002; 22: 10192-10200Crossref PubMed Google Scholar). Subsequent work shows that morphine is a poor inducer of rapid arrestin-dependent endocytosis for both δ and μ (MOPR) receptors (8Arttamangkul S. Quillinan N. Low M.J. von Zastrow M. Pintar J. Williams J.T. Mol. Pharmacol. 2008; 74: 972-979Crossref PubMed Scopus (90) Google Scholar, 9Keith D.E. Anton B. Murray S.R. Zaki P.A. Chu P.C. Lissin D.V. Monteillet-Agius G. Stewart P.L. Evans C.J. von Zastrow M. Mol. Pharmacol. 1998; 53: 377-384Crossref PubMed Scopus (289) Google Scholar, 10Keith D.E. Murray S.R. Zaki P.A. Chu P.C. Lissin D.V. Kang L. Evans C.J. von Zastrow M. J. Biol. Chem. 1996; 271: 19021-19024Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar, 11Whistler J.L. von Zastrow M. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 9914-9919Crossref PubMed Scopus (314) Google Scholar), although one exception is in striatum neurons with high levels of G protein receptor kinases (12Haberstock-Debic H. Kim K.A. Yu Y.J. von Zastrow M. J. Neurosci. 2005; 25: 7847-7857Crossref PubMed Scopus (117) Google Scholar).Two theories predict a relation between lack of endocytosis and the addiction liability of opioids, but the proposed explanations are radically different. One sees rapid endocytosis as a means to quench receptor signaling. Thus, the abnormally sustained signaling pattern produced by a drug that cannot internalize the receptor would promote post-receptor compensatory mechanisms, which may be responsible for the process of tolerance and withdrawal in vivo (13Finn A.K. Whistler J.L. Neuron. 2001; 32: 829-839Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). The other regards endocytosis and the rapid recycling process that follows (more relevant in μ than in δ receptors (14Whistler J.L. Enquist J. Marley A. Fong J. Gladher F. Tsuruda P. Murray S.R. Von Zastrow M. Science. 2002; 297: 615-620Crossref PubMed Scopus (268) Google Scholar)) as a tool for receptor recovery (15Koch T. Widera A. Bartzsch K. Schulz S. Brandenburg L.O. Wundrack N. Beyer A. Grecksch G. Höllt V. Mol. Pharmacol. 2005; 67: 280-287Crossref PubMed Scopus (144) Google Scholar). Thus, a ligand promoting negligible endocytosis (e.g. morphine), even if interacts with arrestin weakly, would cause progressive accumulation of arrestin-bound desensitized receptors on prolonged exposure, as no significant receptor recovery would occur. That might cause tolerance and dependence in vivo (16Bohn L.M. Dykstra L.A. Lefkowitz R.J. Caron M.G. Barak L.S. Mol. Pharmacol. 2004; 66: 106-112Crossref PubMed Scopus (132) Google Scholar, 17Koch T. Höllt V. Pharmacol. Ther. 2008; 117: 199-206Crossref PubMed Scopus (147) Google Scholar).Recent knock-in mice models have established that the loss of signaling due to receptor endocytosis is related to tolerance in vivo. In a transgenic line expressing a fluorescent replacement of DOPR, the loss of analgesic effect upon repeated dosing of an agonist was clearly related to the ability of the agonist to promote receptor endocytosis (18Pradhan A.A. Becker J.A. Scherrer G. Tryoen-Toth P. Filliol D. Matifas A. Massotte D. Gavériaux-Ruff C. Kieffer B.L. PLoS One. 2009; 4: e5425Crossref PubMed Scopus (151) Google Scholar). Likewise, reduced tolerance to morphine was found in mice bearing a MOP/DOPR chimeric receptor that can be internalized by morphine (19Kim J.A. Bartlett S. He L. Nielsen C.K. Chang A.M. Kharazia V. Waldhoer M. Ou C.J. Taylor S. Ferwerda M. Cado D. Whistler J.L. Curr. Biol. 2008; 18: 129-135Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). However, because DOPR and the MOP/DOPR chimera undergo lysosomal down-regulation (20Marie N. Lecoq I. Jauzac P. Allouche S. J. Biol. Chem. 2003; 278: 22795-22804Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 21Tsao P.I. von Zastrow M. J. Biol. Chem. 2000; 275: 11130-11140Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) more than recycling (13Finn A.K. Whistler J.L. Neuron. 2001; 32: 829-839Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 14Whistler J.L. Enquist J. Marley A. Fong J. Gladher F. Tsuruda P. Murray S.R. Von Zastrow M. Science. 2002; 297: 615-620Crossref PubMed Scopus (268) Google Scholar), the data do not dismiss the suspicion that the outcome in vivo of endocytosis in MOPR might be different in DOPRs (18Pradhan A.A. Becker J.A. Scherrer G. Tryoen-Toth P. Filliol D. Matifas A. Massotte D. Gavériaux-Ruff C. Kieffer B.L. PLoS One. 2009; 4: e5425Crossref PubMed Scopus (151) Google Scholar).The role of arrestin in morphine antinociception and tolerance needs clarification. Evidence that β-arrestin 2 plays a role in vivo despite the weak interactions observed in vitro comes from knock-out animals. Targeted deletion of the β-arrestin 2 gene results in an enhanced analgesic effect (22Bohn L.M. Lefkowitz R.J. Gainetdinov R.R. Peppel K. Caron M.G. Lin F.T. Science. 1999; 286: 2495-2498Crossref PubMed Scopus (781) Google Scholar) and reduced tolerance to morphine but not other opioids (23Bohn L.M. Gainetdinov R.R. Lin F.T. Lefkowitz R.J. Caron M.G. Nature. 2000; 408: 720-723Crossref PubMed Scopus (703) Google Scholar). Similarly, delayed tolerance to morphine occurs in rats after antisense targeting of the β-arrestin 2 gene in the spinal cord (24Przewlocka B. Sieja A. Starowicz K. Maj M. Bilecki W. Przewlocki R. Neurosci. Lett. 2002; 325: 107-110Crossref PubMed Scopus (44) Google Scholar). To explain this "morphine paradox," it was proposed that the weak interaction that morphine-bound MOPR establishes with arrestin is the key factor, as it may lead to a progressive build-up of desensitized receptors that cannot be restored by endocytic recycling (16Bohn L.M. Dykstra L.A. Lefkowitz R.J. Caron M.G. Barak L.S. Mol. Pharmacol. 2004; 66: 106-112Crossref PubMed Scopus (132) Google Scholar).Given such background, we thought it useful to measure the differential efficacy for G protein and arrestin of μ and δ receptors, which are the main receptor subtypes involved in tolerance and addiction (25Kieffer B.L. Trends Pharmacol. Sci. 1999; 20: 19-26Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar). We monitored the direct binding interaction between receptors and the two transducers using resonance energy transfer (RET) techniques (26Molinari P. Casella I. Costa T. Biochem. J. 2008; 409: 251-261Crossref PubMed Scopus (50) Google Scholar) to obtain estimates of ligand efficacies unbiased by nonlinear amplification factors and cross-transducer antagonism that are inherent in indirect determinations from second messenger and protein kinase assays. We show that morphine-like ligands are mixed agonist-antagonists for the two transducers; i.e. they can activate G protein but block competitively arrestin.
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