Internalization and Src Activity Regulate the Time Course of ERK Activation by Delta Opioid Receptor Ligands

The present study showed that delta opioid receptor (δOR) ligands Tyr-Ticpsi [CH2-NH]Cha-Phe-OH (TICP) and ICI174864 behaved as inverse agonists in the cyclase pathway but induced agonist responses in the ERK cascade. Unlike ligands that behaved as agonists in both pathways, and whose stimulation of ERK was marked but transient (10 min), ERK activation by ICI174864 and TICP was moderate and sustained, lasting for more than 1 h in the case of TICP. Biochemical experiments showed that duration of ERK activation by agonists and “dual efficacy ligands” was inversely correlated with their ability to trigger receptor phosphorylation and degradation. Thus, although TICP stabilized δORs in a conformation that did not incorporate 32P, was not a substrate for tyrosine kinase Src, and was not down-regulated following prolonged exposure to the drug, the conformation stabilized by d-Pen-2,5-enkephalin (DPDPE) incorporated 32P, was phosphorylated by Src, and suffered degradation within the first 2 h of treatment. Inhibition of endocytosis by sucrose prolonged ERK activation by DPDPE increasing the decay half-life of the response to values that resembled those of dual efficacy ligands (from a 2-min decay t½ increased to 12 min). Src inhibitor PP2 also prolonged ERK stimulation by DPDPE. It did so by maintaining a sustained activation of the kinase at ∼20% of maximum following an initial rapid reduction in the response. These results show that specific kinetics of ERK activation by agonists and dual efficacy ligands are determined, at least in part, by the differential ability of the two types of drugs to trigger mechanisms regulating δOR responsiveness. The present study showed that delta opioid receptor (δOR) ligands Tyr-Ticpsi [CH2-NH]Cha-Phe-OH (TICP) and ICI174864 behaved as inverse agonists in the cyclase pathway but induced agonist responses in the ERK cascade. Unlike ligands that behaved as agonists in both pathways, and whose stimulation of ERK was marked but transient (10 min), ERK activation by ICI174864 and TICP was moderate and sustained, lasting for more than 1 h in the case of TICP. Biochemical experiments showed that duration of ERK activation by agonists and “dual efficacy ligands” was inversely correlated with their ability to trigger receptor phosphorylation and degradation. Thus, although TICP stabilized δORs in a conformation that did not incorporate 32P, was not a substrate for tyrosine kinase Src, and was not down-regulated following prolonged exposure to the drug, the conformation stabilized by d-Pen-2,5-enkephalin (DPDPE) incorporated 32P, was phosphorylated by Src, and suffered degradation within the first 2 h of treatment. Inhibition of endocytosis by sucrose prolonged ERK activation by DPDPE increasing the decay half-life of the response to values that resembled those of dual efficacy ligands (from a 2-min decay t½ increased to 12 min). Src inhibitor PP2 also prolonged ERK stimulation by DPDPE. It did so by maintaining a sustained activation of the kinase at ∼20% of maximum following an initial rapid reduction in the response. These results show that specific kinetics of ERK activation by agonists and dual efficacy ligands are determined, at least in part, by the differential ability of the two types of drugs to trigger mechanisms regulating δOR responsiveness. Occupation of G protein-coupled receptors by agonist ligands has two distinct consequences, the generation of an intracellular signal and the concomitant activation of a series of regulatory mechanisms that modulate receptor responsiveness over time. The chain of regulatory events triggered by agonist occupation of the receptor has been extensively characterized and has led to an established model of desensitization in which phosphorylation of the receptor by G protein-coupled receptor kinases is the first step in the process (1Benovic J.L. Regan J.W. Matsui H. Mayor Jr., F. Cotecchia S. Leeb-Lundberg L.M. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1987; 262: 17251-17253Abstract Full Text PDF PubMed Google Scholar, 2Lohse M.J. Benovic J.L. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1990; 265: 3202-3211Abstract Full Text PDF PubMed Google Scholar). Phosphorylation then promotes the recruitment of βarrestin (3Zhang J. Ferguson S.S. Barak L.S. Bodduluri S.R. Laporte S.A. Law P.Y. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7157-7162Crossref PubMed Scopus (460) Google Scholar, 4Lowe J.D. Celver J.P. Gurevich V.V. Chavkin C. J. Biol. Chem. 2002; 277: 15729-15735Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), which is responsible for uncoupling the receptor from the G protein (5Cheng Z.J. Yu Q.M. Wu Y.L. Ma L. Pei G. J. Biol. Chem. 1998; 273: 24328-24333Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) and for its targeting to clathrin-coated pits. From there receptors will be removed from the cell surface via dynamin-dependent endocytosis (6von Zastrow M. Svingos A. Haberstock-Debic H. Evans C. Curr. Opin. Neurobiol. 2003; 13: 348-353Crossref PubMed Scopus (100) Google Scholar). Once inside the cell the receptor is either degraded or is quickly redirected to the cell membrane (7Tsao P.I. von Zastrow M. J. Biol. Chem. 2000; 275: 11130-11140Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) for a new signaling cycle. Despite the overwhelming evidence supporting this tightly knit model of activation and desensitization, there are also increasing observations indicating that activation and regulatory phenomena can be dissociated. For example, antagonist ligands for cholecystokinin (8Roettger B.F. Ghanekar D. Rao R. Toledo C. Yingling J. Pinon D. Miller L.J. Mol. Pharmacol. 1997; 51: 357-362PubMed Google Scholar) and endothelin receptors (9Bhowmick N. Narayan P. Puett D. Endocrinology. 1998; 139: 3185-3192Crossref PubMed Scopus (55) Google Scholar) selectively induce internalization without causing neither receptor activation nor phosphorylation. Agonists for parathyroid hormone type 1 receptor stabilize an active state that promotes signaling but does not recruit βarrestin or induce internalization (10Bisello A. Chorev M. Rosenblatt M. Monticelli L. Mierke D.F. Ferrari S.L. J. Biol. Chem. 2002; 277: 38524-38530Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In contrast, certain β2-adrenergic receptor (β2AR) 1The abbreviations used are: β2AR, β2-adrenergic receptor; DMEM, Dulbecco's modified Eagle's medium; δOR, delta opioid receptor; DPDPE, d-Pen-2,5-enkephalin; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; PTX, pertussis toxin; TICP, Tyr-Ticpsi [CH2-NH]Cha-Phe-OH; GTPγS, guanosine 5′-O-(thiotriphosphate); ANOVA, analysis of variance; TIPP, H-Tyr-TicPsi-[CH(2)NH]Phe-Phe-OH].1The abbreviations used are: β2AR, β2-adrenergic receptor; DMEM, Dulbecco's modified Eagle's medium; δOR, delta opioid receptor; DPDPE, d-Pen-2,5-enkephalin; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; PTX, pertussis toxin; TICP, Tyr-Ticpsi [CH2-NH]Cha-Phe-OH; GTPγS, guanosine 5′-O-(thiotriphosphate); ANOVA, analysis of variance; TIPP, H-Tyr-TicPsi-[CH(2)NH]Phe-Phe-OH]. ligands that preclude G protein activation are still able to recruit βarrestin to the receptor (11Azzi M. Charest P.G. Angers S. Rousseau G. Kohout T. Bouvier M. Pineyro G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11406-11411Crossref PubMed Scopus (419) Google Scholar). Ligands that stabilize G protein-coupled receptors in a conformation that prevents activation of the G protein are classified as inverse agonists and are commonly thought to induce an inactive conformation of the receptor (12Chidiac P. Hebert T.E. Valiquette M. Dennis M. Bouvier M. Mol. Pharmacol. 1994; 45: 490-499PubMed Google Scholar, 13Samana P. Pei G. Costa T. Cotecchia S. Lefkowitz R.J. Mol. Pharmacol. 1994; 45: 390-394PubMed Google Scholar). More recently, some of these drugs have been described as “proteans” or “dual efficacy ligands,” referring to their ability to display both agonist and inverse agonist behavior (11Azzi M. Charest P.G. Angers S. Rousseau G. Kohout T. Bouvier M. Pineyro G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11406-11411Crossref PubMed Scopus (419) Google Scholar, 14Chidiac P. Nouet S. Bouvier M. Mol. Pharmacol. Google Scholar, F. A. S. R. S. J. H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: PubMed Scopus Google Scholar, G. M. A. Bouvier M. Mol. Pharmacol. PubMed Scopus Google Scholar). For example, have that and two ligands of the β2AR, display dual as inverse agonists in the cyclase pathway but agonist responses in the ERK (11Azzi M. Charest P.G. Angers S. Rousseau G. Kohout T. Bouvier M. Pineyro G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11406-11411Crossref PubMed Scopus (419) Google Scholar). The that some agonist responses that the conformation stabilize is not but a signaling state that is distinct from the stabilized by receptor stabilized by agonists and dual efficacy ligands are then that the responses that also be in a distinct The present study this responses by dual efficacy ligands for the δOR are as agonist responses induced by its show that ERK activation by dual efficacy ligands TICP and ICI174864 was although more than the response induced by agonists as and DPDPE. in with the distinct ability of dual efficacy ligands to stabilize δORs in an conformation that regulatory triggered by DPDPE, PTX, and from and from ICI174864 and from and as described G. C. 51: PubMed Scopus Google Scholar). was from DMEM, and from δOR was the as described M. C. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and was at the to the and the for the The was by and and its signaling to be to those of the type δOR M. A. P. Bouvier M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, G. M. A. P. Bouvier M. Mol. Pharmacol. Google Scholar). of the δOR was by M. von Zastrow of at type and of a from and the and or receptors δORs and type following The of was of cell the δOR as described F. D. J.P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). and in 1 and in a of at Phosphorylation and of for 2 h in DMEM, which was at a of 1 and was to for an this DPDPE TICP or to the for then and as and in 2 2 and 2 at for the was at for and the receptor was from the of in and with used to the receptor at The the was with of and with of modified of The receptor was then by the for at with of modified of was and the and by membrane over was then and used for was used to phosphorylation of and was as described by a and in then from the In the case of first for phosphorylation of at with phosphorylated In both the used to the of receptor protein present in and used to the and of the by phosphorylation was as the phosphorylation and to to the of receptor protein present in For of in and The of the for 2 h in and then to with and cell by in and then for for was by with ERK protein was by and used to by phosphorylation was to protein by results as the and Src in and for the as described for with ligands and in 1 1 1 1 at for of at for in and in at a of was used to the of Src and of protein was by with with 1 of in The of the was with DMEM, and with and was was than then for at in of and drugs at the the of the the was by of and and by and as the of cell membrane as described G. M. A. P. Bouvier M. Mol. Pharmacol. Google Scholar). in 2 and and with a and of at maximum at for and the was at for in and in 1 1 and to of was used at and was in the of The was at a of and was to for at The was by rapid in with and and the was by analysis and of the of δOR Ligands in and ERK has been that certain ligands for display dual inverse agonist responses in the signaling but agonist in the ERK (11Azzi M. Charest P.G. Angers S. Rousseau G. Kohout T. Bouvier M. Pineyro G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11406-11411Crossref PubMed Scopus (419) Google Scholar). this type of dual behavior was specific to receptors or be to receptors to ligands for the δOR in cyclase and ERK signaling In the pathway ligands that the of efficacy from to inverse and DPDPE TIPP, and ICI174864 and TICP inverse agonist these ligands to and of their DPDPE ICI174864 In with the of responses in drugs in the ERK behaved as for that was ERK phosphorylation was induced not by drugs that behaved as agonists in the cyclase but also by TICP and which inverse agonist responses in this to the of their ERK TICP, the inverse agonist in the cyclase pathway was more than agonists and in ERK DPDPE TICP ICI174864 efficacy ligands for the ERK activation via βarrestin recruitment and of G protein (11Azzi M. Charest P.G. Angers S. Rousseau G. Kohout T. Bouvier M. Pineyro G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11406-11411Crossref PubMed Scopus (419) Google Scholar). this was also the case for with PTX, and ERK was the following of the kinase in following ERK stimulation by agonist DPDPE and by dual efficacy TICP These results not that ERK stimulation by DPDPE and TICP protein but also that of signaling for ERK TICP, nor agonists able to ERK activation in that stimulation of ERK signaling was by the δOR Ligands in of ERK the of ERK activation by agonists from that of dual efficacy to a of drug, and ERK phosphorylation was following increasing of time. types of be was of ligands and DPDPE, which and ERK activation that within with a half-life of and The type of induced by agonists and dual efficacy ligands was but more sustained, with a t½ that and and ligands sustained the of TICP be from the of the drugs in the its was more and more sustained and The of ERK by Agonists and Ligands with of the that over is the receptor In δOR signaling efficacy is by phosphorylation of G. J. L.J. Law P.Y. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, P.Y. J. L.J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). in the of ERK activation be to the distinct ability of ligands to trigger phosphorylation of δORs for to DPDPE or TICP in the of and that first for and then used for analysis an two at and to and of the M. A. S. P. Bouvier M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). showed that with DPDPE increased by the but this was for TICP. Thus, at a the ERK response for the agonist was δORs In contrast, ERK activation by the dual efficacy was still at its and phosphorylation of the receptor be Phosphorylation is an initial step in the process of desensitization, but exposure to an agonist is to δORs will to be for degradation (7Tsao P.I. von Zastrow M. J. Biol. Chem. 2000; 275: 11130-11140Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). to the of ERK activation by DPDPE and TICP also correlated with events in the process of desensitization, for 2 h either with the agonist or the dual efficacy the of δOR protein present in membrane was by with TICP in the receptor there was a of the following with DPDPE. These results that in the of ERK activation by DPDPE and TICP is inversely correlated with the ability of to trigger events within the process of in of ERK activation by agonists and dual efficacy ligands to their distinct ability to trigger regulatory mechanisms of receptor with these mechanisms ERK activation by the the more prolonged type of response for the dual efficacy this the of ERK activation by DPDPE was in of which is an inhibitor of sucrose did not ERK activation a prolonged the of DPDPE by increasing the decay t½ of activation from 2 to 12 for a that within the to for dual efficacy to mechanisms regulating δOR responsiveness is to that are in the For in the of the receptor are the for G protein-coupled receptor and their phosphorylation is an step in the desensitization of δORs G. J. L.J. Law P.Y. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, P.Y. J. L.J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, A. Chavkin C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). the of these to the kinetics of ERK activation by DPDPE, experiments a receptor at its also results in which the DPDPE response more than in the receptor of for the of was than that with sucrose the the of sucrose prolonged the decay t½ for DPDPE in receptors of for Agonists but Phosphorylation of that the of ERK activation by DPDPE was prolonged by of the that there be a of regulating ERK activation by agonists in the of In this of a to of the has been to internalization and of the receptor S.A. Mol. 2000; PubMed Scopus Google Scholar). Thus, was of to δORs be phosphorylated at their tyrosine following exposure to agonists and dual efficacy receptors for either with DPDPE or TICP, and receptors and by with that phosphorylated showed that TICP and DPDPE modified of δORs with TICP DPDPE induced an in for in the to the receptor the of Src inhibitor PP2 to exposure to DPDPE the in indicating that a of the Src was in phosphorylation of of TICP to induce phosphorylation of the receptor was to its to Src, the kinase either to DPDPE or TICP and in Src by that both the agonist and the dual efficacy increased for the active of Src, that the in tyrosine phosphorylation of δORs was not to of TICP to The of the series of experiments was to Src ERK responses by DPDPE or TICP. with increasing of Src inhibitor PP2, and ERK stimulation was following exposure to of the two δOR It was that of PP2 ERK activation by the dual efficacy and the the of TICP was that of DPDPE was and was at a of that PP2 with ERK activation by DPDPE The of specific of PP2 the response to TICP is with the that this ERK activation in a the the to the of DPDPE not to Src as an in ERK to this of ERK activation by DPDPE was by with increasing of a Src a as described for PP2, with of the of DPDPE responses and of ERK activation by the The of DPDPE responses by of inactive Src the that of this kinase is for ERK activation by agonists and is with that of Src with ERK activation Pharmacol. 2000; PubMed Scopus Google Scholar). the the that of Src or of PP2 ERK activation by DPDPE that Src also be in the of agonist was by with DPDPE for in the or of PP2 and by then the ability of to induce PP2 for 1 the ability of to but this was by a marked in to of the ability of to as with to the in the efficacy of to induce was following with DPDPE. PP2 was the DPDPE, the ability of to induce was not that of Src a to Src have to the distinct of ERK activation by DPDPE and TICP, the for DPDPE was in of In the of Src ERK phosphorylation by DPDPE within the first of In contrast, in the of PP2, ERK at was still of the response to a ERK in PP2 a ERK activation by DPDPE, following with the phosphorylation of the kinase was not from the of The present study new the of responses induced by efficacy In results that by this type of dual ligands δORs phosphorylation and induce prolonged activation of the ERK cascade. These with those of whose activation of the ERK is transient and correlated with phosphorylation and degradation of the ERK was not by drugs DPDPE, TIPP, and which also in the cyclase but also by ligands TICP and ICI174864 that ERK inverse agonist behavior in and agonists are commonly thought to their via an inactive receptor conformation that G protein signaling (12Chidiac P. Hebert T.E. Valiquette M. Dennis M. Bouvier M. Mol. Pharmacol. 1994; 45: 490-499PubMed Google Scholar, 13Samana P. Pei G. Costa T. Cotecchia S. Lefkowitz R.J. Mol. Pharmacol. 1994; 45: 390-394PubMed Google Scholar). is that activation of the ERK by TICP or ICI174864 be to of the of by to ERK the that ERK activation by TICP to ERK activation by dual efficacy δOR ligands the activation of a is in marked with dual efficacy ligands described for the whose activation of the ERK was not G (11Azzi M. Charest P.G. Angers S. Rousseau G. Kohout T. Bouvier M. Pineyro G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11406-11411Crossref PubMed Scopus (419) Google Scholar). The that TICP to ERK signaling and regulating the cyclase pathway be by the that δORs are of with more than protein G. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, L.J. Evans Law P.Y. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). is for ligands TICP or ICI174864 to display agonist and inverse agonist in ERK and a conformation of the receptor that but In contrast, agonists stabilize a conformation that responsible for the of both with the that agonists and dual efficacy ligands stabilize δORs in is the that the of efficacy with which TICP, TIPP, and modified cyclase signaling was with to their to ERK activation and In in of efficacy for ligands that more than signaling via the receptor is as of the in of a model of active receptor T. Pharmacol. Sci. Full Text PDF PubMed Scopus Google Scholar, S. J. C. P. Mol. Pharmacol. 1998; PubMed Scopus Google Scholar). In the in TICP the and and the to that the conformation via which the dual efficacy ERK signaling is from the in ERK stimulation by the two The observations that receptors stabilized by DPDPE but not those by TICP incorporated and a for the tyrosine kinase Src and the that δORs in and to the that these receptor have distinct desensitization The response for DPDPE is in with that agonists δOR phosphorylation by G protein-coupled receptor kinases and tyrosine kinases P.Y. J. L.J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, A. Chavkin C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Pharmacol. 2000; PubMed Scopus Google Scholar, J. Ferguson S.S. Law P.Y. Barak L.S. Caron M.G. J. PubMed Scopus Google Scholar, J.L. P. von Zastrow M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the TICP in its ability to induce a receptor conformation that is phosphorylated by receptor kinases J. Ferguson S.S. Law P.Y. 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