Internalization and Src Activity Regulate the Time Course of ERK Activation by Delta Opioid Receptor Ligands
2005; Elsevier BV; Volume: 280; Issue: 9 Linguagem: Inglês
10.1074/jbc.m411695200
ISSN1083-351X
AutoresNicolas Audet, Mélanie Paquin‐Gobeil, Olivier Landry-Paquet, Peter W. Schiller, Graciela Piñeyro,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoThe 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. 1996; 50: 662-669PubMed Google Scholar, 15Gbahou F. Rouleau A. Morisset S. Parmentier R. Crochet S. Lin J.S. Ligneau X. Tardivel-Lacombe J. Stark H. Schunack W. Ganelin C.R. Schwartz J.C. Arrang J.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11086-11091Crossref PubMed Scopus (122) Google Scholar, 16Pineyro G. Azzi M. deLe ́an A. Schiller P.W. Bouvier M. Mol. Pharmacol. 2005; 67: 336-348Crossref PubMed Scopus (18) Google Scholar). For example, we have recently shown that ICI118551 and propranolol, two ligands of the β2AR, display dual efficacy, because they behave as inverse agonists in the cyclase pathway but produce agonist responses in the ERK cascade (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 observation that some "inverse agonists" may produce agonist responses indicates that the conformation they stabilize is not inactive, but rather a signaling state that is distinct from the one stabilized by classic agonists. If receptor states stabilized by agonists and dual efficacy ligands are distinct, then one would expect that the responses that they elicit would also be regulated in a distinct manner. The present study focused on this question, assessing whether agonistic responses generated by dual efficacy ligands for the δOR are regulated as agonist responses induced by its classic agonists. Results show that ERK activation by dual efficacy ligands like TICP and ICI174864 was considerably longer, although more modest than the response induced by agonists such as SNC-80 and DPDPE. Differences in time course were associated with the distinct ability of dual efficacy ligands to stabilize δORs in an ERK-stimulating conformation that eluded regulatory steps typically triggered by highly efficacious agonists. Reagents—Buffer chemicals, protease inhibitors, DPDPE, morphine, naloxone, forskolin, isobutylmethylxanthine, PTX, sucrose, anti-FLAG M2 affinity resin, and FLAG peptide were purchased from Sigma. [35S]GTPγS, [3H]adenosine, and [32P]orthophosphoric acid were from PerkinElmer Life Sciences. ICI174864 and SNC-80 were obtained from Tocris Cookson, TIPP and TICPΨ were synthesized as described previously (17Schiller P.W. Weltrowska G. Berezowska I. Nguyen T.M. Wilkes B.C. Lemieux C. Chung N.N. Biopolymers. 1999; 51: 411-425Crossref PubMed Scopus (106) Google Scholar). 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) was from Calbiochem. G418, DMEM, fetal bovine serum, fungizone, glutamine, penicillin, and streptomycin were purchased from Wisent. DNA Constructs—The human δOR cDNA was subcloned into the pcDNA3 expression vector (Invitrogen) as described previously (18Valiquette M. Vu H.K. Yue S.Y. Wahlestedt C. Walker P. J. Biol. Chem. 1996; 271: 18789-18796Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) and was tagged at the C-terminal end using Clontech site-directed mutagenesis kit to remove the stop codon and introduce the sequence coding for the FLAG epitope (DYKDDDDK). The construction was confirmed by restriction enzyme mapping and DNA sequencing, and its signaling properties were shown to be identical to those of the wild type δOR (19Petaja-Repo U.E. Hogue M. Laperriere A. Walker P. Bouvier M. J. Biol. Chem. 2000; 275: 13727-13736Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 20Pineyro G. Azzi M. De Lean A. Schiller P. Bouvier M. Mol. Pharmacol. 2001; 60: 816-827PubMed Google Scholar). A truncated mutant of the murine δOR (δOR344T) was kindly provided by Dr. M. von Zastrow (University of California at San Francisco). Wild type and inhibitory mutant forms of c-Src (K295R/Y527F) were a gift from Dr. Bouvier's laboratory (Université de Montréal). Cell Culture and Transfection—HEK293s cells were transfected using the calcium-phosphate precipitation method and clones stably expressing full-length or truncated receptors were selected using 400 μg/ml G418. Cell lines stably expressing full-length δORs and wild type c-Src were similarly selected, following Lipofectamine transfection (Invitrogen). The dominant inhibitory form of c-Src (K295R/Y527F) was transiently transfected (0.25–3 μg of DNA) onto cell lines expressing the full-length δOR using polyethyleneimine as described previously (21Boussif O. Lezoualc'h F. Zanta M.A. Mergny M.D. Scherman D. Demeneix B. Behr J.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7297-7301Crossref PubMed Scopus (5594) Google Scholar). Cells were grown and maintained in complete DMEM containing 10% (v/v) fetal bovine serum, 1000 units/ml penicillin, 1 mg/ml streptomycin, and 1.5 μg/ml fungizone in a humidified atmosphere of 5% CO2 at 37 °C. Phosphorylation and Immunoprecipitation of FLAG-tagged Receptors—For 32P incorporation assays, cells were incubated for 2 h in phosphate-free DMEM, after which [32P]orthophosphoric acid was added at a final concentration of 1 mCi/ml, and incubation was allowed to proceed for an additional hour. At this time, DPDPE (1 μm), TICP (1 μm), or vehicle (0.01% Me2SO) were added to the incubation medium for 30 min. Cells were then recovered, and membranes were prepared as indicated below and finally suspended in solubilization buffer (0.5% n-dodecyl-maltoside (w/v), 25 mm Tris-HCl, pH 7.4, 2 mm EDTA, 140 mm NaCl, 5 μg/ml leupeptin, 5 μg/ml soybean trypsin inhibitor, 10 μg/ml benzamidine, 2 μg/ml aprotinin, 0.5 mm phenylmethylsulfonyl fluoride, and 2 mm 1,10-phenantroline). Following agitation at 4 °C for 60 min, the solubilized fraction was centrifuged at 12,000 × g for 60 min, and the receptor was immunoprecipitated from the supernatant fraction using anti-FLAG M2 antibody resin. 40 μl of antibody-coupled resin equilibrated in solubilization buffer and supplemented with 0.1% bovine serum albumin (w/v) were used to purify the receptor overnight at 4 °C under gentle agitation. The next morning the resin was pelleted, washed twice with 500 μl of solubilization buffer and four times with 500 μl of modified solubilization buffer (containing 0.1% instead of 0.5% n-dodecyl-maltoside (w/v)). The receptor was then eluted by incubating the resin for 10 min at 4 °C with 100 μl of modified solubilization buffer containing 175 μg of FLAG peptide/ml. This elution was repeated three times, and the eluates were combined and concentrated by membrane filtration over Microcon-30 concentrators (Millipore). SDS sample buffer was then added, and samples were used for SDS-PAGE. A similar immunoprecipitation procedure was used to assess Tyr phosphorylation of δORs. SDS-PAGE and Western Blotting—SDS-PAGE was performed as described by Laemmli using a 4% stacking gel and 10% separating gel. Proteins resolved in SDS-PAGE were then transferred (50 mA, 16 h, Bio-Rad Mini-Trans Blot apparatus) from the gels onto nitrocellulose (Amersham Biosciences). In the case of 32P incorporation studies, membranes were first exposed for autoradiography (BIOMAX films, Eastman Kodak Co.). When assessing Tyr phosphorylation of δORs, membranes were probed overnight at 4 °C with monoclonal antibodies raised against phosphorylated Tyr (1:500, PY99, Santa Cruz Biotechnology, Santa Cruz, CA). In both cases antisera directed against the FLAG M2 antibody (1:1000, Sigma) were used to detect the total amount of receptor protein present in each sample. Horseradish peroxidase-conjugated antimouse secondary antibodies (1:4000, Sigma) and chemiluminescence detection reagents (PerkinElmer Life Sciences) were used to reveal the blotted proteins, and relative intensities of the labeled bands were analyzed by densitometric scanning using MCID (Imaging Research Inc). Receptor phosphorylation was expressed as the ratio between phosphorylation and FLAG signals to normalize to the amount of receptor protein present in each sample. For detection of ERK1/2 activation, cells were grown in 6-well plates and serum-starved overnight. The day of the experiment they were cultured for 2 h in serum-free medium and then exposed to different ligands. Following treatment, cells were washed with ice-cold phosphate-buffered saline, and whole cell extracts were prepared by lysis in SDS sample buffer. Samples were sonicated and then boiled for 5 min before loading for SDS-PAGE. Phospho-ERK1/2 detection was done by probing membranes with antiphospho-ERK1/2 antibody (1:1,000, Santa Cruz Biotechnology). Total ERK protein was determined after stripping by using 1:20,000 dilution anti-ERK1/2 antibody (Santa Cruz Biotechnology). Secondary antimouse (1:5,000, Sigma) and antirabbit (1: 40,000, Amersham Biosciences) horseradish-conjugated antibodies were used to visualize proteins by chemiluminescence. ERK1/2 phosphorylation was normalized according to protein contents by expressing results as the ratio between pERK1/2 and total ERK1/2. To assess Src activation, cells were grown in 100-mm Petri dishes and prepared for the experiment as described for ERK1/2. Following treatment with different ligands cells were washed, harvested, and solubilized in precipitation assay buffer (50 mm Tris-HCl, pH 7.4, 1% Triton X-100, 0.25% deoxycholate acid, 150 mm NaCl, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 5 μg/ml soybean trypsin inhibitor, 10 μg/ml benzamidine, 1 μg/ml aprotinin, 1 mm Na3VO4) at 4 °C for 90 min. After centrifugation of non-solubilized debris at 12,000 × g for 20 min samples were concentrated, suspended in SDS sample buffer, and separated in SDS-PAGE. Anti-phospho-Src (Y416) monoclonal antibody (Upstate Biotechnology Inc.) at a dilution of 1:1000 was used to determine the presence of activated Src and total amount of protein loaded was detected by probing with antibody anti-Src (1:250, Upstate Biotechnology Inc.). cAMP Accumulation Assays—Cells were labeled overnight (16 h) with 1 μCi/ml of [3H]adenine in complete DMEM medium. The day of the experiment radioactive medium was replaced with fresh DMEM, cells were mechanically detached and thoroughly washed (three times) with phosphate-buffered saline (4 °C), and viability was assessed using trypan blue (mortality was never higher than 5%). 5 × 105 cells were then incubated for 20 min at 37 °C in 300 μl of assay mixture containing phosphate-buffered saline, 25 μm forskolin, 2.5 μm isobutylmethylxanthine, and different drugs at the indicated concentrations. At the end of the incubation period, the assay was terminated by adding 600 μl of ice-cold solution containing 5% trichloroacetic acid, 5 mm ATP, and 5 mm cAMP. [3H]ATP and [3H]cAMP were separated by sequential chromatography on Dowex exchange resin and aluminum oxide columns. Results were expressed as the ratio of [3H]cAMP/[3H]ATP plus [3H]cAMP. [35S]GTPγS binding assays were carried out on whole cell membrane preparations as described previously (20Pineyro G. Azzi M. De Lean A. Schiller P. Bouvier M. Mol. Pharmacol. 2001; 60: 816-827PubMed Google Scholar). Cells were suspended in lysis buffer (25 mm Tris-HCl, pH 7.4, 5 mm MgCl2, 2 mm EDTA, 5 μg/ml leupeptin, 5 μg/ml soybean trypsin inhibitor, and 10 μg/ml benzamidine) and homogenized with a Polytron homogenizer (Ultra-Turrax T-25, Janke and Kunkel) using three bursts of 5 s at maximum setting. Homogenates were centrifuged at 700 × g for 5 min, and the supernatant was further centrifuged at 27,000 × g for 20 min. Pellets were washed twice in lysis buffer and were immediately resuspended in [35S]GTPγS assay buffer (50 mm Hepes, 200 mm NaCl, 1 mm EDTA, 5 mm MgCl2, 1 mm dithiothreitol, 0.5% bovine serum albumin, and 3 μm GDP, pH 7.4) to yield 10 μg of protein/tube. [35S]GTPγS was used at 50 nm, and nonspecific binding was determined in the presence of 100 μm GTP. The test compound SNC-80 was introduced at a final concentration of 100 nm and incubation was allowed to proceed for one hour at RT. The reaction was terminated by rapid filtration onto Whatman GF/C glass filters pre-soaked in water. Filters were washed twice with ice-cold wash buffer (pH 7) containing 50 mm Tris, 5 mm MgCl2, and 50 mm NaCl, and the radioactivity retained was determined by liquid scintillation. Data Analysis—Statistical analysis and curve fitting were done using Prism 2.01 (GraphPad, San Diego, CA). Comparison of the Effects of δOR Ligands in cAMP and ERK Signaling Cascades—It has been previously shown that certain ligands for β2ARs display dual efficacy, inducing inverse agonist responses in the cAMP signaling pathway, but producing agonist effects in the ERK cascade (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). To determine whether this type of dual behavior was specific to Gs-coupled receptors or could be extended to receptors coupled to Gi/o proteins, different ligands for the δOR were compared in adenylyl cyclase and ERK signaling pathways. In the cAMP pathway ligands produced effects that spanned the complete spectrum of efficacy ranging from agonism to inverse agonism. At maximally effective concentrations (1 μm) SNC-80 and DPDPE were highly efficacious agonists, morphine, TIPP, and naloxone were partial agonists, while ICI174864 and TICP displayed typical inverse agonist responses. Fig. 1A shows these different ligands ranked according to magnitude and vectorial aspects of their efficacies (SNC-80 ≥ DPDPE > MOR ≥ TIPP ≥ Nx > ICI174864 > TICP). In contrast with the diversity of responses observed in cAMP accumulation assays all drugs tested in the ERK cascade behaved as agonists, except for naloxone that was neutral. Indeed, ERK phosphorylation was induced not only by drugs that behaved as agonists in the cyclase cascade but also by TICP and ICI174864, which had produced inverse agonist responses when tested in this pathway. Moreover, when ranked according to the magnitude of their effect on ERK phosphorylation, TICP, the most efficacious inverse agonist in the cyclase pathway was now more effective than partial agonists TIPP and morphine in activating ERK (SNC-80 > DPDPE > TICP ≥ TIPP ≥ ICI174864 ≥ MOR > Nx). Dual efficacy ligands for the β2AR produce ERK activation via βarrestin recruitment and independently of G protein activity (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). To determine whether this was also the case for Gi/o-coupled receptors, cells were treated overnight with PTX, and ERK activity was assessed the following day. Without modifying basal activity of the kinase (pERK/ERKtotal ratio in controls: 0.6 ± 0.1; following PTX: 0.6 ± 0.1), PTX abolished ERK stimulation by classic agonist DPDPE and by dual efficacy ligand TICP (Fig. 1C). These results indicate not only that ERK stimulation by DPDPE and TICP requires Gi/o protein activity but also that simple inactivation of spontaneous Gi/o signaling cannot account for ERK stimulation. Neither TICP, ICI174864, nor classic agonists were able to evoke ERK activation in non-transfected cells (not shown), confirming that ligand-induced stimulation of ERK signaling was specifically mediated by the δOR. δOR Ligands Differ in Their Kinetics of ERK Activation—To determine whether the time course of ERK activation by classic agonists differed from that of dual efficacy ligands, cells were exposed to a maximally effective concentration (1 μm) of each drug, and ERK phosphorylation was measured following increasing periods of time. Two main types of kinetic profiles could be recognized. One was characteristic of highly efficacious ligands like SNC-80 and DPDPE, which produced quick and pronounced ERK activation that peaked within 5 min (Fig. 2A), decaying right away with a calculated half-life (t½) of ∼2 min (Fig. 2, B and C). The other type of response, induced by partial agonists and dual efficacy ligands was less pronounced but more sustained, decaying with a t½ that ranged between 11 and 14 min (Fig. 2, B and C). Among ligands inducing sustained responses, the effect of TICP could be distinguished from the rest of the drugs in the same category, because its effect was more pronounced and particularly more sustained (p < 0.001 two-way ANOVA; Fig. 2, A and B). The Time Course of ERK Activation by Highly Efficacious Agonists and Dual Efficacy Ligands Is Correlated with Desensitization Parameters—One of the primary checkpoints that controls drug effects over time is the receptor itself. In particular, δOR signaling efficacy is regulated by phosphorylation of C-terminal Ser/Thr residues (22El Kouhen O.M. Wang G. Solberg J. Erickson L.J. Law P.Y. Loh H.H. J. Biol. Chem. 2000; 275: 36659-36664Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 23Law P.Y. Kouhen O.M. Solberg J. Wang W. Erickson L.J. Loh H.H. J. Biol. Chem. 2000; 275: 32057-32065Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). To determine whether differences in the time course of ERK activation could be related to the distinct ability of different ligands to trigger phosphorylation of δORs cells were exposed for 30 min to DPDPE or TICP (1 μm) in the presence of [32P]orthophosphoric acid. Receptors were immunopurified, resolved on SDS-PAGE, and transferred onto nitrocellulose membranes that were first exposed for autoradiography and then used for Western blot analysis using an anti-FLAG M2 antibody. Immunoblots revealed two broad bands at ≅55 and ≅46 kDa, corresponding to mature and immature monomeric forms of the receptor, respectively (24Petaja-Repo U.E. Hogue M. Laperriere A. Bhalla S. Walker P. Bouvier M. J. Biol. Chem. 2001; 276: 4416-4423Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Autoradiograms showed that 30-min incubation with DPDPE increased 32P incorporation by the ≅55-kDa species, but this effect was absent for TICP. Thus, at a time when the ERK response for the agonist was no longer present, δORs were heavily phosphorylated. In contrast, ERK activation by the dual efficacy ligand was still at its maximum, and no phosphorylation of the receptor could be detected. Phosphorylation is an initial step in the process of desensitization, but if exposure to an agonist is allowed to proceed long enough, δORs will start to be targeted 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). Hence, to confirm whether the different time course of ERK activation by DPDPE and TICP also correlated with later events in the process of desensitization, cells were treated for 2 h either with the agonist or the dual efficacy drug. Following treatment the total amount of δOR protein present in membrane preparations was assessed by immunoblot (Fig. 3B). Although incubation with TICP caused no detectable change in the mature receptor species (≅55 kDa), there was a decrease of the corresponding immunoreactive band following treatment with DPDPE. These results confirm that differences in the time course of ERK activation by DPDPE and TICP is inversely correlated with the ability of each ligand to trigger different events within the process of desensitization. If indeed differences in time course of ERK activation by agonists and dual efficacy ligands were due to their distinct ability to trigger regulatory mechanisms of receptor responsiveness, interfering with these mechanisms should transform ERK activation by the agonist, into the more prolonged type of response observed for the dual efficacy ligand. To test this assumption the time course of ERK activation by DPDPE was assessed in presence of sucrose, which is an inhibitor of clathrin-mediated endocytosis. Although sucrose did not turn ERK activation into a stable response, it prolonged the effect of DPDPE by increasing the decay t½ of activation from 2 to 12 min (p < 0.001 for interaction; two-way ANOVA; Fig. 4), a value that falls within the 11- to 15-min range observed for dual efficacy ligands. Another means to modify mechanisms regulating δOR responsiveness is to mutate amino acids that are implicated in the process. For δORs, Ser/Thr residues located in the C-terminal domain of the receptor are the principal target for G protein-coupled receptor kinases, and their phosphorylation is an essential step in the desensitization of full-length δORs (22El Kouhen O.M. Wang G. Solberg J. Erickson L.J. Law P.Y. Loh H.H. J. Biol. Chem. 2000; 275: 36659-36664Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 23Law P.Y. Kouhen O.M. Solberg J. Wang W. Erickson L.J. Loh H.H. J. Biol. Chem. 2000; 275: 32057-32065Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 25Kovoor A. Nappey V. Kieffer B.L. Chavkin C. J. Biol. Chem. 1997; 272: 27605-27611Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). To explore the contribution of these residues to the kinetics of ERK activation by DPDPE, experiments were repeated using a receptor truncated at its C terminus (δOR344T). This approach also yielded results in which the DPDPE response decayed more slowly than in the full-length receptor (t½ of 6 min; two-way ANOVA; p < 0.01 for interaction; Fig. 4). However, the effect of truncation was far less noticeable than that observed with sucrose on the full-length δOR. Moreover, the addition of sucrose further prolonged the decay t½ for DPDPE in truncated receptors (t½ of 38 min; two-way ANOVA; p < 0.02 for interactio
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