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We recently demonstrated that the selective cannabinoid receptor antagonist SR 144528 acts as an inverse agonist that blocks constitutive mitogen-activated protein kinase activity coupled to the spontaneous autoactivated peripheral cannabinoid receptor (CB2) in the Chinese hamster ovary cell line stably transfected with human CB2. In the present report, we studied the effect of SR 144528 on CB2 phosphorylation. The CB2 phosphorylation status was monitored by immunodetection using an antibody specific to the COOH-terminal CB2 which can discriminate between phosphorylated and non-phosphorylated CB2 isoforms at serine 352. We first showed that CB2 is constitutively active, phosphorylated, and internalized at the basal level. By blocking autoactivated receptors, inverse agonist SR 144528 treatment completely inhibited this phosphorylation state, leading to an up-regulated CB2receptor level at the cell surface, and enhanced cannabinoid agonist sensitivity for mitogen-activated protein kinase activation of Chinese hamster ovary-CB2 cells. After acute agonist treatment, serine 352 was extensively phosphorylated and maintained in this phosphorylated state for more than 8 h after agonist treatment. The cellular responses to CP-55,940 were concomitantly abolished. Surprisingly, CP-55,940-induced CB2 phosphorylation was reversed by SR 144528, paradoxically leading to a non-phosphorylated CB2 which could then be fully activated by CP-55,940. The process of CP-55,940-induced receptor phosphorylation followed by SR 144528-induced receptor dephosphorylation kept recurring many times on the same cells, indicating that the agonist switches the system off but the inverse agonist switches the system back on. Finally, we showed that autophosphorylation and CP-55,940-induced serine 352 CB2 phosphorylation involve an acidotropic GRK kinase, which does not use Giβγ. In contrast, SR 144528-induced CB2 dephosphorylation was found to involve an okadaic acid and calyculin A-sensitive type 2A phosphatase. We recently demonstrated that the selective cannabinoid receptor antagonist SR 144528 acts as an inverse agonist that blocks constitutive mitogen-activated protein kinase activity coupled to the spontaneous autoactivated peripheral cannabinoid receptor (CB2) in the Chinese hamster ovary cell line stably transfected with human CB2. In the present report, we studied the effect of SR 144528 on CB2 phosphorylation. The CB2 phosphorylation status was monitored by immunodetection using an antibody specific to the COOH-terminal CB2 which can discriminate between phosphorylated and non-phosphorylated CB2 isoforms at serine 352. We first showed that CB2 is constitutively active, phosphorylated, and internalized at the basal level. By blocking autoactivated receptors, inverse agonist SR 144528 treatment completely inhibited this phosphorylation state, leading to an up-regulated CB2receptor level at the cell surface, and enhanced cannabinoid agonist sensitivity for mitogen-activated protein kinase activation of Chinese hamster ovary-CB2 cells. After acute agonist treatment, serine 352 was extensively phosphorylated and maintained in this phosphorylated state for more than 8 h after agonist treatment. The cellular responses to CP-55,940 were concomitantly abolished. Surprisingly, CP-55,940-induced CB2 phosphorylation was reversed by SR 144528, paradoxically leading to a non-phosphorylated CB2 which could then be fully activated by CP-55,940. The process of CP-55,940-induced receptor phosphorylation followed by SR 144528-induced receptor dephosphorylation kept recurring many times on the same cells, indicating that the agonist switches the system off but the inverse agonist switches the system back on. Finally, we showed that autophosphorylation and CP-55,940-induced serine 352 CB2 phosphorylation involve an acidotropic GRK kinase, which does not use Giβγ. In contrast, SR 144528-induced CB2 dephosphorylation was found to involve an okadaic acid and calyculin A-sensitive type 2A phosphatase. Two cannabinoid receptors have been characterized so far: the central cannabinoid receptor (CB1) 1The abbreviations CB1central cannabinoid receptorCB2peripheral cannabinoid receptorCHOChinese hamster ovaryGPCRG-protein-coupled receptorMAPKmitogen-activated protein kinasePTXBordetella pertussis toxin, Δ9-THC, Δ9-tetrahydrocannabinolTBSTTris-buffered saline with Tween 20PKCprotein kinase CPKAcAMP-dependent protein kinaseGRKG-protein coupled receptor kinase 1The abbreviations CB1central cannabinoid receptorCB2peripheral cannabinoid receptorCHOChinese hamster ovaryGPCRG-protein-coupled receptorMAPKmitogen-activated protein kinasePTXBordetella pertussis toxin, Δ9-THC, Δ9-tetrahydrocannabinolTBSTTris-buffered saline with Tween 20PKCprotein kinase CPKAcAMP-dependent protein kinaseGRKG-protein coupled receptor kinaseprimarily expressed in brain tissue (1Matsuda L.A. Lolait S.J. Brownstein M.J. Young A.C. Bonner T.I. Herkenham M. Lynn A.B. Johnson M.R. Melvin L.S. de Costa B.R. Rice K.C. J. Neurosci. 1990; 346: 561-564Google Scholar, 2Herkenham M. Lynn A.B. Johnson M.R. Melvin L.S. de Costa B.R. Rice K.C. J. Neurosci. 1991; 11: 563-583Crossref PubMed Google Scholar, 3Matsuda L.A. Bonner T.I. Lolait S.J. J. Comp. Neurol. 1993; 327: 535-550Crossref PubMed Scopus (531) Google Scholar) and the peripheral cannabinoid receptor (CB2) expressed in the immune system but not in the brain (4Munro S. Thomas K.L. Abu-Shaar M. J. Neurosci. 1993; 365: 61-65Google Scholar, 5Galiegue S. Mary S. Marchand J. Dussossoy D. Carriere D. Carayon P. Bouaboula M. Shire D. Le Fur G. Casellas P. Eur. J . Biochem. 1995; 232: 54-61Crossref PubMed Scopus (1336) Google Scholar). CB1 is the prime target, accounting for the psychoactive effects of cannabis, while cannabinoid-induced immunomodulation is mainly CB2-mediated. Both CB1 and CB2 receptors belong to the G-protein-coupled receptor (GPCR) superfamily and their stimulation by cannabinoid agonists induces several biological responses, including inhibition of adenylyl cyclase (6Howlett A.C. Mol. Pharmacol. 1985; 27: 429-436PubMed Google Scholar, 7Felder C.C. Joyce K.E. Briley E.M. Mansouri J. Mackie K. Blond O. Lai Ma A.L. Mitchell R.L. Mol. Pharmacol. 1995; 48: 443-450PubMed Google Scholar), activation of mitogen-activated protein kinases (8Bouaboula M. Poinot-Chazel C. Bourrie B. Canat X. Calandra B. Rinaldi-Carmona M. Le Fur G. Casellas P. Biochem. J. 1995; 312: 637-641Crossref PubMed Scopus (456) Google Scholar, 9Bouaboula M. Poinot-Chazel C. Marchand J. Canat X. Bourrie B. Rinaldi-Carmona M. Calandra B. Le Fur G. Casellas P. Eur. J. Biochem. 1996; 237: 704-711Crossref PubMed Scopus (240) Google Scholar), induction of immediate-early gene Krox 24 in vitro (9Bouaboula M. Poinot-Chazel C. Marchand J. Canat X. Bourrie B. Rinaldi-Carmona M. Calandra B. Le Fur G. Casellas P. Eur. J. Biochem. 1996; 237: 704-711Crossref PubMed Scopus (240) Google Scholar, 10Bouaboula M. Bourrie B. Rinaldi-Carmona M. Shire D. Le Fur G. Casellas P. J. Biol. Chem. 1995; 270: 13973-13980Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), the latter has also been observedin vivo (11Mailleux P. Verslype M. Preud'homme X. Vanderhaeghen J.J. Neuroreport. 1994; 5: 1265-1268Crossref PubMed Scopus (54) Google Scholar, 12Glass M. Dragunow M. Neuroreport. 1995; 6: 241-244Crossref PubMed Scopus (31) Google Scholar). All of these actions appear to be exerted through one or more members of the PTX-sensitive Gi family of GTP-binding regulatory proteins that comprises Gi, Go. While synthetic (CP-55, 940, WIN55212–2) cannabinoid ligands cannot discriminate between CB1 and CB2receptors, selective antagonists have recently been developed that specifically target either CB1 (SR141716) (13Rinaldi-Carmona M. Barth F. Heaulme M. Shire D. Calandra B. Congy C. Martinez S. Maruani J. Neliat G. Caput D. Pei G. Samama P. Lohse M. Wang M. Codina J. Lefkowitz R.J. FEBS Lett. 1994; 350: 240-244Crossref PubMed Scopus (1627) Google Scholar, 14Rinaldi-Carmona M. Pialot F. Congy C. Redon E. Barth F. Bachy A. Breliere J.C. Soubrie P. Le Fur G. Life Sci. 1996; 58: 1239-1247Crossref PubMed Scopus (117) Google Scholar) or CB2 receptors (SR 144528) (15Rinaldi-Carmona M. Barth F. Millan J. Derocq J.M. Casellas P. Congy C. Oustric D. Sarran M. Bouaboula M. Calandra B. Portier M. Shire D. Breliere J.C. Le Fur G.L. J. Pharmacol. Exp. Ther. 1998; 284: 644-650PubMed Google Scholar).Several studies revealed that receptor activation can occur spontaneously in the absence of an agonist. This discovery led to a reclassification of antagonists as neutral antagonists or inverse agonists. Neutral antagonists block agonist action without any effect on constitutive activity (16Costa T. Herz A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7321-7325Crossref PubMed Scopus (477) Google Scholar, 17Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (754) Google Scholar, 18Chidiac P. Hebert T.E. Valiquette M. Dennis M. Bouvier M. Mol. Pharmacol. 1994; 45: 490-499PubMed Google Scholar, 19Bond R.A. Leff P. Johnson T.D. Milano C.A. Rockman H.A. McMinn T.R. Apparsundaram S. Hyek M.F. Kenakin T.P. Allen L.F. J. Neurosci. 1995; 374: 272-276Google Scholar), whereas agonists block agonist action but also suppress constitutive activity.We recently demonstrated the agonist-independent activity of CB1 and CB2 receptors expressed in mammalian cells following transfection (20Bouaboula M. Perrachon S. Milligan L. Canat X. Rinaldi-Carmona M. Portier M. Barth F. Calandra B. Pecceu F. Lupker J. Maffrand J.P. Le, Fur G. Casellas P. J. Biol. Chem. 1997; 272: 22330-22339Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 21Bouaboula M. Desnoyer N. Carayon P. Combes T. Casellas P. Mol. Pharmacol. 1999; 55: 473-480PubMed Google Scholar). We also showed that the CB1 antagonist SR141716 and the CB2 antagonist SR 144528 not only block the actions of cannabinoid agonists but they also suppress the constitutive activity of these receptors, indicating that these molecules act as inverse agonists. Furthermore, we revealed for the first time a novel property of these inverse agonists. We demonstrated that they also switch off the activation induced by other unrelated Gi-dependent receptors such as insulin or insulin-like growth factor-1 receptors, strongly suggesting that the biological functions of inverse agonists have been underestimated.In the present study, we investigated the effect of inverse agonists on the desensitization process. It is clearly established that GPCR functionality and expression are dynamically regulated after agonist exposure. Cell exposure to agonists causes the desensitization and sequestration of the receptors. Phosphorylation by serine/threonine kinases and their subsequent binding to members of a family of cytosolic proteins were shown to be key factors for uncoupling the receptor and its cognate G protein and receptor sequestration (22Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar). Here we used the constitutively active CB2 stably expressed in the CHO cell line as a cellular model to study the effects of inverse agonists on phosphorylation, cell surface receptor modulation, and CB2 biological responses. The CB2phosphorylation status was monitored by immunodetection using a phosphorylation state-specific antibody. We showed that the inverse agonist SR 144528 inhibits phosphorylation of the constitutively autoactivated CB2. In addition, we found that SR 144528 induced extensive CB2 dephosphorylation of agonist-induced CB2 phosphorylation. These data provide new insight into the relationship between inverse agonists and the phosphorylation/desensitization process. Two cannabinoid receptors have been characterized so far: the central cannabinoid receptor (CB1) 1The abbreviations CB1central cannabinoid receptorCB2peripheral cannabinoid receptorCHOChinese hamster ovaryGPCRG-protein-coupled receptorMAPKmitogen-activated protein kinasePTXBordetella pertussis toxin, Δ9-THC, Δ9-tetrahydrocannabinolTBSTTris-buffered saline with Tween 20PKCprotein kinase CPKAcAMP-dependent protein kinaseGRKG-protein coupled receptor kinase 1The abbreviations CB1central cannabinoid receptorCB2peripheral cannabinoid receptorCHOChinese hamster ovaryGPCRG-protein-coupled receptorMAPKmitogen-activated protein kinasePTXBordetella pertussis toxin, Δ9-THC, Δ9-tetrahydrocannabinolTBSTTris-buffered saline with Tween 20PKCprotein kinase CPKAcAMP-dependent protein kinaseGRKG-protein coupled receptor kinaseprimarily expressed in brain tissue (1Matsuda L.A. Lolait S.J. Brownstein M.J. Young A.C. Bonner T.I. Herkenham M. Lynn A.B. Johnson M.R. Melvin L.S. de Costa B.R. Rice K.C. J. Neurosci. 1990; 346: 561-564Google Scholar, 2Herkenham M. Lynn A.B. Johnson M.R. Melvin L.S. de Costa B.R. Rice K.C. J. Neurosci. 1991; 11: 563-583Crossref PubMed Google Scholar, 3Matsuda L.A. Bonner T.I. Lolait S.J. J. Comp. Neurol. 1993; 327: 535-550Crossref PubMed Scopus (531) Google Scholar) and the peripheral cannabinoid receptor (CB2) expressed in the immune system but not in the brain (4Munro S. Thomas K.L. Abu-Shaar M. J. Neurosci. 1993; 365: 61-65Google Scholar, 5Galiegue S. Mary S. Marchand J. Dussossoy D. Carriere D. Carayon P. Bouaboula M. Shire D. Le Fur G. Casellas P. Eur. J . Biochem. 1995; 232: 54-61Crossref PubMed Scopus (1336) Google Scholar). CB1 is the prime target, accounting for the psychoactive effects of cannabis, while cannabinoid-induced immunomodulation is mainly CB2-mediated. Both CB1 and CB2 receptors belong to the G-protein-coupled receptor (GPCR) superfamily and their stimulation by cannabinoid agonists induces several biological responses, including inhibition of adenylyl cyclase (6Howlett A.C. Mol. Pharmacol. 1985; 27: 429-436PubMed Google Scholar, 7Felder C.C. Joyce K.E. Briley E.M. Mansouri J. Mackie K. Blond O. Lai Ma A.L. Mitchell R.L. Mol. Pharmacol. 1995; 48: 443-450PubMed Google Scholar), activation of mitogen-activated protein kinases (8Bouaboula M. Poinot-Chazel C. Bourrie B. Canat X. Calandra B. Rinaldi-Carmona M. Le Fur G. Casellas P. Biochem. J. 1995; 312: 637-641Crossref PubMed Scopus (456) Google Scholar, 9Bouaboula M. Poinot-Chazel C. Marchand J. Canat X. Bourrie B. Rinaldi-Carmona M. Calandra B. Le Fur G. Casellas P. Eur. J. Biochem. 1996; 237: 704-711Crossref PubMed Scopus (240) Google Scholar), induction of immediate-early gene Krox 24 in vitro (9Bouaboula M. Poinot-Chazel C. Marchand J. Canat X. Bourrie B. Rinaldi-Carmona M. Calandra B. Le Fur G. Casellas P. Eur. J. Biochem. 1996; 237: 704-711Crossref PubMed Scopus (240) Google Scholar, 10Bouaboula M. Bourrie B. Rinaldi-Carmona M. Shire D. Le Fur G. Casellas P. J. Biol. Chem. 1995; 270: 13973-13980Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), the latter has also been observedin vivo (11Mailleux P. Verslype M. Preud'homme X. Vanderhaeghen J.J. Neuroreport. 1994; 5: 1265-1268Crossref PubMed Scopus (54) Google Scholar, 12Glass M. Dragunow M. Neuroreport. 1995; 6: 241-244Crossref PubMed Scopus (31) Google Scholar). All of these actions appear to be exerted through one or more members of the PTX-sensitive Gi family of GTP-binding regulatory proteins that comprises Gi, Go. While synthetic (CP-55, 940, WIN55212–2) cannabinoid ligands cannot discriminate between CB1 and CB2receptors, selective antagonists have recently been developed that specifically target either CB1 (SR141716) (13Rinaldi-Carmona M. Barth F. Heaulme M. Shire D. Calandra B. Congy C. Martinez S. Maruani J. Neliat G. Caput D. Pei G. Samama P. Lohse M. Wang M. Codina J. Lefkowitz R.J. FEBS Lett. 1994; 350: 240-244Crossref PubMed Scopus (1627) Google Scholar, 14Rinaldi-Carmona M. Pialot F. Congy C. Redon E. Barth F. Bachy A. Breliere J.C. Soubrie P. Le Fur G. Life Sci. 1996; 58: 1239-1247Crossref PubMed Scopus (117) Google Scholar) or CB2 receptors (SR 144528) (15Rinaldi-Carmona M. Barth F. Millan J. Derocq J.M. Casellas P. Congy C. Oustric D. Sarran M. Bouaboula M. Calandra B. Portier M. Shire D. Breliere J.C. Le Fur G.L. J. Pharmacol. Exp. Ther. 1998; 284: 644-650PubMed Google Scholar). central cannabinoid receptor peripheral cannabinoid receptor Chinese hamster ovary G-protein-coupled receptor mitogen-activated protein kinase Bordetella pertussis toxin, Δ9-THC, Δ9-tetrahydrocannabinol Tris-buffered saline with Tween 20 protein kinase C cAMP-dependent protein kinase G-protein coupled receptor kinase central cannabinoid receptor peripheral cannabinoid receptor Chinese hamster ovary G-protein-coupled receptor mitogen-activated protein kinase Bordetella pertussis toxin, Δ9-THC, Δ9-tetrahydrocannabinol Tris-buffered saline with Tween 20 protein kinase C cAMP-dependent protein kinase G-protein coupled receptor kinase Several studies revealed that receptor activation can occur spontaneously in the absence of an agonist. This discovery led to a reclassification of antagonists as neutral antagonists or inverse agonists. Neutral antagonists block agonist action without any effect on constitutive activity (16Costa T. Herz A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7321-7325Crossref PubMed Scopus (477) Google Scholar, 17Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (754) Google Scholar, 18Chidiac P. Hebert T.E. Valiquette M. Dennis M. Bouvier M. Mol. Pharmacol. 1994; 45: 490-499PubMed Google Scholar, 19Bond R.A. Leff P. Johnson T.D. Milano C.A. Rockman H.A. McMinn T.R. Apparsundaram S. Hyek M.F. Kenakin T.P. Allen L.F. J. Neurosci. 1995; 374: 272-276Google Scholar), whereas agonists block agonist action but also suppress constitutive activity. We recently demonstrated the agonist-independent activity of CB1 and CB2 receptors expressed in mammalian cells following transfection (20Bouaboula M. Perrachon S. Milligan L. Canat X. Rinaldi-Carmona M. Portier M. Barth F. Calandra B. Pecceu F. Lupker J. Maffrand J.P. Le, Fur G. Casellas P. J. Biol. Chem. 1997; 272: 22330-22339Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 21Bouaboula M. Desnoyer N. Carayon P. Combes T. Casellas P. Mol. Pharmacol. 1999; 55: 473-480PubMed Google Scholar). We also showed that the CB1 antagonist SR141716 and the CB2 antagonist SR 144528 not only block the actions of cannabinoid agonists but they also suppress the constitutive activity of these receptors, indicating that these molecules act as inverse agonists. Furthermore, we revealed for the first time a novel property of these inverse agonists. We demonstrated that they also switch off the activation induced by other unrelated Gi-dependent receptors such as insulin or insulin-like growth factor-1 receptors, strongly suggesting that the biological functions of inverse agonists have been underestimated. In the present study, we investigated the effect of inverse agonists on the desensitization process. It is clearly established that GPCR functionality and expression are dynamically regulated after agonist exposure. Cell exposure to agonists causes the desensitization and sequestration of the receptors. Phosphorylation by serine/threonine kinases and their subsequent binding to members of a family of cytosolic proteins were shown to be key factors for uncoupling the receptor and its cognate G protein and receptor sequestration (22Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar). Here we used the constitutively active CB2 stably expressed in the CHO cell line as a cellular model to study the effects of inverse agonists on phosphorylation, cell surface receptor modulation, and CB2 biological responses. The CB2phosphorylation status was monitored by immunodetection using a phosphorylation state-specific antibody. We showed that the inverse agonist SR 144528 inhibits phosphorylation of the constitutively autoactivated CB2. In addition, we found that SR 144528 induced extensive CB2 dephosphorylation of agonist-induced CB2 phosphorylation. These data provide new insight into the relationship between inverse agonists and the phosphorylation/desensitization process.
Bouaboula et al. (Thu,) studied this question.