Phosphorylation of a Carboxyl-terminal Serine within the κ-Opioid Receptor Produces Desensitization and Internalization

It is demonstrated that GRK-mediated phosphorylation of serine 369 mediates rat KOR desensitization and internalization.

Abstract

G-protein receptor kinase and β-arrestin mediated desensitization of the rat κ-opioid receptor (KOR) was previously shown using Xenopus oocyte expression to require serine 369 within the C terminus of KOR. To define the effects of phosphorylation of this residue in desensitization and internalization processes in mammalian expression systems, wild-type KOR-green fluorescent protein (KOR-GFP) and KOR(S369A)-GFP were stably expressed in AtT-20 and HEK293 cells. Using whole-cell patch clamp recording in transfected AtT-20 cells, agonist activation of either κ receptor form produced equivalent activation of the intrinsic G-protein-gated inwardly rectifying potassium channel. Incubation for 60 min with the κ agonist U50,488 (100 nm) desensitized the response in cells expressing wild-type KOR-GFP by 86% but had no effect on KOR(S369A)-GFP-expressing cells. Phosphorylation of serine 369 was detected using a phosphospecific antibody (KOR-P) able to distinguish the phosphorylated form of the receptor. The agonist-induced increase in KOR-P labeling was dose-dependent, blocked by co-treatment with the κ antagonist norbinaltorphimine, and prevented by co-expression of the dominant negative form of the G-protein receptor kinase, GRK2(K220R). In contrast, agonist-induced increase in KOR-P labeling was not evident in KOR(S369A) expressing cells. Prolonged activation resulted in receptor internalization that was also blocked by KOR(S369A) substitution, but interestingly, KOR-P labeling was evident at lower agonist concentrations than required to induce internalization. Following the removal of agonist, receptor dephosphorylation detected by loss of KOR-P labeling was complete within 60 min, could be blocked by okadaic acid, and was not blocked by sucrose inhibition of receptor internalization. These results demonstrate that GRK-mediated phosphorylation of serine 369 mediates rat KOR desensitization and internalization. G-protein receptor kinase and β-arrestin mediated desensitization of the rat κ-opioid receptor (KOR) was previously shown using Xenopus oocyte expression to require serine 369 within the C terminus of KOR. To define the effects of phosphorylation of this residue in desensitization and internalization processes in mammalian expression systems, wild-type KOR-green fluorescent protein (KOR-GFP) and KOR(S369A)-GFP were stably expressed in AtT-20 and HEK293 cells. Using whole-cell patch clamp recording in transfected AtT-20 cells, agonist activation of either κ receptor form produced equivalent activation of the intrinsic G-protein-gated inwardly rectifying potassium channel. Incubation for 60 min with the κ agonist U50,488 (100 nm) desensitized the response in cells expressing wild-type KOR-GFP by 86% but had no effect on KOR(S369A)-GFP-expressing cells. Phosphorylation of serine 369 was detected using a phosphospecific antibody (KOR-P) able to distinguish the phosphorylated form of the receptor. The agonist-induced increase in KOR-P labeling was dose-dependent, blocked by co-treatment with the κ antagonist norbinaltorphimine, and prevented by co-expression of the dominant negative form of the G-protein receptor kinase, GRK2(K220R). In contrast, agonist-induced increase in KOR-P labeling was not evident in KOR(S369A) expressing cells. Prolonged activation resulted in receptor internalization that was also blocked by KOR(S369A) substitution, but interestingly, KOR-P labeling was evident at lower agonist concentrations than required to induce internalization. Following the removal of agonist, receptor dephosphorylation detected by loss of KOR-P labeling was complete within 60 min, could be blocked by okadaic acid, and was not blocked by sucrose inhibition of receptor internalization. These results demonstrate that GRK-mediated phosphorylation of serine 369 mediates rat KOR desensitization and internalization. The use of opioid agonists to produce clinical analgesia is limited by their propensity to induce drug tolerance and dependence (1Gutstein H.B. Akil H. Goodman Gilman A. Hardman J.G. Limbird L.E. Goodman and Gilman's The Pharmacological Basis of Therapeutics. 10th Ed. McGraw-Hill Book Co., New York2001: 569-619Google Scholar). Therefore, regulatory mechanisms responsible for opioid tolerance are of therapeutic interest. Opioid receptor desensitization and internalization are likely to play significant roles in the control of receptor signaling, but the biochemical steps underlying these mechanisms are uncertain. Agonistinduced receptor phosphorylation is thought to mediate both G-protein coupled receptor desensitization and internalization (for a recent review, see Ref. 2Kohout T.A. Lefkowitz R.J. Mol. Pharmacol. 2003; 63: 9-18Crossref PubMed Scopus (348) Google Scholar). However, establishing a direct link between these receptor mechanisms and the phosphorylation of individual amino acid residues on the opioid receptors has been confounded by the large number of potential phosphorylation sites and the practical difficulty of identifying specific phosphorylated sites within the opioid receptors. Direct evidence of κ-opioid receptor (KOR) 1The abbreviations used are: KOR, κ-opioid receptor; GFP, green fluorescent protein; KOR-P, affinity-purified rabbit polyclonal antibody directed to phosphoserine 369 in the rodent κ-opioid receptor; KT-2, affinity-purified rabbit anti-rat/mouse κ-opioid receptor polyclonal antibody; KE-4, affinity-purified rabbit anti-rat/mouse κ-opioid receptor polyclonal antibody; MOR, μ-opioid receptor; GRK, G-protein receptor kinase; nor-BNI, norbinaltorphimine; U50,488, (±)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methane sulfonate hydrate.1The abbreviations used are: KOR, κ-opioid receptor; GFP, green fluorescent protein; KOR-P, affinity-purified rabbit polyclonal antibody directed to phosphoserine 369 in the rodent κ-opioid receptor; KT-2, affinity-purified rabbit anti-rat/mouse κ-opioid receptor polyclonal antibody; KE-4, affinity-purified rabbit anti-rat/mouse κ-opioid receptor polyclonal antibody; MOR, μ-opioid receptor; GRK, G-protein receptor kinase; nor-BNI, norbinaltorphimine; U50,488, (±)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methane sulfonate hydrate. phosphorylation was obtained by immunoprecipitation of 32P-labeled KOR from guinea pig hippocampal slices treated with a KOR-selective agonist (3Appleyard S.M. Patterson T.A. Jin W. Chavkin C. J. Neurochem. 1997; 69: 2405-2412Crossref PubMed Scopus (51) Google Scholar). This increase in KOR phosphorylation after agonist treatment coincided with an equivalent reduction in electrophysiological response to the KOR agonist and a significant reduction in the amount of immunoprecipitated protein detected, possible examples of agonist-induced receptor desensitization and internalization, respectively. However, determining the site of 32P incorporation into the KOR was not possible with this ex vivo model because of the low abundance of receptor in brain and uncertain stoichiometry of 32P labeling. Further evidence correlating KOR control mechanisms to agonist-induced phosphorylation of specific amino acids has come from site-directed mutagenesis and subsequent expression of receptors lacking potential phosphorylation sites in cell lines and heterologous gene expression systems, but this work has yielded contradictory results. For example, electrophysiological studies of Xenopus oocytes expressing wild-type rat KOR (rKOR) demonstrated a G-protein receptor kinase (GRK)and β-arrestin-2-mediated desensitization of the κ agonist response that was blocked by substitution of KOR(S369A) but not KOR(T363A) or KOR(S356A,T357A) (4Appleyard S.M. Celver J. Pineda V. Kovoor A. Wayman G.A. Chavkin C. J. Biol. Chem. 1999; 274: 23802-23807Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). These results suggest that rKOR desensitization results from an agonist-induced phosphorylation of serine 369. Consistent with this finding Joseph and Bidlack (5Joseph D.B. Bidlack J.M. J. Pharmacol. Exp. Ther. 1995; 272: 970-976PubMed Google Scholar) reported that prolonged exposure to κ agonists can produced a substantial reduction in KOR expression in thymoma cells. However, others have reported that κ agonist treatment can produce rKOR desensitization without an increase in phosphorylation or receptor internalization (6Li J.-G. Luo L.Y. Krupnick J.G. Benovic J.L. Liu-Chen L.-Y. J. Biol. Chem. 1999; 274: 12087-12094Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 7Li J. Li J.-G. Chen C. Zhang F. Liu-Chen L.-Y. Mol. Pharmacol. 2002; 61: 73-84Crossref PubMed Scopus (50) Google Scholar). Taken together, the studies cited above suggest that the relationship between agonist-induced phosphorylation of specific sites within KOR and either receptor desensitization or internalization remains unclear. In addition, the direct identification of agonist-induced phosphorylation sites responsible for the desensitization or internalization of KOR has not been achieved. To address this question, we expressed rat KOR-GFP and KOR(S369A)-GFP fusion proteins in AtT-20 cells for electrophysiology studies of receptor desensitization and in HEK293 cells to examine receptor internalization under confocal microscopy. Moreover, we used immunocytochemistry and a newly generated antibody (KOR-P) selective for the phosphorylated form of serine 369 in the rKOR to directly determine whether agonist-induced phosphorylation of rKOR(S369) mediates either κ receptor desensitization or internalization. Chemicals—(±)-U50,488 was obtained from the National Institute on Drug Abuse drug supply program (National Institutes of Health, Bethesda, MD) and Sigma. Dynorphin A-(1–13) was obtained from Phoenix Pharmaceuticals, Inc. (Belmont, CA). All other drugs were purchased from Sigma. Peptides and Antibody Production—A phosphorylated peptide corresponding to the carboxyl-terminal domain of KOR designated KOR-P (RVRNTVQDPAS(PO4)MRD; matching amino acids 359–372) was synthesized and purified by PeptidoGenic Research and Co. (Livermore, CA). This peptide sequence was compared with known proteins using the GenBank™ data base, and no sequences in other proteins were found having >50% homology or four identical amino acids in a row. peptide was coupled to the and KOR-P antibody was produced and affinity-purified (3Appleyard S.M. Patterson T.A. Jin W. Chavkin C. J. Neurochem. 1997; 69: 2405-2412Crossref PubMed Scopus (51) Google Scholar). The the of the and the of the rat were previously (3Appleyard S.M. Patterson T.A. Jin W. Chavkin C. J. Neurochem. 1997; 69: 2405-2412Crossref PubMed Scopus (51) Google Scholar, Patterson T.A. Chavkin C. T.A. J. PubMed Scopus Google Scholar). were at with of either KOR-P peptide peptide identical to or or peptide from the sequence of the μ-opioid in sites were blocked with in for at and the were with with KOR-P to from was to and at under was in were with and for in in the with the in and was and to for min in the The was with the of and was at for of KOR to KOR and and of The for number was into the site of to produce sites for and were to the and of the rat KOR sequence the with the This KOR was and into the and sites of the with Chavkin C. C. Mol. Pharmacol. Google Scholar). The KOR-GFP the to the carboxyl-terminal of KOR. were in KOR-GFP to produce using previously S.M. A. Chavkin C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). All were by for the κ receptors was used to previously S.M. A. Chavkin C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and of KOR-GFP or cells were in with and AtT-20 cells were in with and cell lines expressing KOR-GFP or KOR(S369A)-GFP protein in both of cells were by both cells with of for KOR-GFP or KOR(S369A)-GFP for using and the and expressing under with for HEK293 for AtT-20 expressing were under and under for a of a of Xenopus oocytes were with for the or receptors with expressing the inwardly rectifying potassium previously S.M. A. Chavkin C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and the of KOR-GFP and KOR κ-opioid Xenopus oocytes expressing either fusion protein or rKOR with were used in clamp previously S.M. A. Chavkin C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). AtT-20 cells stably expressing KOR-GFP or KOR(S369A)-GFP were on for were used in patch clamp in the whole-cell with a and in an and potassium were with an and and with In the whole-cell of the patch cells were at and to for the agonist treatment was the in response to agonist after a exposure to and stably expressing KOR-GFP or KOR(S369A)-GFP were in for that for using the dominant negative of HEK293 cells were transfected with both of KOR-GFP and of using the of protein cells were treated at and for immunocytochemistry and confocal previously J. PubMed Google Scholar). or KOR-P was used the antibody with cells, to at in with was used a antibody to the from the expressed protein and KOR-P were or using a confocal with the of internalization by agonist treatment of HEK293 cells, with the confocal were with (National Institutes of Health, Bethesda, MD) for the of within the of cells expressing KOR-GFP or or agonist To the of KOR-GFP receptors by the KOR-P by confocal were into green and antibody using CA). was for number of and a of to green was generated for a of complete and of antibody labeling and green this was by by the of 369 in KOR work with rat KOR expressed in Xenopus oocytes demonstrated the of serine 369 in desensitization of the receptor (4Appleyard S.M. Celver J. Pineda V. Kovoor A. Wayman G.A. Chavkin C. J. Biol. Chem. 1999; 274: 23802-23807Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). However, the of this residue in a mammalian expression was not and the of tolerance in Xenopus oocytes is limited by a of internalization of receptor in this expression not Therefore, we rat KOR and KOR(S369A) receptors and whether the receptor of receptor were expressed in Xenopus activation of wild-type KOR, and KOR(S369A)-GFP and produced and for cells at previously S.M. A. Chavkin C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). These were not and the results suggest that the of to the C terminus of the KOR receptor activation of Moreover, the KOR-GFP expressed in cells receptor expression and cell KOR-GFP expressed in cells that were in potassium under produced a potassium in response to with the κ agonist, U50,488 that was by the activation of KOR(S369A)-GFP produced an equivalent potassium in response to U50,488 and at a to that of the wild-type receptor prolonged agonist (100 U50,488, the potassium response to U50,488 by KOR-GFP was by the KOR(S369A)-GFP response the treatment was This that the KOR(S369A) residue was also for receptor desensitization in a mammalian cell internalization of KOR-GFP was evident with concentrations of U50,488 the lower (100 that produced receptor desensitization not increase receptor internalization compared with cells that desensitization was not to internalization. of to the a to phosphorylation of serine 369 in KOR, we generated and affinity-purified a polyclonal antibody (KOR-P) a acid peptide corresponding to rat a phosphorylated serine 369 KOR-P antibody to in an the than the sequence The KOR-P antibody also to corresponding to the other of the κ receptor and phosphorylated of of and KOR(S369A)-GFP by of labeling in the of KOR-GFP or KOR(S369A)-GFP expressing AtT-20 cells of receptor Therefore, HEK293 cells stably expressing either KOR-GFP or KOR(S369A)-GFP were used in immunocytochemistry with either or KOR-P the The lower evident in HEK293 cells of receptor that both and KOR(S369A)-GFP the antibody labeling the green fluorescent Incubation of cells with U50,488 produced internalization of KOR-GFP but not detected by and of and In contrast, cells expressing KOR-GFP were by KOR-P KOR-P labeling was evident in cells treated with U50,488 min at KOR-P labeling on the cell treatment min, with and However, these concentrations of U50,488 to produce of KOR-GFP internalization, with U50,488 resulted in and internalization of KOR-GFP evident by both KOR-P antibody labeling and These effects were with the KOR-selective antagonist not produce either KOR-P antibody labeling or evidence of receptor In addition, of with U50,488 prevented both receptor and KOR-P antibody labeling in KOR-P labeling and receptor internalization were also evident after treatment with the opioid A-(1–13) and labeling was blocked by with the KOR-P antibody with the corresponding KOR-P peptide prevented antibody labeling U50,488 In addition, HEK293 cells stably expressing KOR(S369A)-GFP to demonstrate KOR-P antibody either or after with U50,488 KOR-P antibody labeling was not evident in HEK293 cells treated with U50,488 The results suggest that KOR-P antibody could the form of KOR and that phosphorylated receptor could be detected at both the and in agonist serine 369 is phosphorylated in a by κ-opioid agonists to mediate receptor internalization. HEK293 cells were stably transfected with KOR-GFP or KOR(S369A)-GFP and were for min at with the drugs above were with affinity-purified KOR-P antibody and with a rabbit antibody using the confocal the of fluorescent at nm) and fluorescent antibody the KOR-P antibody labeling. KOR-P antibody not cells However, with U50,488 produced a increase in KOR-P antibody labeling that with the KOR-GFP receptor of U50,488 also KOR-GFP receptor internalization In contrast, with the KOR-selective antagonist not in KOR-P labeling but blocked the KOR-P antibody labeling and receptor by U50,488 to the effects of U50,488, with A-(1–13) resulted in KOR-P antibody labeling of receptor and internalization of with the KOR-selective antagonist also blocked agonist-induced receptor and the increase in KOR-P antibody labeling of KOR-GFP U50,488 KOR-P labeling of KOR-GFP was by the KOR-P antibody with the peptide corresponding to to with the cells, but agonist-induced internalization of KOR-GFP was agonist-induced in KOR-P antibody labeling of the receptor KOR(S369A)-GFP were not evident in either or to U50,488 Moreover, KOR-P antibody labeling was not detected in cells not transfected with after U50,488 treatment All were on with shown of from of the Phosphorylation of KOR-GFP in HEK293 by a cells stably expressing KOR-GFP with U50,488 for min demonstrated KOR-GFP internalization and KOR-P antibody labeling receptor KOR-P labeling was prevented by with either the protein kinase kinase C the or the C However, in HEK293 cells expressing both KOR-GFP and the dominant negative U50,488 exposure was to induce receptor and KOR-P antibody labeling was These results suggest that agonist-induced phosphorylation of KOR-GFP serine 369 was mediated by a in HEK293 cells. by KOR in HEK293 results that receptor internalization required the serine 369 phosphorylation site and phosphorylation by receptor internalization was for both and the KOR(S369A)-GFP with U50,488 receptor with KOR-GFP on the cell and within the a significant increase in the agonist treatment of cells expressing KOR-GFP In contrast, KOR(S369A)-GFP not agonist-induced receptor and a of the receptor to be in the of KOR(S369A)-GFP no increase in the U50,488 treatment of KOR 369 Phosphorylation by in the and at the phosphorylation of receptors is thought to be removal of the agonist by receptor internalization and dephosphorylation J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C. C. J. Neurochem. 1999; PubMed Scopus Google Scholar). To HEK293 cells stably expressing KOR-GFP were treated with U50,488 at of agonist after min, and to in for to 60 min to KOR-P antibody labeling. to results and in KOR-P labeling and KOR-GFP internalization were evident min after agonist removal KOR-P antibody labeling was evident min after agonist was KOR-P antibody labeling of cells was min after agonist removal and was 60 min after agonist removal of and green that KOR-P labeling the 60 min U50,488 To determine whether receptor internalization was required for cells were treated with sucrose to receptor internalization, and the of KOR-P labeling was of of sucrose not increase in KOR-P antibody internalization was blocked In addition, dephosphorylation was not blocked by KOR-P labeling in cell sucrose However, the of loss of KOR-P labeling was by sucrose that dephosphorylation of KOR be by receptor internalization of KOR-GFP is but not receptor internalization. The of treatment in were to in sucrose okadaic acid or sucrose and okadaic acid and treated with U50,488 and to to 60 min in the in the to the of to green fluorescent from under cells from U50,488 demonstrated of in the min agonist a number of κ-opioid receptors. This of to within 60 cells with sucrose and to U50,488 demonstrated an of agonist removal that to within 60 In contrast, U50,488 treatment of cells with okadaic acid produced a of the of after agonist but the not to in the of the Moreover, okadaic acid and sucrose prevented the in KOR-P antibody labeling a reduction in the of The labeling of KOR-GFP by KOR-P antibody to 60 min after agonist in the of both sucrose and okadaic acid that receptor dephosphorylation have also at the cell of receptor internalization. All were on with of from of To the HEK293 cells stably expressing KOR-GFP and with U50,488 were treated with okadaic acid to the protein and or with a of sucrose and okadaic okadaic acid the of sucrose and okadaic acid treatment the increase in KOR-P antibody labeling after the removal of agonist and acid but not the in KOR-P labeling and In contrast, the of sucrose and okadaic acid prevented the loss of KOR-P antibody labeling 60 min after agonist removal and these results suggest that receptor internalization the dephosphorylation of KOR serine 369 and that receptor dephosphorylation by either protein or at the without internalization. The finding of this is that the rat κ opioid receptor was desensitized by phosphorylation of serine 369 by a phosphorylation was detected by a phosphospecific KOR-P that of the κ-opioid receptor. of for serine 369 blocked both receptor and receptor internalization, processes that Moreover, KOR dephosphorylation was by receptor internalization but could at the This directly demonstrated phosphorylation at a specific site within the opioid receptor and to the steps in the desensitization and KOR in mammalian cells. These are with other an agonist-induced phosphorylation underlying the desensitization of KOR J. Li J.-G. Chen C. Zhang F. Liu-Chen L.-Y. Mol. Pharmacol. 2002; 61: 73-84Crossref PubMed Scopus (50) Google Scholar) and μ-opioid receptors J. Kovoor A. Chavkin C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H.B. H. W. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). receptor desensitization is thought to be a to the internalization and of opioid receptors Mol. Pharmacol. PubMed Scopus Google Scholar, Mol. Pharmacol. Google Scholar). However, the between agonist-induced KOR desensitization and internalization is at unclear. For example, a of rat with U50,488 produced complete desensitization and inhibition of J. Neurochem. PubMed Scopus Google Scholar). subsequent demonstrated that a with U50,488 or to produce a of KOR number in PubMed Scopus Google Scholar). KOR expressed in HEK293 cells to after treatment with J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). In contrast, U50,488 treatment produced a reduction in KOR number on thymoma cells without significant receptor desensitization (5Joseph D.B. Bidlack J.M. J. Pharmacol. Exp. Ther. 1995; 272: 970-976PubMed Google Scholar). in using KOR expressed in cells, KOR internalization and agonists U50,488 not Biol. PubMed Scopus Google Scholar). work with cells expressing KOR demonstrated that U50,488 an increase in 32P incorporation that with desensitization of the but not KOR J. Li J.-G. Chen C. Zhang F. Liu-Chen L.-Y. Mol. Pharmacol. 2002; 61: 73-84Crossref PubMed Scopus (50) Google Scholar). demonstrated that U50,488 internalization of the but not KOR F. Li J. Li J.-G. Liu-Chen L.-Y. J. Pharmacol. Exp. Ther. 2002; PubMed Scopus Google the of KOR phosphorylation in internalization was not directly The that the of agonist-induced rKOR was to the of in the of the agonist-induced phosphorylation in cells J. Li J.-G. Chen C. Zhang F. Liu-Chen L.-Y. Mol. Pharmacol. 2002; 61: 73-84Crossref PubMed Scopus (50) Google Scholar, F. Li J. Li J.-G. Liu-Chen L.-Y. J. Pharmacol. Exp. Ther. 2002; PubMed Scopus Google Scholar). However, direct 32P incorporation into specific phosphorylation sites was not and the substitution of not an agonist-induced desensitization be that phosphorylation site receptor desensitization and The results of the with the of studies on the with lower concentrations of U50,488 to produce KOR internalization but significant κ receptor desensitization in the concentrations of U50,488 KOR internalization. these data receptor internalization was not in the agonist exposure a direct with studies the receptor in this were by fusion are not to the internalization of the KOR. the the KOR-P antibody labeling in an agonist that was specific for the KOR and with receptor to the cell These results suggest that agonist-induced phosphorylation of KOR serine 369 agonist-induced receptor detected by KOR-P antibody with in cell lines Mol. Pharmacol. PubMed Scopus Google Scholar, Mol. Pharmacol. Google Scholar). The agonist-induced phosphorylation of KOR detected by KOR-P labeling was prevented by of KOR-GFP with the dominant negative is thought to in cells Benovic J.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). selective for the protein kinase protein kinase or were in agonist-induced KOR-GFP the possible of other with the KOR be the of both of and substitution of KOR(S369A) to agonist-induced receptor internalization that agonist-induced phosphorylation of KOR-GFP serine 369 in HEK293 cells was mediated by a These results the that a agonist-induced phosphorylation of is required for receptor internalization. dephosphorylation of was by the loss of KOR-P labeling with the of KOR-GFP to the cell is known the or of receptor dephosphorylation in the internalization of opioid to the difficulty in specific identification of receptors with 32P incorporation using transfected that in cell dephosphorylation of agonist H. V. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of or receptors after agonist exposure was shown to require was prevented by okadaic J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C. C. J. Neurochem. 1999; PubMed Scopus Google Scholar). The results suggest that a dephosphorylation at serine 369 was required for the of a dephosphorylation of agonist of KOR-GFP after the of KOR internalization with sucrose that serine receptor at the cell of this is that dephosphorylation can without internalization of the KOR. This with on this on studies of other receptors. For example, agonist-induced desensitization of receptors was by receptor and the of receptor to the cell within of agonist removal Lefkowitz R.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). These were shown to be on receptor phosphorylation and thought to subsequent with the to induce receptor internalization PubMed Scopus Google Scholar). of receptor internalization and have been demonstrated to on agonist-induced phosphorylation of specific receptor with receptor subsequent receptor dephosphorylation in studies receptors J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google receptors A. J. J. Neurochem. PubMed Scopus Google Scholar, A. F. J. J. Pharmacol. Exp. Ther. Google and μ-opioid receptors H. V. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, V. A. J. W. J. Neurochem. 1995; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. W. Mol. Pharmacol. PubMed Scopus Google Scholar). However, at in the of the MOR, this effect to be and the were shown to induce desensitization and internalization, could not induce internalization J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). These results suggest that mechanisms to receptor internalization in the of receptor a recent with desensitized receptors a receptor that receptor J. Pharmacol. Exp. Ther. 2002; PubMed Scopus Google with the the of the of opioid into the of tolerance on a of the the agonist-induced phosphorylation of be to to the of opioid tolerance the of receptor and internalization. The phosphorylation of KOR from guinea pig this (3Appleyard S.M. Patterson T.A. Jin W. Chavkin C. J. Neurochem. 1997; 69: 2405-2412Crossref PubMed Scopus (51) Google Scholar). a with reported a increase in incorporation of 32P that with the desensitization of in H.B. H. W. PubMed Scopus Google Scholar). demonstrate a of U50,488 the to KOR. and C. for the direct of an agonist-induced phosphorylation of underlying κ-opioid tolerance studies using the KOR-P antibody be directed at the phosphorylation of KOR in The results from the demonstrate that agonist-induced phosphorylation of rat KOR serine 369 mediated by a is required for KOR desensitization and internalization in mammalian cell Moreover, dephosphorylation of is in the of the receptor to the cell Taken with these results suggest that the of agonist-induced receptor phosphorylation the regulatory response to agonist that in the of opioid the and rabbit a in the a of the the from a number by The rKOR was obtained from number The was obtained from Benovic