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, 73 (4), 1319-30

Agonist-promoted Lys63-linked Polyubiquitination of the Human Kappa-Opioid Receptor Is Involved in Receptor Down-Regulation

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Agonist-promoted Lys63-linked Polyubiquitination of the Human Kappa-Opioid Receptor Is Involved in Receptor Down-Regulation

Jian-Guo Li et al. Mol Pharmacol.

Abstract

Ubiquitination of the human kappa opioid receptor (hKOR) expressed in Chinese hamster ovary (CHO) cells was observed in the presence of the proteasomal inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) and enhanced by the agonists (-)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny) cyclohexyl] benzeneacetamide (U50,488H) and dynorphin A (Dyn A). The dominant-negative (DN) mutants GRK2-K220R and beta-arrestin (319-418), but not dynamin I-K44A, reduced Dyn A-stimulated hKOR ubiquitination, and a phosphorylation-defective hKOR mutant (hKOR-S358N) did not undergo Dyn A-stimulated ubiquitination, indicating that hKOR ubiquitination is enhanced by receptor phosphorylation but not by receptor internalization. A hKOR mutant (hKOR-10 KR) in which all 10 intracellular Lys residues were changed to Arg showed greatly reduced basal and agonist-promoted receptor ubiquitination and substantially decreased Dyn A-induced receptor down-regulation, without changing ligand binding affinity, receptor-G protein coupling, or receptor internalization or desensitization. The ubiquitination sites were further determined to be the three Lys residues in the C-terminal domain. The K63R ubiquitin mutant decreased Dyn A-induced hKOR ubiquitination and down-regulation, but the K48R mutant did not. Expression of HN-CYLD, a DN mutant of deubiquitination enzyme cylindromatosis tumor suppressor gene (CYLD) that breaks Lys63-linked polyubiquitin chain, increased Dyn A-induced hKOR ubiquitination and down-regulation. These results indicate that ubiquitinated hKOR after agonist treatment contains predominantly Lys63-linked polyubiquitin chains and ubiquitination of the hKOR involved in agonist-induced down-regulation.

Figures

Fig. 1
Fig. 1. Ubiquitination of the hKOR was enhanced by U50,488H and Dyn A
CHO cells were transiently transfected with HA-Ub and FLAG-hKOR or the vector pcDNA3. Approximately two days later, cells were treated with 50 μM MG132 for 3 h and then without or with 10 μM U50,488H or 1 μM Dyn A for another 30 min. (A) Cells were lysed, solubilized and immunoprecipitated with anti-FLAG M2 affinity gel and western blot was performed using rabbit anti-HA antibody (upper) or rabbit anti-FLAG antibody (lower) as described in Materials and Methods. (B) Cells were lysed, solubilized and immunoprecipitated with anti-HA monoclonal antibody and western blot was performed using rabbit anti-FLAG antibody (upper) or rabbit anti-HA antibody (lower) as described in Materials and Methods. (C) Cells were lysed, solubilized and immunoprecipitated with anti-FLAG M2 affinity gel, treated with 2% SDS Laemmli sample buffer followed by addition of excess Triton and then immunoprecipitation with anti-FLAG M2 affinity gel again. Immnoblotting was performed using rabbit anti-HA antibody (upper) or rabbit anti-FLAG antibody (lower) as described in Materials and Methods. Each figure represents one of three independent experiments performed with similar results.
Fig. 2
Fig. 2. Ubiquitination of the hKOR was regulated by receptor phosphorylation, but not by receptor internalization
(A) (B) CHO-FLAG-hKOR cells were transiently transfected with HA-Ub and dynamin I-K44A, β-arrestin (319-418), GRK2-K220R or pcDNA3. (C) CHO cells were transiently transfected with HA-Ub and FLAG-hKOR or FLAG-hKOR-S358N to similar receptor expression levels. (A) (C) Approximately two days later, cells were treated with 50 μM MG132 for 3 h and then 1 μM Dyn A was added for another 30 min. Cells were lysed, solubilized and immunoprecipitated with anti-FLAG M2 affinity gel and western blot was performed using rabbit anti-HA antibody (upper) or rabbit anti-FLAG antibody (lower) as described in Materials and Methods. Each figure represents one of three independent experiments performed with similar results. (B) Quantitation of results from (A). Densitometric analysis was performed using the OptiQuant software. Each point represents the mean ± s.e.m. of three independent experiments. * P<0.05, compared with the pcDNA3 control group by two-tailed Student’s t test.
Fig. 3
Fig. 3. Mutations of the intracellular Lys residues abolished ubiquitination of the hKOR
(A) Schematic representation of the hKOR and the intracellular Lys residues mutated. A hKOR mutant (hKOR-10 KR) was generated by changing all ten Lys residues to Arg (at positions 89, 91, 165, 174, 176, 254, 265, 338, 349, and 378) denoted by Asterisks in the intracellular loops and C-terminal domain. (B) CHO cells were transiently transfected with HA-Ub and FLAG-hKOR or FLAG-hKOR-10KR 3 as indicated. Approximately two days later, cells were treated with 50 μM MG132 for 3 h and then without or with 1 μM U50,488H or 1 μM Dyn A for another 30 min. The cells were lysed, solubilized and immunoprecipitated with anti-FLAG M2 affinity gel and western blot was performed using rabbit anti-HA antibody (upper) or rabbit anti-FLAG antibody (lower) as described in Materials and Methods. Each figure represents one of three independent experiments performed with similar results.
Fig. 4
Fig. 4. Mutation of the hKOR ubiquitination sites did not change agonist-induced [35S]GTP γS binding and desensitization
CHO-FLAG-hKOR or CHO-FLAG-hKOR-10KR cells were incubated without or with 1 μM U50,488H for 30 min and washed. Membranes were prepared and U50,488H-stimulated [35S]GTPγS binding were determined as described in Materials and Methods. The EC50 and Emax values were showed in the table. Each value represents the mean ± s.e.m. of three independent experiments. * P<0.05, compared with the control group by two-tailed Student’s t test.
Fig. 5
Fig. 5. Mutation of the hKOR ubiquitination sites decreased agonist-induced receptor down-regulation, but did not affect agonist-induced internalization
CHO-FLAG-hKOR or CHO-FLAG-hKOR-10KR cells were treated without or with 1 μM Dyn A for (A) 4 h or (B) indicated time periods for down-regulation or (C) with 10 μM U50,488H for 30 min for internalization. Receptor down-regulation was determined by (A) immunoblotting with anti-FLAG antibody followed by quantitation of the 55-kDa form or (B) fluorescence flow cytometry of cell surface receptors as described in Materials and Methods. (C) For agonist-induced internalization, cell surface receptors were determined using the fluorescence flow cytometry assay. Each value represents the mean ± s.e.m. of three independent experiments. * P<0.05, compared with the control group by two-tailed Student’s t test.
Fig. 6
Fig. 6. Mutations of Lys residues in C-tail of hKOR greatly reduced Dyn A-induced receptor ubiquitination and down-regulation
(A) CHO cells were transiently transfected with HA-Ub and FLAG-hKOR or FLAG-hKOR-3KR in which all three Lys residues in the C-terminal domain (at positions 338, 349, and 378) were changed to Arg residues. Approximately two days later, cells were treated with 50 μM MG132 for 3 h and then cells were treated with 1 μM Dyn A for another 30 min. The cells were lysed, solubilized and immunoprecipitated with anti-FLAG M2 affinity gel and western blot was performed using rabbit anti-HA antibody (upper) or rabbit anti-FLAG antibody (lower) as described in Materials and Methods. Each figure represents one of three independent experiments performed with similar results. (B) CHO-FLAG-hKOR and CHO cells stably expressing FLAG-hKOR-3KR (CHO-FLAG-hKOR-3KR) were treated without or with 1 μM Dyn A for 4 h and then receptor down-regulation was determined fluorescence flow cytometry of cell surface receptors as described in Materials and Methods. Each value represents the mean ± s.e.m. of three independent experiments. * P<0.05, compared with the control group by two-tailed Student’s t test.
Fig. 7
Fig. 7. Effects of Ub-K63R and Ub-K48R mutants on ubiquitination and down-regulation of the hKOR
CHO-FLAG-hKOR cells were transiently transfected with HA-Ub wildtype or its K63R (HA-Ub-K63R) or K48R mutant (HA-Ub-K48R). (A) Approximately two days later, cells were treated with 50 μM MG132 for 3 h and then 1 μM Dyn A was added for another 30 min. Cells were lysed, solubilized and immunoprecipitated with anti-FLAG M2 affinity gel and western blot was performed using rabbit anti-HA antibody (upper and middle) or rabbit anti-FLAG antibody (lower) as described in Materials and Methods. Each figure represents one of three independent experiments performed with similar results. (B) About two days after transfection, cells were treated without or with 1 μM Dyn A for 4 h. Receptor down-regulation was determined by fluorescence flow cytometry of cell surface receptors as described in Materials and Methods. Each value represents the mean ± s.e.m. of three independent experiments. * P<0.05, compared with the control group by two-tailed Student’s t test.
Fig. 8
Fig. 8. Effect of HN-CYLD mutant on ubiquitination and down-regulation of the hKOR
CHO-FLAG-hKOR cells were transiently transfected with HA-Ub and HN-CYLD or pcDNA3. (A) Approximately two days later, cells were treated with 50 μM MG132 for 3 h and then 1 μM Dyn A was added for another 30 min. Cells were lysed, solubilized and immunoprecipitated with anti-FLAG M2 affinity gel and western blot was performed using rabbit anti-HA antibody (upper) or rabbit anti-FLAG antibody (lower) as described in Materials and Methods. Each figure represents one of three independent experiments performed with similar results. (B) About two days after transfection, cells were treated without or with 1 μM Dyn A for 2 or 4 hrs. Receptor down-regulation was determined by fluorescence flow cytometry as described in Materials and Methods. Each value represents the mean ± s.e.m. of three independent experiments. * P<0.05, compared with the control group by two-tailed Student’s t test.

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