Proteasome Involvement in Agonist-induced Down-regulation of μ and δ Opioid Receptors*

This study investigated the mechanism of agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged δ and μ receptors with agonists caused a time-dependent decrease in opioid receptor levels assayed by immunoblotting. Pulse-chase experiments using [35S]methionine metabolic labeling indicated that the turnover rate of δ receptors was accelerated 5-fold following agonist stimulation. Inactivation of functional Gi and Go proteins by pertussis toxin-attenuated down-regulation of the μ opioid receptor, while down-regulation of the δ opioid receptor was unaffected. Pretreatment of cells with inhibitors of lysosomal proteases, calpain, and caspases had little effect on μ and δ opioid receptor down-regulation. In marked contrast, pretreatment with proteasome inhibitors attenuated agonist-induced μ and δ receptor down-regulation. In addition, incubation of cells with proteasome inhibitors in the absence of agonists increased steady-state μ and δ opioid receptor levels. Immunoprecipitation of μ and δ opioid receptors followed by immunoblotting with ubiquitin antibodies suggested that preincubation with proteasome inhibitors promoted accumulation of polyubiquitinated receptors. These data provide evidence that the ubiquitin/proteasome pathway plays a role in agonist-induced down-regulation and basal turnover of opioid receptors.

[1]  A. Ciechanover,et al.  The ubiquitin system , 2000, Nature Medicine.

[2]  E. J. Simon,et al.  Tyrosine phosphorylation of the delta-opioid receptor. Evidence for its role in mitogen-activated protein kinase activation and receptor internalization*. , 2000, Biochemical pharmacology.

[3]  G. Dondio,et al.  Pharmacological profiles of selective non-peptidic δ opioid receptor ligands , 2000 .

[4]  M. von Zastrow,et al.  Type-specific Sorting of G Protein-coupled Receptors after Endocytosis* , 2000, The Journal of Biological Chemistry.

[5]  K. Chaturvedi,et al.  μ Opioid receptor: role for the amino terminus as a determinant of ligand binding affinity , 2000 .

[6]  A. Strosberg,et al.  Beta(2)-adrenergic receptor down-regulation. Evidence for a pathway that does not require endocytosis. , 1999, The Journal of biological chemistry.

[7]  Lakshmi A. Devi,et al.  G-protein-coupled receptor heterodimerization modulates receptor function , 1999, Nature.

[8]  H. Loh,et al.  Visualization of time-dependent redistribution of delta-opioid receptors in neuronal cells during prolonged agonist exposure. , 1999, Brain research. Molecular brain research.

[9]  L. Hicke.  Gettin' down with ubiquitin: turning off cell-surface receptors, transporters and channels. , 1999, Trends in cell biology.

[10]  P. Penela,et al.  Degradation of the G Protein-coupled Receptor Kinase 2 by the Proteasome Pathway* , 1998, The Journal of Biological Chemistry.

[11]  P. Breuer,et al.  Stabilization of Mutant 46-kDa Mannose 6-Phosphate Receptors by Proteasomal Inhibitor Lactacystin* , 1998, The Journal of Biological Chemistry.

[12]  J. Wess,et al.  Molecular basis of receptor/G-protein-coupling selectivity. , 1998, Pharmacology & therapeutics.

[13]  A. Gingras,et al.  μ-Opioid Receptor Activates Signaling Pathways Implicated in Cell Survival and Translational Control* , 1998, The Journal of Biological Chemistry.

[14]  J. Whistler,et al.  Morphine-activated opioid receptors elude desensitization by β-arrestin , 1998 .

[15]  Robert J. Lefkowitz,et al.  G Protein-coupled Receptors , 1998, The Journal of Biological Chemistry.

[16]  D. Lissin,et al.  mu-Opioid receptor internalization: opiate drugs have differential effects on a conserved endocytic mechanism in vitro and in the mammalian brain. , 1998, Molecular pharmacology.

[17]  A. Cavalli,et al.  mu Opioid receptor knockout in mice: effects on ligand-induced analgesia and morphine lethality. , 1998, Brain research. Molecular brain research.

[18]  B. Roques,et al.  Disruption of the κ‐opioid receptor gene in mice enhances sensitivity to chemical visceral pain, impairs pharmacological actions of the selective κ‐agonist U‐50,488H and attenuates morphine withdrawal , 1998, The EMBO journal.

[19]  K. Tanaka,et al.  Proteasomes: structure and biology. , 1998, Journal of biochemistry.

[20]  F. Medzihradsky,et al.  Down-regulation of mu-opioid receptor by full but not partial agonists is independent of G protein coupling. , 1997, Molecular pharmacology.

[21]  L. Devi,et al.  Dimerization of the delta opioid receptor: implication for a role in receptor internalization. , 1997, The Journal of biological chemistry.

[22]  D. Lissin,et al.  δ and κ Opioid Receptors Are Differentially Regulated by Dynamin-dependent Endocytosis When Activated by the Same Alkaloid Agonist* , 1997, The Journal of Biological Chemistry.

[23]  Douglas K. Miller,et al.  The Interleukin-1 Receptor-associated Kinase Is Degraded by Proteasomes following Its Phosphorylation* , 1997, The Journal of Biological Chemistry.

[24]  H. Loh,et al.  The μ-opioid receptor down-regulates differently from the δ-opioid receptor: Requirement of a high affinity receptor/G protein complex formation , 1997 .

[25]  M. Y. Khan,et al.  Molecular biology of opioid receptors: recent advances. , 1997, Neuroimmunomodulation.

[26]  S. Law,et al.  Changes in the association of G protein subunits with the cloned mouse delta opioid receptor on agonist stimulation. , 1997, The Journal of pharmacology and experimental therapeutics.

[27]  H. Jansen,et al.  Altered Hematopoiesis, Behavior, and Sexual Function in μ Opioid Receptor–deficient Mice , 1997, The Journal of experimental medicine.

[28]  I. Sora,et al.  Opiate receptor knockout mice define mu receptor roles in endogenous nociceptive responses and morphine-induced analgesia. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[29]  S. Ōmura,et al.  Degradation of the Met tyrosine kinase receptor by the ubiquitin-proteasome pathway , 1997, Molecular and cellular biology.

[30]  E. Kornilova,et al.  Lysosomal Targeting of Epidermal Growth Factor Receptors via a Kinase-dependent Pathway Is Mediated by the Receptor Carboxyl-terminal Residues 1022-1123* , 1996, The Journal of Biological Chemistry.

[31]  G. Milligan,et al.  Agonist activation of p42 and p44 mitogen-activated protein kinases following expression of the mouse delta opioid receptor in Rat-1 fibroblasts: effects of receptor expression levels and comparisons with G-protein activation. , 1996, The Biochemical journal.

[32]  L. Devi,et al.  Sequestration of the delta opioid receptor. Role of the C terminus in agonist-mediated internalization. , 1996, The Journal of biological chemistry.

[33]  Brigitte L. Kieffer,et al.  Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the µ-opioid-receptor gene , 1996, Nature.

[34]  S. Ōmura,et al.  Lactacystin increases LDL receptor level on HepG2 cells. , 1996, Biochemical and biophysical research communications.

[35]  Christopher J. Evans,et al.  Morphine Activates Opioid Receptors without Causing Their Rapid Internalization* , 1996, The Journal of Biological Chemistry.

[36]  M. Obin,et al.  Ubiquitinylation and Ubiquitin-dependent Proteolysis in Vertebrate Photoreceptors (Rod Outer Segments) , 1996, The Journal of Biological Chemistry.

[37]  Howard Riezman,et al.  Ubiquitination of a Yeast Plasma Membrane Receptor Signals Its Ligand-Stimulated Endocytosis , 1996, Cell.

[38]  S. Ōmura,et al.  Degradation Process of Ligand-stimulated Platelet-derived Growth Factor β -Receptor Involves Ubiquitin-Proteasome Proteolytic Pathway * , 1995, The Journal of Biological Chemistry.

[39]  R. Lefkowitz,et al.  Agonist-dependent phosphorylation of the mouse delta-opioid receptor: involvement of G protein-coupled receptor kinases but not protein kinase C. , 1995, Molecular pharmacology.

[40]  R F Standaert,et al.  Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin , 1995, Science.

[41]  Stanley J. Watson,et al.  Opioid-receptor mRNA expression in the rat CNS: anatomical and functional implications , 1995, Trends in Neurosciences.

[42]  L. Sepp-Lorenzino,et al.  Inhibition of the proteolytic activity of the multicatalytic proteinase complex (proteasome) by substrate-related peptidyl aldehydes. , 1994, The Journal of biological chemistry.

[43]  A. Goldberg,et al.  Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules , 1994, Cell.

[44]  Tom Maniatis,et al.  The ubiquitinproteasome pathway is required for processing the NF-κB1 precursor protein and the activation of NF-κB , 1994, Cell.

[45]  H. Loh,et al.  Ability of delta-opioid receptors to interact with multiple G-proteins is independent of receptor density. , 1994, The Journal of biological chemistry.

[46]  Q. Deveraux,et al.  A 26 S protease subunit that binds ubiquitin conjugates. , 1994, The Journal of biological chemistry.

[47]  Lei Yu,et al.  Molecular cloning and functional expression of a mu opioid receptor from rat brain , 1994, Regulatory Peptides.

[48]  G. Bell,et al.  Cloning and functional comparison of kappa and delta opioid receptors from mouse brain. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[49]  B. Carter,et al.  Go mediates the coupling of the mu opioid receptor to adenylyl cyclase in cloned neural cells and brain. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[50]  G. Schultz,et al.  μ and δ opioid receptors differentially couple to G protein subtypes in membranes of human neuroblastoma SH-SY5Y cells , 1993, Neuron.

[51]  R. Edwards,et al.  Cloning of a delta opioid receptor by functional expression. , 1992, Science.

[52]  K. Befort,et al.  The delta-opioid receptor: isolation of a cDNA by expression cloning and pharmacological characterization. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[53]  J. Shabanowitz,et al.  Identification of the regulatory phosphorylation sites in pp42/mitogen‐activated protein kinase (MAP kinase). , 1991, The EMBO journal.

[54]  A. Goldstein,et al.  Multiple opioid receptors: ligand selectivity profiles and binding site signatures. , 1989, Molecular pharmacology.

[55]  K. Chaturvedi,et al.  Structure and regulation of opioid receptors. , 2000, Biopolymers.

[56]  H. Loh,et al.  Molecular mechanisms and regulation of opioid receptor signaling. , 2000, Annual review of pharmacology and toxicology.

[57]  S. Mehdi,et al.  Cell-penetrating inhibitors of calpain. , 1991, Trends in biochemical sciences.