title: Molecular basis of differences in (-)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)-cyclohexyl]benzeneacetamide-induced desensitization and phosphorylation between human and rat kappa-opioid receptors expressed in Chinese hamster ovary cells.
abstract: The agonist (-)(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)-cyclohexyl]benzeneacetamide [(-)U50,488H] caused desensitization of the human kappa-opioid receptor (hkor) and Flag-tagged hkor (Flag-hkor) but not the rat kappa-opioid receptor (rkor) and Flag-tagged rkor (Flag-rkor) stably expressed in CHO cells as assessed by guanosine 5'-O-(3-[35S]thiotriphosphate) binding. In addition, (-)U50,488H stimulation enhanced phosphorylation of the Flag-hkor, but not Flag-rkor. (-)U50,488H-induced phosphorylation of the Flag-hkor was reduced by expression of the dominant negative mutant GRK2-K220R, demonstrating the involvement of G protein-coupled receptor kinases (GRKs). However, expression of GRK2 and arrestin-2 or GRK3 and arrestin-3 did not result in desensitization or phosphorylation of the Flag-rkor after (-)U50,488H pretreatment. To understand the molecular basis of the species differences, we constructed two Flag-tagged chimeric receptors, Flag-h/rkor and Flag-r/hkor, in which the C-terminal domains of Flag-hkor and Flag-rkor were switched. When stably expressed in CHO cells, Flag-r/hkor, but not Flag-h/rkor, was desensitized and phosphorylated after exposure to (-)U50,488H, indicating that the C-terminal domain plays a critical role in the differences. We then generated a Flag-hkor mutant, in which S358 was mutated to N (Flag-hkorS358N) and a Flag-rkor mutant, in which N358 was substituted with S (Flag-rkorN358S). Although Flag-hkorS358N was not phosphorylated or desensitized by (-)U50,488H stimulation, Flag-rkorN358S underwent (-)U50,488H-induced desensitization with slightly increased phosphorylation. These results indicate that there are differences in (-)U50,488H-induced desensitization and phosphorylation between the hkor and the rkor. In addition, the C-terminal domain plays a crucial role in these differences and the 358 locus contributes to the differences. Our findings suggest caution in extrapolating studies on kappa-opioid receptor regulation from rats to humans.
authors.affiliation: Department of Pharmacology and Center for Substance Abuse Research, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Li
authors.forename: Jian-Guo
authors.initials: JG
authors.lastname: Li
authors.forename: Chongguang
authors.initials: C
authors.lastname: Chen
authors.forename: Fengqin
authors.initials: F
authors.lastname: Zhang
authors.forename: Lee-Yuan
authors.initials: LY
authors.lastname: Liu-Chen
mesh_terms: D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer / Q000494:pharmacology*; D000595:Amino Acid Sequence; D018712:Analgesics, Non-Narcotic / Q000494:pharmacology*; D000818:Animals; D019390:Arrestins / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004174:Diprenorphine / Q000494:pharmacology; D054770:G-Protein-Coupled Receptor Kinase 3; D015870:Gene Expression / Q000187:drug effects; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D017473:Receptors, Opioid, kappa / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013462:Sulfur Radioisotopes; D014162:Transfection; D014316:Tritium; D051552:beta-Adrenergic Receptor Kinases
chemical_list: D018712:Analgesics, Non-Narcotic; D019390:Arrestins; D010750:Phosphoproteins; D017473:Receptors, Opioid, kappa; D011993:Recombinant Fusion Proteins; D013462:Sulfur Radioisotopes; C082333:arrestin3; D014316:Tritium; D004174:Diprenorphine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D054770:G-Protein-Coupled Receptor Kinase 3; C517848:GRK3 protein, human; C517851:Grk3 protein, rat; D051552:beta-Adrenergic Receptor Kinases