Phosphorylation of a carboxyl-terminal serine within the kappa-opioid receptor produces desensitization and internalization.
G-protein receptor kinase and beta-arrestin mediated desensitization of the rat kappa-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 kappa receptor form produced equivalent activation of the intrinsic G-protein-gated inwardly rectifying potassium channel. Incubation for 60 min with the kappa 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 kappa 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.
McLaughlin
Jay P
JP
Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington 98195-7280, USA.
Xu
Mei
M
Mackie
Ken
K
Chavkin
Charles
C
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D004797:Enzyme-Linked Immunosorbent Assay; D049452:Green Fluorescent Proteins; D006801:Humans; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D015221:Potassium Channels / Q000737:chemistry; D017434:Protein Structure, Tertiary; D051381:Rats; D017473:Receptors, Opioid, kappa / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013696:Temperature; D013997:Time Factors; D014162:Transfection
D018076:DNA, Complementary; D008164:Luminescent Proteins; D010455:Peptides; D015221:Potassium Channels; D017473:Receptors, Opioid, kappa; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012694:Serine