Post-endocytic fates of delta-opioid receptor are regulated by GRK2-mediated receptor phosphorylation and distinct beta-arrestin isoforms.
Once internalized, some G protein-coupled receptors (GPCRs) can recycle back to the cell surface, while some of them are delivered to lysosomes for degradation. Because recycling and degradation represent two opposing receptor fates, understanding the mechanisms that determine post-endocytic fate of GPCRs is of great importance. Our recent work has verified that agonist-induced internalization of delta-opioid receptor (DOR) employs both phosphorylation-dependent and -independent mechanisms in HEK293 cells. To investigate whether these two internalization mechanisms work differently in receptor regulation, we monitored receptor post-endocytic fates using flow cytometry, surface receptor biotinylation and radioligand binding assays. Results showed that the internalized wild type DOR could either recycle to the cell surface or be degraded. Mutant DOR M4/5/6, which lacks all three G protein-coupled receptor kinase 2 (GRK2) phosphorylation sites, could also internalize upon agonist challenge although in a reduced level as compared with the wild type counterpart. However, the internalized mutant DOR could not recycle back to the cell surface and all mutant DOR was degraded after internalization. Inhibition of GRK2 expression by GRK2 RNAi also strongly attenuated recycling of DOR. Furthermore, overexpression of GRK2, which significantly increased receptor phosphorylation and internalization, also targeted more internalized receptors to the recycling pathway. These data suggest that GRK2-catalyzed receptor phosphorylation is critically involved in DOR internalization and recycling, and the phosphorylation-independent internalization leads to receptor degradation. Data obtained from beta-arrestin1 and beta-arrestin2 RNAi experiments indicated that both beta-arrestin1 and beta-arrestin2 participate in phosphorylation-dependent internalization and the subsequent recycling of DOR. However, phosphorylation-independent internalization and degradation of DOR were strongly blocked by beta-arrestin2 RNAi, but not beta-arrestin1 RNAi. Taken together, these data demonstrate for the first time that GRK2 phosphorylation-dependent internalization mediated by both beta-arrestin1 and beta-arrestin2 leads DOR to recycle, whereas GRK2-independent internalization mediated by beta-arrestin2 alone leads to receptor degradation. Thus, the post-endocytic fate of internalized DOR can be regulated by GRK2-catalyzed receptor phosphorylation as well as distinct beta-arrestin isoforms.
Zhang
Xiaoqing
X
Pharmacology Research Center and National Laboratory of Medical Neurobiology, Shanghai Medical College and Institutes of Brain Science, Fudan University, Shanghai, China.
Wang
Feifei
F
Chen
Xiaoqing
X
Chen
Yuejun
Y
Ma
Lan
L
D000701:Analgesics, Opioid / Q000494:pharmacology; D019390:Arrestins / Q000235:genetics / Q000378:metabolism*; D001710:Biotin / Q000378:metabolism; D002461:Cell Line, Transformed; D004174:Diprenorphine / Q000493:pharmacokinetics; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D020881:Enkephalin, D-Penicillamine (2,5)- / Q000494:pharmacology; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D009154:Mutation / Q000502:physiology; D009292:Narcotic Antagonists / Q000493:pharmacokinetics; D010766:Phosphorylation / Q000187:drug effects; D020033:Protein Isoforms / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D017465:Receptors, Opioid, delta / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013997:Time Factors; D014316:Tritium / Q000493:pharmacokinetics; D000071557:beta-Arrestins
D000701:Analgesics, Opioid; D019390:Arrestins; D009292:Narcotic Antagonists; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D017465:Receptors, Opioid, delta; D000071557:beta-Arrestins; D014316:Tritium; D049452:Green Fluorescent Proteins; D004174:Diprenorphine; D012694:Serine; D001710:Biotin; D020881:Enkephalin, D-Penicillamine (2,5)-; C496567:GRK2 protein, human; D054769:G-Protein-Coupled Receptor Kinase 2