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Post‐endocytic fates of δ‐opioid receptor are regulated by GRK2‐mediated receptor phosphorylation and distinct β‐arrestin isoforms
Author(s) -
Zhang Xiaoqing,
Wang Feifei,
Chen Xiaoqing,
Chen Yuejun,
Ma Lan
Publication year - 2008
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2008.05431.x
Subject(s) - internalization , endocytic cycle , microbiology and biotechnology , phosphorylation , receptor , g protein coupled receptor , beta adrenergic receptor kinase , g protein coupled receptor kinase , enzyme linked receptor , clathrin , biology , arrestin , chemistry , signal transduction , biochemistry , endocytosis
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 δ‐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 β‐arrestin1 and β‐arrestin2 RNAi experiments indicated that both β‐arrestin1 and β‐arrestin2 participate in phosphorylation‐dependent internalization and the subsequent recycling of DOR. However, phosphorylation‐independent internalization and degradation of DOR were strongly blocked by β‐arrestin2 RNAi, but not β‐arrestin1 RNAi. Taken together, these data demonstrate for the first time that GRK2 phosphorylation‐dependent internalization mediated by both β‐arrestin1 and β‐arrestin2 leads DOR to recycle, whereas GRK2‐independent internalization mediated by β‐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 β‐arrestin isoforms.