Abstract
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.
Highlights
The use of opioid agonists to produce clinical analgesia is limited by their propensity to induce drug tolerance and dependence (1)
Prolonged activation resulted in receptor internalization that was blocked by KOR(S369A) substitution, but interestingly, KOR-P labeling was evident at lower agonist concentrations than required to induce internalization
Electrophysiological studies of Xenopus oocytes expressing wild-type rat KOR demonstrated a G-protein receptor kinase (GRK)and -arrestin-2-mediated desensitization of the agonist response that was blocked by substitution of KOR(S369A) but not KOR(T363A) or KOR(S356A,T357A) (4)
Summary
The use of opioid agonists to produce clinical analgesia is limited by their propensity to induce drug tolerance and dependence (1). Electrophysiological studies of Xenopus oocytes expressing wild-type rat KOR (rKOR) demonstrated a G-protein receptor kinase (GRK)and -arrestin-2-mediated desensitization of the agonist response that was blocked by substitution of KOR(S369A) but not KOR(T363A) or KOR(S356A,T357A) (4). Agonist-induced Phosphorylation of KOR-GFP in HEK293 Cells Is Mediated by a GRK—HEK293 cells stably expressing KOR-GFP incubated with 10 M U50,488 for 30 min demonstrated robust KOR-GFP internalization and strong KOR-P antibody labeling (Fig. 5A).
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