Determination of sites of U50,488H-promoted phosphorylation of the mouse κ opioid receptor (KOPR): disconnect between KOPR phosphorylation and internalization

Abstract

Phosphorylation sites of KOPR following treatment with the selective agonist U50,488H were identified after affinity purification, SDS-PAGE, in-gel digestion with Glu-C and LC-MS/MS. Single- and double-phosphorylated peptides were identified containing phosphorylated S356, T357, T363 and S369 in the C-terminal domain. Antibodies were generated against three phosphopeptides containing pS356/pT357, pT363, and pS369, respectively, and affinity-purified antibodies were found to be highly specific for phospho-KOPR. U50,488H markedly enhanced staining of the KOPR by pT363, pS369 and pS356/pT357 antibodies in immunoblotting, which was blocked by the selective KOPR antagonist norbinaltorphimine. S369 phosphorylation affected T363 phosphorylation and vice versa and T363 or S369 phosphorylation was important for S356/T357 phosphorylation, revealing phosphorylation hierarchy. U50,488H, but not etorphine, promoted robust KOPR internalization, although both were full agonists. U50,488H induced higher degrees of phosphorylation than etorphine at S356/T357, T363 and S369 by immunoblotting. Using SILAC (stable isotope labeling by amino acids in cell culture) and LC-MS/MS, we found that compared with control (C), U50,488H (U) and etorphine (E) KOPR promoted single phosphorylation primarily at T363 and S369 with U/E ratio of 2.5 and 2, respectively. Both induced double phosphorylation at T363+S369 and T357+S369 with ratios of U/E=3.3 and 3.4, respectively. Only U50,488H induced triple phosphorylation at S356+T357+S369. An unphosphorylated KOPR(354–372) fragment containing all the phosphorylation sites was detected with a ratio of C/E/U =1/0.7/0.4, indicating that ~60% and ~30% of the mKOPR are phosphorylated following U50,488H and etorphine, respectively. Thus, KOPR internalization requires receptor phosphorylation above a certain threshold and higher-order KOPR phosphorylation may be disproportionally important.

Agonist-selective recruitment of engineered protein probes and of GRK2 by opioid receptors in living cells

“…In particular, because agonist-induced internalization of KOR requires multi-site phosphorylation on its cytoplasmic tail, and ET is known to stimulate this phosphorylation less strongly than DynA 41 , we considered the possibility that differential biosensor recruitment occurs secondarily to differential phosphorylation. To test this, we measured biosensor recruitment by a mutant KOR lacking all relevant phosphorylation sites in the cytoplasmic tail (KOR-TPD for 'total phosphorylation defective', Figure 2H).…”

Modulation of serotonin transporter function by kappa-opioid receptor ligands

“…U69,593 or U50,488 at 10 μM concentration was chosen for subsequent experiments. Several studies have used this concentration of U50,488 and U69,593 to delineate KOR-triggered signaling in heterologous transfected cell models as well as primary striatal neurons and astrocytes (Bruchas et al, 2006; Chen et al, 2016; Clayton et al, 2009; McLaughlin et al, 2003; Schmid et al, 2013). …”

Agonist-Dependent And -Independent Kappa Opioid Receptor Phosphorylation Showed Distinct Phosphorylation Patterns And Resulted In Different Cellular Outcomes

“…The mKOPR tagged with FLAG at the N-terminus and 6×His-tag at the Cterminus (FmK6H) was cloned into the vector pcDNA6. FmK6H was stably transfected into N2A cells and stable clonal N2A-FmK6H cells were selected (Chen et al, 2016).…”

“…Chavkin and colleagues reported that U50,488H induced rat KOPR (rKOPR) phosphorylation at S369 in human embryonic kidney 293 cells (HEK293 cells) by immunoblotting (McLaughlin et al, 2003). Recently, by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, we determined the phosphorylation sites of the mouse KOPR (mKOPR) following U50,488H treatment to be S356, T357, T363 and S369 in the C-terminal domain (Chen et al, 2016). Antibodies were generated against three phosphopeptides containing pS356/pT357, pT363 or pS369, respectively.…”

“…Antibodies were generated against three phosphopeptides containing pS356/pT357, pT363 or pS369, respectively. Affinity-purified antibodies were extensively characterized and found to be highly specific in immunoblotting for the mouse KOPR phosphorylated at S356/T357, T363 or S369 (Chen et al, 2016). U50,488H markedly enhanced staining intensity of the KOPR by immunoblotting with these three phospho-specific antibodies and the U50,488H effect was blocked by the selective KOPR antagonist norbinaltorphimine, indicating that U50,488H-induced KOPR phosphorylation at these sites is KOPR-mediated (Chen et al, 2016).…”