Identification of phosphorylation sites in the COOH-terminal tail of the μ-opioid receptor

Authors


Address correspondence and reprint requests to Eamonn Kelly, School of Physiology and Pharmacology, Medical Sciences Building, University Walk, University of Bristol, Bristol BS8 1TD, UK. E-mail: e.kelly@bristol.ac.uk

Abstract

Phosphorylation is considered a key event in the signalling and regulation of the μ opioid receptor (MOPr). Here, we used mass spectroscopy to determine the phosphorylation status of the C-terminal tail of the rat MOPr expressed in human embryonic kidney 293 (HEK-293) cells. Under basal conditions, MOPr is phosphorylated on Ser363 and Thr370, while in the presence of morphine or [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO), the COOH terminus is phosphorylated at three additional residues, Ser356, Thr357 and Ser375. Using N-terminal glutathione S transferase (GST) fusion proteins of the cytoplasmic, C-terminal tail of MOPr and point mutations of the same, we show that, in vitro, purified G protein-coupled receptor kinase 2 (GRK2) phosphorylates Ser375, protein kinase C (PKC) phosphorylates Ser363, while CaMKII phosphorylates Thr370. Phosphorylation of the GST fusion protein of the C-terminal tail of MOPr enhanced its ability to bind arrestin-2 and -3. Hence, our study identifies both the basal and agonist-stimulated phospho-acceptor sites in the C-terminal tail of MOPr, and suggests that the receptor is subject to phosphorylation and hence regulation by multiple protein kinases.

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