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

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 HEK-293 cells. Under basal conditions, MOPr is phosphorylated on Ser(363) and Thr(370) , while in the presence of morphine or [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO), the COOH-terminus is phosphorylated at three additional residues, Ser(356) , Thr(357) , and Ser(375) . 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 Ser(375) , PKC phosphorylates Ser(363) whilst CaMKII phosphorylates Thr(370) . 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.