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

Chen YJ, Oldfield S, Butcher AJ, Tobin AB, Saxena K, Gurevich VV, Benovic JL, Henderson G, Kelly E
J Neurochem. 2013 124 (2): 189-99

PMID: 23106126 · PMCID: PMC4226418 · DOI:10.1111/jnc.12071

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 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), protein kinase C (PKC) phosphorylates Ser(363), while 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.

© 2012 International Society for Neurochemistry.

MeSH Terms (12)

5' Flanking Region Amino Acid Sequence Animals HEK293 Cells Humans Molecular Sequence Data Phosphorylation Protein Kinases Protein Structure, Tertiary Protein Transport Rats Receptors, Opioid, mu

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