Document Type

Article

Publication Date

1-2013

Comments

This is the peer reviewed version of the following article: Chen, Y. -., Oldfield, S., Butcher, A. J., Tobin, A. B., Saxena, K., Gurevich, V. V., . . . Kelly, E. (2013). Identification of phosphorylation sites in the COOH-terminal tail of the μ-opioid receptor. Journal of Neurochemistry, 124(2), which has been published in final form at DOI: 10.1111/jnc.12071.. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.

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 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.

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