Identification of phosphorylation sites in the COOH‐terminal tail of the μ‐opioid receptor. (30th November 2012)

• Record Type: Journal Article
• Authors: Chen, Ying‐Ju; Oldfield, Sue; Butcher, Adrian J.; Tobin, Andrew B.; Saxena, Kunal; Gurevich, Vsevolod V.; Benovic, Jeffrey L.; Henderson, Graeme; Kelly, Eamonn
• Is Part Of: Journal of neurochemistry. Volume 124:Number 2(2013:Jan.)
• Page Start: 189
• Page End: 199
• Publication Date: 2012-11-30
• Subjects: Neurochemistry -- Periodicals; 616.8042
• DOI: 10.1111/jnc.12071
• Languages: English
• ISSNs: 0022-3042

Abstract

<abstract abstract-type="main" id="jnc12071-abs-0001"> <title>Abstract</title> <p>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, <italic>in vitro</italic>, 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.</p> </abstract>