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

Ying-Ju Chen, Sue Oldfield, Adrian J. Butcher, Andrew B. Tobin, Kunal Saxena, Vsevolod V. Gurevich, Jeffrey L. Benovic, Graeme Henderson, Eamonn Kelly*

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)

29 Citations (Scopus)

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 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.
Original languageEnglish
Pages (from-to)189-199
Number of pages11
JournalJournal of Neurochemistry
Volume124
Issue number2
Early online date26 Oct 2012
DOIs
Publication statusPublished - Jan 2013

Keywords

  • arrestins
  • desensitization
  • kinases
  • mass spectrometry
  • phosphorylation
  • mu-opioid receptor
  • PROTEIN-COUPLED RECEPTOR
  • MASS-SPECTROMETRIC ANALYSIS
  • AGONIST-SELECTIVE MECHANISMS
  • BETA-ARRESTIN
  • AMINO-ACIDS
  • KINASE-II
  • 2-MEDIATED DESENSITIZATION
  • BRAIN NEURONS
  • THREONINE 394
  • C-TERMINUS

Fingerprint Dive into the research topics of 'Identification of phosphorylation sites in the COOH-terminal tail of the μ-opioid receptor'. Together they form a unique fingerprint.

  • Projects

    Cite this

    Chen, Y-J., Oldfield, S., Butcher, A. J., Tobin, A. B., Saxena, K., Gurevich, V. V., Benovic, J. L., Henderson, G., & Kelly, E. (2013). Identification of phosphorylation sites in the COOH-terminal tail of the μ-opioid receptor. Journal of Neurochemistry, 124(2), 189-199. https://doi.org/10.1111/jnc.12071