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Examining Site-Specific GPCR Phosphorylation

  • Adrian J. Butcher
  • Andrew B. TobinEmail author
  • Kok Choi Kong
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 746)

Abstract

Phosphorylation of G protein-coupled receptors (GPCRs) is one of the most prominent post-translation modifications mediated by agonist stimulation. This process has been shown to result not only in receptor desensitisation but also, via the recruitment of arrestin adaptor proteins, to promote receptor coupling to numerous signalling pathways. Furthermore, there is now a growing body of evidence suggesting that GPCRs may employ phosphorylation as a mechanism to regulate their cell-type-specific signalling, hence generating tissue-specific functions. These advances have resulted partly from improved methods used in the determination of phospho-acceptor sites on GPCRs and improved analysis of the consequences of phosphorylation. This chapter aims to describe the methods used in our laboratory for the investigation of site-specific phosphorylation of the M3-muscarinic receptor. These methods could easily be applied in the study of other receptors.

Key words

Antibodies G protein-coupled receptor Immunoprecipitation Phosphorylation Proteomics 2D phosphopeptide maps SDS–PAGE Mass spectrometry 

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Adrian J. Butcher
  • Andrew B. Tobin
    • 1
    Email author
  • Kok Choi Kong
  1. 1.Department of Cell Physiology and Pharmacology and the Protein and Nucleic Acid Chemistry LaboratoryUniversity of LeicesterLeicesterUK

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