Examining Site-Specific GPCR Phosphorylation
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 wordsAntibodies G protein-coupled receptor Immunoprecipitation Phosphorylation Proteomics 2D phosphopeptide maps SDS–PAGE Mass spectrometry
- 8.Tran, T. M., Friedman, J., Qunaibi, E., Baameur, F., Moore, R. H., and Clark, R. B. (2004) Characterization of agonist stimulation of cAMP-dependent protein kinase and G protein-coupled receptor kinase phosphorylation of the beta2-adrenergic receptor using phosphoserine-specific antibodies. Mol. Pharmacol. 65, 196–206.PubMedCrossRefGoogle Scholar
- 9.Kara, E., Crepieux, P., Gauthier, C., Martinat, N., Piketty, V., Guillou, F., and Reiter, E. (2006) A phosphorylation cluster of five serine and threonine residues in the C-terminus of the follicle-stimulating hormone receptor is important for desensitization but not for beta-arrestin-mediated ERK activation. Mol. Endocrinol. 20, 3014–3026.PubMedCrossRefGoogle Scholar