Discovering Cell Type-Specific Patterns of G Protein-Coupled Receptor Phosphorylation
Phosphorylation of G protein-coupled receptors (GPCRs) occurs within seconds of agonist stimulation and is one of the most prevalent mechanisms through which signalling of this super receptor family is regulated. Although traditionally associated with receptor desensitisation and internalisation, there is an increasing body of evidence that suggests that GPCRs employ phosphorylation as a mechanism of coupling the receptor to non-G protein signalling pathways. Recently, it has become clear that GPCRs can be differentially phosphorylated in different cell types or tissues, possibly by tissue- or cell type-specific employment of a variety of receptor kinases to generate specific “phosphorylation patterns” that might encode signalling properties on the receptor. Although hampered by low levels of expression and high hydrophobicity, GPCR phosphorylation can be studied using various methods including two-dimensional (2D) phosphopeptide mapping, mass spectrometry, production of phospho-specific antibodies and site-directed mutagenesis. In this chapter, we discuss the first three methods which are employed in our laboratory for the studies of M3-muscarinic receptor phosphorylation.
Key wordsAntibodies G-protein-coupled receptor Immunoprecipitation Phosphorylation Proteomics 2D phosphopeptide maps SDS-PAGE Mass spectrometry
- 17.Kara E, Crepieux P, Gauthier C et al (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
- 23.Benovic JL, Pike LJ, Cerione RA et al (1985) Phosphorylation of the mammalian beta-adrenergic receptor by cyclic AMP-dependent protein kinase. Regulation of the rate of receptor phosphorylation and dephosphorylation by agonist occupancy and effects on coupling of the receptor to the stimulatory guanine nucleotide regulatory protein. J Biol Chem 260:7094–7101.Google Scholar