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.