Determination of GPCR Phosphorylation Status: Establishing a Phosphorylation Barcode.
G protein-coupled receptors (GPCRs) are rapidly phosphorylated following agonist occupation in a process that mediates receptor uncoupling from its cognate G protein, a process referred to as desensitization. In addition, this process provides a mechanism by which receptors can engage with arrestin adaptor molecules and couple to downstream signaling pathways. The importance of this regulatory process has been highlighted recently by the understanding that ligands can direct receptor signaling along one pathway in preference to another, the phenomenon of signaling bias that is partly mediated by the phosphorylation status or phosphorylation barcode of the receptor. Methods to determine the phosphorylation status of a GPCR in vitro and in vivo are necessary to understand not only the physiological mechanisms involved in GPCR signaling, but also to fully examine the signaling properties of GPCR ligands. This unit describes detailed methods for determining the overall phosphorylation pattern on a receptor (the phosphorylation barcode), as well as mass spectrometry approaches that can define the precise sites that become phosphorylated. These techniques, coupled with the generation and characterization of receptor phosphorylation-specific antibodies, provide a full palate of techniques necessary to determine the phosphorylation status of any given GPCR subtype.
MRC Toxicology Unit, University of Leicester, Leicester, United Kingdom.
Rudi
R
Prihandoko
MRC Toxicology Unit, University of Leicester, Leicester, United Kingdom.
Sophie J
SJ
Bradley
MRC Toxicology Unit, University of Leicester, Leicester, United Kingdom.
Andrew B
AB
Tobin
MRC Toxicology Unit, University of Leicester, Leicester, United Kingdom.
Adrian J
AJ
Butcher
D000818:Animals; D045163:Antibodies, Phospho-Specific / Q000378:metabolism; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D020962:Heterotrimeric GTP-Binding Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D010449:Peptide Mapping; D010600:Pharmacology / Q000379:methods*; D010748:Phosphopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D053719:Tandem Mass Spectrometry
D045163:Antibodies, Phospho-Specific; D008024:Ligands; D010748:Phosphopeptides; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D020962:Heterotrimeric GTP-Binding Proteins