Immunoprecipitation and phosphorylation of G protein-coupled receptors.
G protein-coupled receptors (GPCRs) are integral membrane proteins with seven transmembrane-spanning alpha-helices. Following ligand activation, many GPCRs are rapidly phosphorylated on serine/threonine residues in their cytoplasmic domains, principally the carboxyl-terminus. GPCR phosphorylation recruits arrestin proteins to the activated receptor leading to receptor internalization and desensitization. Arrestins also act as scaffolds to recruit other regulatory and signaling molecules to the receptor. The low level of expression of GPCRs in tissues, the difficulty in developing antibodies that can specifically detect and harvest receptor protein, and the hydrophobic and heterogeneric nature of GPCRs makes examination of their structure, function, and biology an interesting challenge. Receptor phosphorylation is typically performed in cells transfected with wild and mutated receptors usually bearing an epitope tag and equilibrated with [(32)Pi] to radiolabel cellular ATP pools. Following ligand stimulation, receptor protein is extracted using a detergent lysis buffer and immunoprecipitated with antibodies raised against the epitope tag; following separation on SDS-polyacrylamide gel electrophoresis, phosphorylated receptors are quantified using phosphorimaging.
Thomas
Walter G
WG
School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D015153:Blotting, Western / Q000379:methods*; D002478:Cells, Cultured; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*
D043562:Receptors, G-Protein-Coupled; D000255:Adenosine Triphosphate