Using Bioluminescence Resonance Energy Transfer (BRET) to Characterize Agonist-Induced Arrestin Recruitment to Modified and Unmodified G Protein-Coupled Receptors.
G protein-coupled receptors (GPCRs) represent ∼25% of current drug targets. Ligand binding to these receptors activates G proteins and arrestins, which are involved in differential signaling pathways. Because functionally selective or biased ligands activate one of these two pathways, they may be superior medications for certain diseases states. The identification of such ligands requires robust drug screening assays for both G protein and arrestin activity. This unit describes protocols for two bioluminescence resonance energy transfer (BRET)-based assays used to monitor arrestin recruitment to GPCRs. One assay requires modification of GPCRs by fusion to a BRET donor or acceptor moiety, whereas the other can detect arrestin recruitment to unmodified GPCRs.
Departments of Psychiatry and Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York.|Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York.
Prashant
P
Donthamsetti
Departments of Psychiatry and Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York.|Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York.
Jose Rafael
JR
Quejada
Departments of Psychiatry and Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York.|Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York.
Jonathan A
JA
Javitch
Department of Pharmacology, Vanderbilt University, Nashville, Tennessee.
Vsevolod V
VV
Gurevich
Department of Pharmacology and Toxicology, Medical College of Georgia, Georgia Regents University, Augusta, Georgia.
Nevin A
NA
Lambert
D019393:Arrestin / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D008163:Luminescent Measurements / Q000379:methods*; D011094:Polyethyleneimine / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000187:drug effects*; D014178:Translocation, Genetic
D019393:Arrestin; D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D011094:Polyethyleneimine; D019204:GTP-Binding Proteins
G protein-coupled receptors (GPCRs); arrestin; bioluminescence resonance energy transfer (BRET)