Dissecting the pharmacology of G protein-coupled receptor signaling complexes using bimolecular fluorescence complementation.
The affinity of G protein-coupled receptors (GPCRs) for particular ligands is altered by allosteric regulation with other proteins, for example signaling partners such as G proteins or β-arrestins, or multimeric receptor complexes. Studying the ways in which such interactions modulate pharmacology requires techniques that report these events at the molecular level. Options include bimolecular fluorescence complementation (BiFC), an imaging-based method that can directly demonstrate protein-protein association in living cells. Commonly used fluorescent proteins are split into two nonfluorescent halves, which then tag the protein partners under investigation. Interaction between the partners brings the complementary fragments together, allowing refolding and regeneration of the fluorescent protein to indicate that association has occurred. BiFC is irreversible and is not a real-time technique, yet the simplicity of its fluorescent signal holds key advantages for quantification and cellular localization of the resultant complexes.This review introduces general experimental considerations for using the BiFC approach, and describes specific protocols to develop a BiFC assay for GPCR-β-arrestin association, quantified using high content imaging and analysis. A further application of BiFC is to identify a particular protein-protein complex, thereby allowing investigation of its functional properties. This is illustrated in a protocol to quantify ligand-induced internalization of GPCR dimers of precise composition.
Cell Signalling Research Group, School of Biomedical Sciences, University of Nottingham, The Medical School, Nottingham, UK.
Laura E
LE
Kilpatrick
Nicholas D
ND
Holliday
D019390:Arrestins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D010600:Pharmacology / Q000379:methods*; D011485:Protein Binding; D055503:Protein Multimerization; D021381:Protein Transport; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection; D000071557:beta-Arrestins
D019390:Arrestins; D008164:Luminescent Proteins; D043562:Receptors, G-Protein-Coupled; D000071557:beta-Arrestins