Visualization of G protein-coupled receptor (GPCR) interactions in living cells using bimolecular fluorescence complementation (BiFC).
Members of the G protein-coupled receptor (GPCR) superfamily have been shown to homo- and hetero-oligomerize both in vitro and in vivo. Although the functional and pharmacological significance of GPCR oligomerization is far from being completely understood, evidence suggests that, depending on the receptor, oligomerization may influence ligand binding, G protein coupling, and receptor targeting. Bimolecular fluorescence complementation (BiFC) is a technique based on the complementation of fragments from fluorescent proteins that allows the measurement and visualization of protein interactions in living cells. It can be extended to the simultaneous detection of distinct protein-protein interactions using a multicolor setup. This unit describes the application of BiFC and multicolor BiFC to the visualization of GPCR oligomerization in a neuronal cell model. Oligomerization of GPCR fusions to BiFC tags is visualized and measured using fluorescence microscopy and fluorometry. The effect of ligands on the relative formation of distinct oligomeric species is monitored with a ratiometric multicolor BiFC approach.
Vidi
Pierre-Alexandre
PA
Purdue University, West Lafayette, Indiana.
Przybyla
Julie A
JA
Hu
Cheng-Deng
CD
Watts
Val J
VJ
D000818:Animals; D018929:Cell Culture Techniques; D005453:Fluorescence; D005455:Fluorescent Antibody Technique / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008856:Microscopy, Fluorescence / Q000379:methods*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism*
D005456:Fluorescent Dyes; D008024:Ligands; D010446:Peptide Fragments; D043562:Receptors, G-Protein-Coupled