Bimolecular Fluorescence Complementation Methodology to Study G Protein-Coupled Receptor Dimerization in Living Cells
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Proteins, such as G protein-coupled receptors (GPCRs), can interact with each other to form dimeric or higher order oligomeric complexes with novel pharmacological properties. GPCRs play a crucial role in numerous physiological processes and diseases, and much research has been performed to prove the existence of GPCR heterodimerization and to investigate the physiological role of the heterodimers. GPCRs are targeted by roughly 25% of all FDA-approved drugs, but heterodimers may represent an untapped additional source of novel drug targets. However, study of GPCR heteromers is not trivial, with most methods having distinct strengths and weaknesses. One method to study GPCR dimerization in living cells is through bimolecular fluorescence complementation (BiFC). The BiFC technique is based on the complementation of two nonfluorescent fragments of a fluorescent protein that is facilitated by fusing the fragments to two interacting proteins. The advantage of BiFC over alternative resonance energy transfer techniques is a high signal-to-noise ratio due to its strong intrinsic fluorescence without exogenous fluorogenic or chromogenic agents required. Here we provide a detailed description of protocols to measure dimerization-induced BiFC in a low-throughput, high-resolution approach using confocal microscopy and in a medium-throughput, low-resolution approach using an automated cell imaging multimode plate reader (Biotek Cytation 3). In this chapter, we use mu and delta opioid receptor heterodimerization to provide a step-by-step BiFC protocol; however, the protocol can be adapted for use with other receptors as well as other confocal or automated microscopes.
Key wordsBimolecular fluorescence complementation G protein-coupled receptor Dimerization Screening Confocal microscopy
This work was supported by funding from the National Institute on Mental Health (R33MH101673) to Dr. Watts.
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