Combining SRET2 and BiFC to Study GPCR Heteromerization and Protein-Protein Interactions

Methods Mol Biol. 2019:1947:199-215. doi: 10.1007/978-1-4939-9121-1_11.

Abstract

G protein-coupled receptors (GPCRs) are the target for many drugs. Evidence continues to accumulate demonstrating that multiple receptors form homo- and heteromeric complexes, which in turn dynamically couple with G proteins, and other interacting proteins. Here, we describe a method to simultaneously determine the identity of up to four distinct constituents of GPCR complexes using a combination of sequential bioluminescence resonance energy transfer 2-fluorescence resonance energy transfer (SRET2) with bimolecular fluorescence complementation (BiFC). The method is amenable to moderate throughput screening of changes in response to ligands and time-course analysis of protein-protein oligomerization.

Keywords: BRET2, Bioluminescence energy transfer 2; BiFC; Bimolecular fluorescence complementation; FRET, Fluorescence resonance energy transfer; SRET2, Sequential BRET2-FRET.

MeSH terms

  • Bioluminescence Resonance Energy Transfer Techniques / methods*
  • Fluorescence
  • Fluorescence Resonance Energy Transfer / methods*
  • Fluorescent Antibody Technique / methods*
  • Humans
  • Luciferases, Renilla / metabolism*
  • Microscopy, Fluorescence
  • Protein Multimerization*
  • Receptors, G-Protein-Coupled / chemistry*
  • Receptors, G-Protein-Coupled / metabolism*

Substances

  • Receptors, G-Protein-Coupled
  • Luciferases, Renilla