Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization

Nat Methods. 2008 Jun;5(6):561-7. doi: 10.1038/nmeth.1213. Epub 2008 May 18.

Abstract

Cell-surface proteins are important in cell-cell communication. They assemble into heterocomplexes that include different receptors and effectors. Elucidation and manipulation of such protein complexes offers new therapeutic possibilities. We describe a methodology combining time-resolved fluorescence resonance energy transfer (FRET) with snap-tag technology to quantitatively analyze protein-protein interactions at the surface of living cells, in a high throughput-compatible format. Using this approach, we examined whether G protein-coupled receptors (GPCRs) are monomers or assemble into dimers or larger oligomers--a matter of intense debate. We obtained evidence for the oligomeric state of both class A and class C GPCRs. We also observed different quaternary structure of GPCRs for the neurotransmitters glutamate and gamma-aminobutyric acid (GABA): whereas metabotropic glutamate receptors assembled into strict dimers, the GABA(B) receptors spontaneously formed dimers of heterodimers, offering a way to modulate G-protein coupling efficacy. This approach will be useful in systematic analysis of cell-surface protein interaction in living cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biophysics / methods*
  • COS Cells
  • Cell Membrane / metabolism*
  • Chlorocebus aethiops
  • Dimerization
  • Fluorescence Resonance Energy Transfer / methods*
  • Humans
  • Models, Biological
  • Protein Interaction Mapping / methods*
  • Protein Structure, Quaternary
  • Receptors, G-Protein-Coupled / metabolism*
  • Receptors, GABA-B / chemistry
  • Surface Properties
  • gamma-Aminobutyric Acid

Substances

  • Receptors, G-Protein-Coupled
  • Receptors, GABA-B
  • gamma-Aminobutyric Acid