Measurement of heterotrimeric G-protein and regulators of G-protein signaling interactions by time-resolved fluorescence resonance energy transfer

Anal Biochem. 2006 Aug 15;355(2):201-12. doi: 10.1016/j.ab.2006.04.042. Epub 2006 May 11.


G-protein-coupled receptors transduce their signals through G-protein subunits which in turn are subject to modulation by other intracellular proteins such as the regulators of G-protein signaling (RGS) proteins. We have developed a cell-free, homogeneous (mix and read format), time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor heterotrimeric G-protein subunit interactions and the interaction of the G alpha subunit with RGS4. The assay uses a FRET pair consisting of a terbium cryptate chelate donor spectrally matched to an Alexa546 fluor acceptor, each of which is conjugated to separate protein binding partners, these being G alpha(i1):beta4gamma2 or G alpha(i1):RGS4. Under conditions favoring specific binding between labeled partners, high-affinity interactions were observed as a rapid increase (>fivefold) in the FRET signal. The specificity of these interactions was demonstrated using denaturing or competitive conditions which caused significant reductions in fluorescence (50-85%) indicating that labeled proteins were no longer in close proximity. We also report differential binding effects as a result of altered activation state of the G alpha(i1) protein. This assay confirms that interactions between G-protein subunits and RGS4 can be measured using TR-FRET in a cell- and receptor-free environment.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Fluorescence
  • Fluorescence Resonance Energy Transfer / methods*
  • Heterotrimeric GTP-Binding Proteins / analysis*
  • Heterotrimeric GTP-Binding Proteins / chemistry
  • Heterotrimeric GTP-Binding Proteins / metabolism
  • Organometallic Compounds / chemistry
  • Organometallic Compounds / metabolism
  • Protein Binding
  • Protein Subunits / chemistry
  • Protein Subunits / metabolism
  • Quinolinium Compounds / chemistry
  • Quinolinium Compounds / metabolism
  • RGS Proteins / analysis*
  • RGS Proteins / chemistry
  • RGS Proteins / metabolism
  • Receptors, Cell Surface / metabolism
  • Reproducibility of Results
  • Signal Transduction
  • Terbium / chemistry
  • Terbium / metabolism
  • Time Factors


  • Alexa fluor 546
  • Organometallic Compounds
  • Protein Subunits
  • Quinolinium Compounds
  • RGS Proteins
  • Receptors, Cell Surface
  • Terbium
  • Heterotrimeric GTP-Binding Proteins