Measurement of heterotrimeric G-protein and regulators of G-protein signaling interactions by time-resolved fluorescence resonance energy transfer.
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.
Leifert
Wayne R
WR
CSIRO Molecular and Health Technologies, Adelaide, SA 5000, Australia. wayne.leifert@csiro.au
Bailey
Kelly
K
Cooper
Tamara H
TH
Aloia
Amanda L
AL
Glatz
Richard V
RV
McMurchie
Edward J
EJ
D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D020962:Heterotrimeric GTP-Binding Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D009942:Organometallic Compounds / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011806:Quinolinium Compounds / Q000737:chemistry / Q000378:metabolism; D020710:RGS Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D015203:Reproducibility of Results; D015398:Signal Transduction; D013725:Terbium / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
C481052:Alexa fluor 546; D009942:Organometallic Compounds; D021122:Protein Subunits; D011806:Quinolinium Compounds; D020710:RGS Proteins; D011956:Receptors, Cell Surface; D013725:Terbium; D020962:Heterotrimeric GTP-Binding Proteins