A fluorescence resonance energy transfer activation sensor for Arf6.
The involvement of the small GTPase Arf6 in Rac activation, cell migration, and cancer invasiveness suggests that it is activated in a spatially and temporally regulated manner. Small GTPase activation has been imaged in cells using probes in which the GTPase and a fragment of a downstream effector protein are fused to fluorescent reporter proteins that constitute a fluorescence resonance energy transfer (FRET) donor/acceptor pair. Unlike other Ras family GTPases, the N terminus of Arf6 is critical for membrane targeting and, thus, cannot be modified by fusion to a fluorescent protein. We found that the previously described C-terminal green fluorescent protein (GFP) derivative also shows diminished membrane targeting. Therefore, we inserted a fluorescent protein into an inert loop within the Arf6 sequence. This fusion showed normal membrane targeting, nucleotide-dependent interaction with the downstream effector GGA3, and normal regulation by a GTPase-activating protein (GAP) and a guanine nucleotide exchange factor (GEF). Using the recently developed CyPET/YPET fluorescent proteins as a FRET pair, we found that Arf6-CyPET underwent efficient energy transfer when bound to YPET-GGA3 effector domain in intact cells. The addition of platelet-derived growth factor (PDGF) to fibroblasts triggered a rapid and transient increase in FRET, indicative of Arf6 activation. These reagents should be useful for investigations of Arf6 activation and function.
Hall
Brian
B
Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908, USA.
McLean
Mark A
MA
Davis
Kathryn
K
Casanova
James E
JE
Sligar
Steven G
SG
Schwartz
Martin A
MA
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002460:Cell Line; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D010446:Peptide Fragments / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000378:metabolism
D000089982:ADP-Ribosylation Factor 6; D033942:Adaptor Proteins, Vesicular Transport; C485182:Cyan Fluorescent Protein; C407306:GGA adaptor proteins; D010446:Peptide Fragments; D049452:Green Fluorescent Proteins; D020558:GTP Phosphohydrolases; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; C000715727:Arf6 protein, mouse; D020744:rac GTP-Binding Proteins