GxcC is a specific RapA interaction partner. (A) Dynamics of Rac activation visualized with CRIB-GFP. Cells were developed in shaken suspension for 3 hours and pulsed with 50 nM cAMP for another 2 hours. Developed cells were transferred to a glass bottom dish and overlayed with PB + 0.4% agarose. Images were collected using TIRF microscopy (original experiments are shown in Additional file 1: Movie 1 and Additional file 2: Movie 2). Scale bar is 10 μM. (B) Domain composition of GxcC. ARM – Armadillo repeats, CC – Coiled Coil region, RacGEF – Guanine Nucleotide Exchange factor for Rac, PH – Pleckstrin Homology domain. Arrows indicate the boundaries of truncated constructs. (C) Pull down in Dictyostelium lysate with GST-GxcC as bait and GFP-RapACA as prey. (D) Pull down with GST-ARM or GST-DH-PH as bait and GFP-RapACA as prey. The amount of prey was detected by western blotting using antibody specific for GFP (E) SDS PAGE analysis of recombinant GST-ARM (95 kDa) purified from E coli. (F) GDI assay in which the dissociation rate of mGppNHP from RapA was measured in the presence of varying concentrations of GST-ARM. The addition of increasing concentrations of the effector results in a concentration-dependent decrease of the observed rate constant (kobs).
Plak et al. BMC Cell Biology 2013 14:6 doi:10.1186/1471-2121-14-6