The proper function of Rho GTPases requires precise spatial and temporal regulation of effector interactions. Integrin-mediated cell adhesion modulates the interaction of GTP-Rac with its effectors by controlling GTP-Rac membrane targeting. Here, we show that the translocation of GTP-Rac to membranes is independent of effector interactions, but instead requires the polybasic sequence near the carboxyl terminus. Cdc42 also requires integrin-mediated adhesion for translocation to membranes. A recently developed fluorescence resonance energy transfer (FRET)-based assay yields the surprising result that, despite its uniform distribution, the interaction of activated V12-Rac with a soluble, cytoplasmic effector domain is enhanced at specific regions near cell edges and is induced locally by integrin stimulation. This enhancement requires Rac membrane targeting. We show that Rho-GDI, which associates with cytoplasmic GTP-Rac, blocks effector binding. Release of Rho-GDI after membrane translocation allows Rac to bind to effectors. Thus, Rho-GDI confers spatially restricted regulation of Rac–effector interactions.
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We thank P. Read for providing plasmids for purification of Rho-GDI and prenylated Rac, A. Hall for Rac effector mutants, Y. Takai for pEFBOS-myc–Rho-GDI, J.H. Jackson for pRK5-myc–V12-Rac-6Q, K. Moriyoshi for the GAP-43–GFP plasmid and A. Woods for the 150.9 monoclonal anti-syndecan-4. We also thank C. Chamberlain for technical advice establishing the FRET assays, and S. Shattil and E. Tzima for their critical comments. This work was supported by United States Public Health Service grant RO1 GM47214 (to M.A.S) and GM39434 (to K.M.H). M.A.d.P. was supported by the Human Frontier Science Program (LT0019/1998-M) and then by a Lady Tata Memorial Trust International Award for Research in Leukemia.
Figure S1 Activated GTPases localize at the basal level of the plasma membrane. Confocal z-section series of cells expressing various GFP fusions from Fig. 2 A and C. (PDF 1139 kb)
Figure S2 Time course of integrin-induced FRET in GFP-V12 Rac cells.
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Journal of the Indian Institute of Science (2017)
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