RESUMO
We demonstrate using Ca2+-dependent calmodulin (CaM)-affinity chromatography and overlay with biotinylated CaM that the adaptor proteins growth factor receptor bound (Grb)7 and Grb7V (a naturally occurring variant lacking the Src homology 2 (SH2) domain) are CaM-binding proteins. Deletion of an amphiphilic basic amino-acid sequence (residues 243-256) predicted to form an alpha-helix located in the proximal region of its pleckstrin homology (PH) domain demonstrates the location of the CaM-binding domain. This site is identical in human and rodents Grb7, and shares great homology with similar regions of Grb10 and Grb14, and the Mig10 protein from Caenorhabditis elegans. We show that Grb7 and Grb7V are present in the cytosol and bound to membranes, while the deletion mutants (Grb7Delta and Grb7VDelta) have less capacity to be associated to membranes. Grb7Delta maintains in part the capacity to bind phosphoinositides, and CaM competes for phosphoinositide binding. Activation of ErbB2 by heregulin beta1 decreases the pool of Grb7 associated to membranes. The cell-permeable CaM antagonist W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), but not the CaM-dependent protein kinase II inhibitor KN93, prevents this effect. Highly specific cell-permeable CaM inhibitory peptides decrease the association of Grb7 to membranes. This suggests that CaM regulates the intracellular mobilization of Grb7 in living cells. Direct interaction between enhanced yellow fluorescent protein (EYFP)-Grb7 and enhanced cyan fluorescent protein (ECFP)-CaM chimeras at the plasma membrane of living cells was demonstrated by fluorescence resonance energy transfer (FRET). The FRET signal dramatically decreased in cells loaded with a cell-permeable Ca2+ chelator, and was significantly attenuated when enhanced yellow fluorescent protein-Grb7 chimera (EYFP-Grb7)Delta instead of EYFP-Grb7 was used. Finally, we show that conditioned media from cells transiently transfected with Grb7Delta and Grb7VDelta lost its angiogenic activity, in contrast to those from cells transiently transfected with their wild-type counterparts.
