RalGDS binds CaM in a Ca²⁺-dependent manner. (A) A putative calmodulin-binding domain (CaMBD) was identified in the amino-terminal region of RalGDS (aa's 75-97). An axial helical projection was generated from this amino acid sequence as outlined in the calmodulin target database.²⁷  Hydrophobic residues are shown in gray. (B) CaM pulldown (CaM-PD) from lysates of HEK293 cells expressing GFP or GFP-RalGDS using CaM-agarose as described in “CaM pull-down assays.” The CaM-agarose specifically pulled down GFP-RalGDS. One percent of the total lysate was used to show expression levels (left). Vertical lines have been inserted to indicate repositioned gel lanes. (C) GFP-RalGDS binding to CaM is Ca²⁺ dependent, as the presence of 5 mM EDTA/EGTA completely abolished this interaction. (D) CaM pulldown of endogenous RalGDS from HUVEC lysates. Endogenous RalGDS was precipitated by CaM-agarose in a Ca²⁺-dependent manner.