Previously, we have shown that RalA, a calmodulin (CaM)-binding protein, binds to the C2 region in the C-terminal of PLC-delta 1, and increases its enzymatic activity. Since PLC-delta 1 contains a CaM-like region in its N-terminus, we have investigated if RalA can also bind to the N-terminus of PLC-delta 1. Therefore, we created a GST-PLC-delta 1 construct consisting of the first 294 amino acids of PLC-delta 1 (GST-PLC-delta 1 (1-294)). In vitro binding experiments Confirmed that PLC-delta 1 (1-294) was capable of binding directly to RalA. W-7 coupled to polyacrylamide beads bound pure PLC-delta 1, demonstrating that PLC-delta 1 contains a CaM-like region. Competition assays with W-7, peptides representing RalA and the newly identified RalB CaM-binding regions, or the IQ peptide from PLC-delta 1 were able to inhibit RalA binding to PLC-delta 1 (1-294). This study demonstrates that there are two binding sites for RalA in PLC-delta 1 and provides further insight into the role of Ral GTPase in the regulation of PLC-delta 1 function. (C) 2009 Elsevier Inc. All rights reserved.