FLJ00018/PLEKHG2 is a guanine nucleotide exchange factor for the Rho family small GTPases. FLJ00018 is directly activated by heterotrimeric G protein G beta gamma subunits. Using two-hybrid screening, we have identified non-muscle cytosolic actin as a binding partner of FLJ00018. We found that there were two actin-binding regions in FLJ00018 at the N-terminal region (150-283 amino acids) and at the C-terminal region (465-1386 amino acids). The overexpression of non-muscle cytosolic actin attenuated the FLJ00018-induced serum response element-dependent gene transcription. These results suggest that non-muscle cytosolic actin may be a negative regulator of FLJ00018 through its interaction with the Dbl homology domain. (C) 2012 Elsevier Inc. All rights reserved.