Determination of the activated Cdc42-binding region of Cupidin and generation of deletion mutants lacking binding activities. (A-D) Top, the domain structure of Cupidinα/Homer2a (CPDα) and its mutants used in the ligand overlay assay. The Cdc42 binding activities are summarized on the right, presented as normalized values corresponding to CPD (A) or CPD191–283 (B, C, and D) set to 1.00. Asterisks indicate values over 0.70, which was arbitrarily considered to represent positive values. CPDα is composed of 343 amino-acid residues. The EVH1 domain in the N-terminal 110 residues, the coiled-coil domain (CC) predicted within the amino acid stretch 173–317, and two Leu zipper motifs A (LZA, 238–296 aa) and B (LZB, 311–339 aa) are indicated. The deletion constructs consist of the regions shown by solid thick bars; Middle, autoradiograms resulting from the [35S]-GTPγS-bound Cdc42 ligand overlay assay; Bottom, Coomassie Brilliant Blue (CBB)-stained gel corresponding to the ligand overlay assay, in which all GST-fused CPD-relating proteins migrated as top bands by SDS-PAGE. (A) First trial to determine the Cdc42-binding region using rough deletion of CPD C. (B) Second trial to narrow down the Cdc42-binding region by making mutants of CPD 191–283. (C) Third trial to narrow down the Cdc42-binding region by using shortened fragments of CPD 191–283. (D) Final trial to determine the deletion mutants of Cupidin lacking Cdc42-binding ability.