Fig. 2. The DHR-2 domain of DOCK180 binds to and activates Rac in vitro and is necessary and sufficient for DOCK180-mediated Rac activation in vivo. (A) Schematic representation of the DOCK180 DHR-2 domain. The boundaries of the fusion proteins that were used in GTPase binding and GEF assays are indicated. (B) Lysates from untransfected COS-1 cells (for RhoA detection) or cells transfected with Myc-Rac or Myc-Cdc42 were incubated with the indicated GST-fusion proteins bound to Glutathione beads. The precipitated proteins were detected by immunoblotting with anti-Myc or anti-RhoA antibodies. TCL, total cell lysate. (C) Bacterially produced, purified DHR-2 domain of DOCK180 and the DPC domain of Vav2 were used in an in vitro GEF assay toward [³H] GDP-loaded Rac, Cdc42 or RhoA. Reactions were stopped at 0, 15 and 30 minute time points. Radiolabelled GDP bound to the GTPases was measured by using a filter-binding assay, as described in Materials and Methods. (D) 293-T cells were transfected with a control vector or with vectors coding for Flag-DOCK180, DOCK180DHR-2, Myc-DHR-2, Myc-DHR-2/DH domain or Myc-Docker (see text for details). Rac-GTP was pulled down from cell lysates using the p21-binding domain of PAK fused to GST (`PBD' assay). The amount of Rac in pull-downs and in total cell lysates (`TCL') was detected by anti-Rac immunoblotting. Expression levels of the various DOCK180 proteins were analyzed by anti-DOCK180 and anti-Myc immunoblotting in total cell lysates.