NRG1 activation of the ErbB2 and 3 heterodimer stimulates the GEF activity of Dock7. (A–C) 125 ng of immobilized FLAG-Dock7-DHR-2 was incubated with 16 ng/μl GST-Rac1, Cdc42, or RhoA and 3 μM [³H]GDP in 30 μl of reaction buffer for 0–30 min, and the guanine nucleotide binding activities were measured (n = 10). (D–F) The release of [³H]GDP from GST-Rac1-[³H]GDP, Cdc42-[³H]GDP, or GST-RhoA-[³H]GDP by FLAG–Dock7–DHR-2 was measured (n = 10). Immunoprecipitated FLAG-Dbs-DHPH was used as the positive control for the RhoA-GEF. Dock7–DHR-2, closed circle; control, open circle; Dbs-DHPH, closed square. (G–L) 293T cells were transfected with pCMV–FLAG–Dock7–DHR-2 or pCMV-FLAG-Dbs-DHPH. The cell lysates were affinity precipitated with 20 μg each of nucleotide-free GST-Rho GTPase (Rac1G15A, Cdc42G15A, or RhoAG17) or the wild type (Rac1, Cdc42, or RhoA) and immunoblotted with an anti-FLAG antibody. The total FLAG–Dock7–DHR-2 or FLAG-Dbs-DHPH is also shown. Each GST-Rho GTPase was immobilized in the same experimental conditions, subjected to SDS-PAGE, and stained with Coomassie brilliant blue. (M and O) 293T cells were cotransfected with pCMV-FLAG-Dock7, pCMV-ErbB2, and pCMV-ErbB3 and stimulated with or without NRG1 for 30 min. The expression of ErbB2 and 3 in 293T cells was below the detection level of immunoblotting (not depicted). The release of [³H]GDP from GST-Rac1-[³H]GDP or Cdc42-[³H]GDP by immunoprecipitated FLAG-Dock7 was measured (n = 3). (N and P) Cells were cotransfected with pCMV-FLAG-Dock7, pCMV-ErbB2, and pCMV-ErbB3. The affinity precipitation of the cell lysates with GST-Rac1G15A or Cdc42G15A was performed. The total FLAG-Dock7 is also shown. Error bars show ±SD. Data were evaluated by using one-way ANOVA (*, P < 0.01).