FIGURE 3.

Functional roles of the RH domain and C-terminal region for DH/PH domains-mediated GEF activation of LARG. A, Enhancement of the Gα₁₃-stimulated RhoGEF activity of LARG-DH/PH by LARG-RH in HeLa cells. HeLa cells were transiently transfected with the indicated plasmids: Gα₁₃QL (1 μg), myc-tagged LARG-DH/PH (1.5 μg), myc-LARG-RH (2 μg), or myc-p115-RH (0.02 μg). GTP-bound form of RhoA was isolated as described in Fig. 2B. The left of the panel is a representative result from three independent experiments. The right panel shows the mean ± S.E. of values of amount of GTP-RhoA scanned using ImageJ program. B, potentiation of the Gα₁₃-induced RhoGEF activity of LARG-DH/PH by LARG-RH in vitro. GTPγS binding to RhoA (200 nm) was measured at 20 °C after 5-min incubation in the presence of the indicated proteins: AMF-activated Gα₁₃ (100 nm), RH (600 nm), DH/PH (120 nm). (Fisher's protected least significant difference test: n = 3, ^*, p < 0.001). C, enhancement of the binding of LARG-RH and LARG-DPC with Gα₁₃ immobilized on Ni-NTA-agarose. Equal amounts of cell lysates of COS1 cells transfected with myc-LARG-DPC (8 μg) or myc-LARG-RH (4 μg) were incubated with 1.4 μg of immobilized His-Gα_i/13 or His-Gα_i/13KA proteins onto Ni-NTA agarose beads in the presence or absence of AMF. The relative amount of LARG fragments bound to the activated- or deactivated-Gα₁₃ was visualized by Western blot analysis using anti-Myc antibody. The expression of the LARG proteins in total cell lysates and the Gα₁₃ protein in the reaction mixtures are also shown. D, the role of LARG C terminus in RhoGEF activation of LARG by Gα₁₃. GTPγS binding to RhoA (200 nm) was measured at 20 °C after 5-min incubation in the presence of the indicated proteins: AMF-activated Gα₁₃ (100 nm), RDP (20 nm), C (200 nm), and RDPC (20 nm) (Student's t test: n = 3, ^*, p = 0.0001).