FIGURE 2.

RhoGEF activation of LARG by the direct interaction of the DH/PH domains with Gα₁₃. A, potentiation of SRF activation by LARG with Gα₁₃. HeLa cells were cotransfected with 0.1 μg of SRE-luciferase reporter plasmid and the indicated constructs: Gα₁₃QL (0.01 μg), myc-LARG-RDPC (0.1 μg), myc-LARG-DPC (0.1 μg), myc-LARG-DH/PH (0.01 μg), and myc-LARG-C (0.1 μg). SRF activities of cell lysates were measured 24 h after transfection as described in the supplemental materials. B, RhoA activation by LARG-DH/PH with Gα₁₃ in HeLa cells. HeLa cells were transiently transfected with the indicated plasmids: Gα₁₃QL (1 μg), myc-tagged RDPC (10 μg), myc-DPC (1 μg), and myc-DH/PH (0.5 μg). The cells were serum-starved for 24 h after 5 h of transfection. Endogenous RhoA in the GTP-bound form was isolated using GST-Rhotekin RBD from the cell lysates as described in the supplemental materials. The expression of Myc-tagged LARG constructs and Gα₁₃ were analyzed by Western blotting using anti-Myc and anti-Gα₁₃ antibodies. C, in vitro RhoGEF assays of LARG-DH/PH and -RDPC. GDP dissociation from RhoA was measured at 20 °C after 2-min incubation in the presence of the indicated proteins: LARG-RDPC (20 nm), LARG-DH/PH (30 nm), -activated Gα₁₃WT (100 nm). (Student's t test: n = 4, ^*, p < 0.001). D, recruitment of LARG with RH domain to the plasma membrane by constitutively active Gα₁₃. HeLa cells were cotransfected in the same procedure as in Fig. 2A. The expression of LARG constructs in total cell lysate (T), crude cytosolic (C), and membrane (M) fractions, and Gα₁₃ in total lysates was detected by immunoblotting using anti-Myc antibody or anti-Gα₁₃ antibody. E, cell rounding induced by LARG constructs with the constitutively active Gα₁₃ in MDCKII cells. MDCKII cells were transiently transfected with the indicated myc-tagged LARG constructs in the presence or absence of Gα₁₃QL, or FLAG-tagged V14RhoA alone. The cells were serum-starved for 24 h after 5 h of transfection, then fixed, and triply stained with anti-Myc antibody, anti-Gα₁₃ antibody, and phalloidin for filamentous actin. Transfected cells were visualized by fluorescence microscopy, identified, and scored for rounding indicating the involvement of RhoA activation as described under the supplemental materials. The values were calculated from four independent experiments. The images are in supplemental Fig. S4. F, stimulation of the RhoGEF activity of LARG by Gα_i/13KA. GTPγS binding to RhoA (200 nm) was measured at 20 °C after 5-min incubation in the presence of the following proteins: ○, control; ▵, -activated Gα_i/13 (100 nm); □, AMF-activated Gα_i/13KA (100 nm); •, RDPC (10 nm); ▴, RDPC (10 nm) plus AMF-activated Gα_i/13 (100 nm); ⋄, RDPC (10 nm) plus AMF-activated Gα_i/13KA (100 nm).