Figure 6.

Preferential coupling of p114RhoGEF to Gα12 in signaling to SRE. (A) Effects of an ectopically expressed region of p114RhoGEF on Gα12- and Gα13-mediated signaling. HEK293 cells grown in 12-well plates were transfected with myc-tagged, constitutively active Gα12 or Gα13, plus either a construct encoding the Gα12-interacting 257 amino acids of p114RhoGEF with an N-terminal FLAG-tag (p114²⁵⁷) or empty vector. All cells were co-transfected with the firefly luciferase reporter SRE-L (0.2 µg) and the Renilla luciferase reporter pRL-TK (0.02 µg). Approximately 48 h post-transfection, cells were harvested and assayed for firefly and Renilla luciferase activity as described in Methods. For Gα12 and Gα13 transfections, effects of the p114RhoGEF construct are presented as % of vector control results, which were set at 100% for each α subunit. Graphs indicate mean ± s.e.m. for three independent experiments. (B) Effects of p114RhoGEF titration on serum dependence of its signaling to SRE. HEK293 cells grown in 12-well plates were transfected in duplicate with decreasing amounts of plasmid encoding myc-tagged, full-length p114RhoGEF (X-axis), plus uniform amounts of SRE-L and pRL-TK. After 32 h, one sample per transfection condition was washed twice with DMEM and serum-starved in the same medium for 12 h, whereas the duplicate sample received washes and addition of DMEM containing 10% FBS. Cell lysates were assayed for firefly and Renilla luciferase activity. Data shown are the mean of two independent experiments, with error bars indicating range. (C) Effects of dominant-negative Gα12 and Gα13 on serum-dependent signaling through p114RhoGEF. HEK293 cells were transfected with 5 ng plasmid encoding myc-tagged, full-length p114RhoGEF (myc-p114) or 5 ng empty vector, along with 50 ng plasmid encoding myc-tagged, constitutively GDP-bound variants of Gα12 (12GA-myc) or Gα13 (13GA-myc). All cells were co-transfected with SRE-L and pRL-TK and grown in DMEM + 10% serum for approximately 48 h, then assayed by luminometry. Data presented are mean ± s.e.m. for three independent experiments. At bottom are results of SDS-PAGE/immunoblot analysis of cell lysates, with expression of myc-p114RhoGEF (upper panel) and myc-tagged, dominant-negative G12/13 α subunits (lower panel) tracked using an anti-myc antibody. Immunoblots shown are a representative of three independent experiments.