Figure 2: Gβ subunit binds to Tctex-1 and activates GEF-H1.

(a) Gβ subunit binds Tctex-1 in HEK293T cells. Flag-Tctex-1 was expressed in HEK293T cells alone or co-expressed with Gβ or a Gβ variant with mutations at the Tctex-1-binding site (Gβ_mut, R48A, R49A and R52A). Cells were lysed and Gβ protein complexes precipitated with anti-Gβ antibody were probed with anti-Gβ, anti-Tctex-1 and anti-GEF-H1 antibodies. (b) Gβ displaces GEF-H1 from Tctex-1. Myc-GEF-H1 and Flag-Tctex-1 were expressed with Gβ₁₂ or Gβ_mut in HEK293T cells, and following lysis, GEF-H1 complexes were immunoprecipitated with anti-Myc antibody, and probed with anti-Gβ, anti-Tctex-1 or anti-GEF-H1 antibodies, respectively. (c) Gβ₁₂γ₁₂ overexpresion induces GEF-H1 relocalization. Confocal images of GEF-H1^−/− cells expressing GEF-H1–eGFP or co-expressing GEF-H1–eGFP and Gβ₁₂γ₁₂, with or without βARKct (Gβγ-specific antagonist). Cells were fixed 1 day after transfection and stained with anti-Gβ or α-tubulin antibodies. Scale bar, 20 μm. (d) Gβ₁₂γ₁₂ expression induces stress fibre formation in a GEF-H1-dependent manner. Wild-type or GEF-H1^−/−MEFs, expressing Gβ₁₂γ₁₂ proteins, were fixed 1 day after transfection, stained with anti-Gβ and anti-phalloidin antibodies and visualized by confocal microscopy. Scale bar, 20 μm. (e) Gβγ expression activates GEF-H1 exchange activity in a specific manner. RhoA nucleotide exchange rates in the presence of lysates derived from HEK293T cells overexpressing eGFP alone, GEF-H1–eGFP or co-expressing GEF-H1–eGFP and Flag-Tctex-1 with or without co-transfection of Gβγ, Gβ_mutγ or Gβγ and the inhibitory βARKct peptide. GEF-H1, Tctex-1 and Gβ protein levels in cell lysates were detected by western blots. Error bars represent s.d. of at least three independent replicates, and P values derived from pairwise t-tests are indicated.