Figure 4: The Gα_12/13 subunit binds to GEF-H1, displaces Tctex-1 and stimulates exchange activity.

(a) The Gα-binding site resides in the N-terminal region of GEF-H1. His-GEF-H1 (a.a. 1–985) or a series of N- and C-terminal truncations and deletions were expressed in HEK293T cells and protein complexes were immunoprecipitated with α-His antibodies. GEF-H1 immune complexes were immunoblotted with anti-GEF-H1 or anti-Gα₁₃ antibodies. (b) Optimal binding to endogenous Gα₁₃ requires the first 240 amino acids of GEF-H1. Flag-GEF-H1 or a series of N-terminal fragments were expressed in HEK293T cells and protein complexes were immunoprecipitated with anti-Flag antibodies. GEF-H1 immune complexes were immunoblotted with anti-GEF-H1, anti-Gα₁₃ or anti-Tctex-1 antibodies to detect endogenous Gα₁₃ and Tctex-1, respectively. Total Gα₁₃ levels were assessed by western blot. (c) Constitutively active Gα₁₃ inhibits the association of Tctex-1 with GEF-H1. Flag complexes were immunoprecipitated from HEK293T lysates expressing Flag-GEF-H1 with or without Myc-Tctex-1 and Gα_13QL, and immunoblotted with anti-GEF-H1, anti-Tctex-1 or anti-Gα₁₃ antibodies. Western blot of GEF-H1, Gα₁₃ and Tctex-1 are shown. (d) Gα_13QL antagonizes Tctex-1-mediated inhibition of GEF-H1 exchange activity. RhoA nucleotide exchange rates were measured in lysates from HEK293T cells expressing combinations of eGFP, GEF-H1–eGFP, Flag-Tctex-1, constitutively active Gα_13QL and the inhibitory rgRGS domain, as indicated. GEF-H1, Tctex-1 and Gα_QL expression were confirmed by western blots. The s.d. derived from at least three independent replicate experiments are shown along with P values from pairwise t-tests.