FIGURE 7:

Inactive Gαs directly binds GIV. (A) Endogenous GIV and Gβ (used as a positive control) preferentially bind inactive GST-Gαs-GDP (lane 3, upper panels). Little binding to active GST-Gαs-GDP/AlF₃⁻ (lane 5) or GST alone (lanes 2 and 4) is observed. Purified GST-Gαs (lanes 3 and 5) and GST alone (lanes 2 and 4) were preloaded with GDP (lanes 2 and 3) or GDP/AlF₃⁻ (lanes 4 and 5) and incubated overnight with ∼1 mg Cos7 cell lysate. Lane 1, 5% input. Recombinant proteins were visualized by Ponceau S staining (lower panel), and bound proteins were analyzed by immunoblotting for GIV and Gβ (top). (B, C) Endogenous GIV coimmunoprecipitates with Gαs-GFP-GDP (B, lane 2) but not Gαs-GFP-GDP/AlF₃⁻ (B, lane 3) or GFP alone (C; lanes 2 and 3), indicating that GIV preferentially interacts with GDP-bound Gαs. Cos7 cells expressing Gαs-GFP (B) or GFP alone (C) were lysed in the presence of GDP (lanes 1 and 2) or GDP/AlF₃⁻, (lanes 3 and 4) and incubated with anti-GFP IgG, and bound proteins were analyzed by immunoblotting for GFP and endogenous GIV. Lanes 1 and 4, 1% input. (D) GST-Gαs-GDP (lane 4) but not GST-Gαs-GDP/AlF₃⁻ (lane 5) or GST alone (lanes 2 and 3) binds His-GIV-CT (amino acids 1623–1870), indicating that GIV-CT binds directly to inactive Gαs. Purified GST (lanes 2 and 3) or GST-Gαs (lane 4 and 5) was preloaded with GDP (lanes 2 and 4) or GDP/AlF₃⁻ (lanes 3 and 5) as in A and incubated with purified His-GIV-CT. Lane 1, 10% input. (E) GST-Gαs-GDP binds FLAG-GIV-wt (lane 3), but binding of the mutant FLAG-GIV-FA (lane 5) is greatly decreased, indicating that the GEF motif in the C-terminus of GIV binds inactive Gαs. Purified GST (lanes 2 and 4) or GST-Gαs (lanes 3 and 5) was preloaded with GDP and incubated with lysates from Cos7 cells expressing either FLAG-GIV-wt (lanes 1–3) or FLAG-GIV-FA (lanes 4–6). Lanes 1 and 6, 1% input. Recombinant proteins were visualized by Ponceau S staining (bottom), and bound proteins were analyzed by immunoblotting (top) for FLAG (FLAG-GIV) and Gβ.