Figure 2.

KB-752 is a selective guanine nucleotide exchange factor for Gα_i subunits. (A) KB-752 (10 μM) enhances the GTPγS binding rate of Gα_i1·GDP (50 nM); rate constants at 30 °C: Gα_i1 alone = 0.029 ± 0.006 min^-1, Gα_i1 + KB-752 = 0.086 ± 0.008 min^-1. (B) KB-752 is equipotent as a GEF on all three Gα_i members. 50 nM Gα_i1, Gα_i2, or Gα_i3 was incubated with indicated concentrations of KB-752 and the amount of [³⁵S]GTPγS binding was measured after 10 min at 30°C, expressed as percent of maximal GTPγS binding. KB-752 does not alter the rate of GTPγS binding by (C) Gα_i-heterotrimer Gα_i2·GDP/Gβ₁γ₂ (peptide and protein amounts as in panel A), nor (D) isolated Gα_o·GDP. For the dose-response curve of panel (D), 50 nM Gα_i1 or Gα_o was incubated in the presence of the indicated concentrations of KB-752 and the amount of [³⁵S]GTPγS binding was measured (after 10 min at 30 °C for Gα_i1; after 5 min at 20 °C for Gα_o) as described in Experimental Procedures and is expressed as the percent of GTPγS bound in the absence of KB-752. The EC₅₀ value for GEF activity on Gα_i1 was 5.6 ± 1.1 μM. Data shown are from a representative experiment of 3-5 independent experiments.