FIGURE 1.
GIV is a bona fide GEF for Gαi3. A, His-GIV-CT (aa 1623–1870) and His-GIV-CTs (aa 1660–1870) are equally efficient in increasing the steady-state GTPase activity of Gαi3. The steady-state GTPase activity of purified His-Gαi3 (50 nm) was determined in the presence of the indicated amounts (0, 0.1, 0.25, 0.5, 1, and 2 μm) of purified His-GIV-CT (aa 1623–1870, closed circles) or His-GIV-CTs (aa 1660–1870, open circles) by quantification of the amount of [γ-32P]GTP (0.5 μm, ∼50 cpm/fmol) hydrolyzed in 10 min. Data are expressed as % of GTP hydrolyzed by the G protein alone (0 μm His-GIV-CT or His-GIV-CTs). Results are shown as mean ± S.E. of n = 3 independent experiments. B, His-GIV-CTs does not affect the rate of GTP hydrolysis by Gαi3. Single-turnover GTPase assays for Gαi3 (50 nm) were performed as described under “Experimental Procedures” in the presence of His-GIV-CTs (2 μm, open circles), GST-GAIP (1 μm, “x”), or buffer (closed circles). GST-GAIP increases the rate of GTP hydrolysis by Gαi3, whereas His-GIV-CTs has no effect. One representative experiment of four is shown (C) His-GIV-CTs increases GTPγS binding to Gαi3. Nucleotide binding activity of purified His-Gαi3 (50 nm) was determined in the presence of the indicated amounts (0, 0.1, 0.5, 1, and 2 μm) of purified wild-type His-GIV-CTs (closed circles) or His-GIV-CTs F1685A mutant (open circles) by quantification of the amount of [35S]GTPγS (0.5 μm, ∼50 cpm/fmol) bound in 15 min. Data are expressed as % of GTPγS bound by the G protein alone in the absence of His-GIV-CTs. His-GIV-CTs increases GTPγS binding up to 2.2-fold over basal binding, whereas His-GIV-CTs F1685A has no significant effect. Results are shown as mean ± S.E. of n = 3 independent experiments.