Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2011 Mar 1;22(5):673–686. doi: 10.1091/mbc.E10-08-0738

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2011 Garcia-Marcos et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

“ASCB®,“ “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology.

PMC Copyright notice

FIGURE 2: — Gα_i3 activity is reversibly modulated by GIV (a GEF) and AGS3 (a GDI). (A) Preparation of Gα_i3–AGS3 complexes. His-Gα_i3:GST-AGS3 (465–650) complexes were purified (4:1 stoichiometry) by glutathione-agarose affinity chromatography. An aliquot of the purified complex was separated by SDS–PAGE and stained with Coomassie blue. (B, C) GIV efficiently promotes G protein activation in the Gα_i3–AGS3 complex. The steady-state GTPase activity of purified His-Gα_i3 (50 nM) alone (closed circles) or in complex with GST-AGS3 (open circles) was determined in the presence of the indicated amounts (0, 0.1, 0.25, 0.5, 1, and 2 μM) of purified His-GIV-CTs (aa 1660–1870, containing the GEF motif responsible for Gα_i binding) by quantification of the amount of [γ-³²P]GTP (0.5 μM, ∼50 cpm/fmol) hydrolyzed in 10 min. Data are expressed as fmol of radioactive phosphate (³²Pi) released (absolute GTPase activity, B). The same data were normalized to the GTPase activity in the absence of His-GIV-CTs and expressed as % of ³²Pi released (fold activation, C) by the G protein alone (closed circles) or in complex with GST-AGS3 (open circles) in the absence of His-GIV-CTs. Results are shown as mean ± SD of a representative experiment out of three performed in duplicate. (D) Preparation of Gα_i3:GIV-CT complexes. His-Gα_i3:His-GIV-CTs (aa 1660–1870) complexes were purified (1:1 stoichiometry) by gel filtration chromatography. An aliquot of the purified complex was separated by SDS–PAGE and stained with Coomassie blue. (E, F) AGS3 efficiently blocks G protein activation in the Gα_i3–GIV complex. The steady-state GTPase activity of purified His-Gα_i3 (50 nM) alone (closed circles) or in complex with His-GIV-CTs (open circles) was determined in the presence of the indicated amounts (0, 0.11, 0.055, 0.11, 0.275, 0.55, 1.1, and 2.2 μM) of purified His-AGS3 (aa 424–650) by quantification of the amount of [γ-³²P]GTP (0.5 μM, ∼50 cpm/fmol) hydrolyzed in 10 min. Data are expressed as fmol radioactive phosphate (³²Pi) released (absolute GTPase activity, E). The same data were normalized to the GTPase activity in the absence of His-AGS3 and expressed as % of ³²Pi released (fold activation, F) by the G protein alone (closed circles) or in complex with His-GIV-CTs (open circles) in the absence of His-AGS3. Results are shown as mean ± SD of a representative experiment out of three performed in duplicate. (G) Schematic representation of the reversible regulation of Gα_i3 activity by AGS3 GDI and GIV GEF.