Activator of G protein signaling 3 is a guanine dissociation inhibitor for gα subunits

Activator of G protein signaling 3 (AGS3) is a newly identified protein shown to act at the level of the G protein itself. AGS3 belongs to the GoLoco family of proteins, sharing the 19-aa GoLoco motif that is a Gα_i/o binding motif. AGS3 interacts only with members of the Gα_i/o subfamily, By surface plasmon resonance, we found that AGS3 binds exclusively to the GDP-bound form of Gα_i3. In GTPγS binding assays, AGS3 behaves as a guanine dissociation inhibitor (GDI), inhibiting the rate of exchange of GDP for GTP by Gα_i3. AGS3 interacts with both Gα_i3 and Gα_o subunits, but has GDI activity only on Geα_i3, not on Gα_o. The fourth GoLoco motif of AGS3 is a major contributor to this activity. AGS3 stabilizes Gα_i/3 in its GDP-bound form, as it inhibits the increase in tryptophan fluorescence of the Gα_i3-GDP subunit stimulated by AIF₄/^-. AGS3 is widely expressed as it is detected by immunoblotting in brain, testis, liver, kidney, heart, pancreas, and in PC-12 cells. Several different sizes of the protein are detected. By Northern blotting, AGS3 shows 2.3-kb and 3.5-kb mRNAs in heart and brain, respectively, suggesting tissue-specific alternative splicing. Taken together, our results demonstrate that AGS3 is a GDI. To the best of our knowledge, no other GDI has been described for heterotrimeric G proteins. Inhibition of the Gα subunit and stimulation of heterotrimeric G protein signaling, presumably by stimulating Gβγ, extend the possibilities for modulating signal transduction through heterotrimeric G proteins.