Activator of G protein signaling 3 is a guanine dissociation inhibitor for Gα(i) 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 Gα(i3), not on Gα(o). The fourth GoLoco motif of AGS3 is a major contributor to this activity. AGS3 stabilizes Gα(i3) in its GDP-bound form, as it inhibits the increase in tryptophan fluorescence of the Gα(i3)-GDP subunit stimulated by AlF(4)(−). 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.