AGS3-LONG and AGS3-SHORT contain G-protein regulatory motifs that interact with and stabilize the GDP-bound conformation of Galpha(i) > Galpha(o). AGS3 and related proteins may influence signal strength or duration as well as the adaptation of the signaling system associated with sustained stimulation. To address these issues, we determined the effect of AGS3 on the integration of stimulatory (Galpha(s)-mediated vasoactive intestinal peptide receptor) and inhibitory (Galpha(i)-mediated alpha(2)-adrenergic receptor (alpha(2)-AR)) signals to adenylyl cyclase in Chinese hamster ovary cells. AGS3-SHORT and AGS3-LONG did not alter the VIP-induced increase in cAMP or the inhibitory effect of alpha(2)-AR activation. System adaptation was addressed by determining the influence of AGS3 on the sensitization of adenylyl cyclase that occurs following prolonged activation of a Galpha(i)-coupled receptor. Incubation of cells with the alpha(2)-AR agonist UK14304 (1 microm) for 18 h resulted in a approximately 1.8-fold increase in the vasoactive intestinal peptide-induced activation of adenylyl cyclase, and this was associated with a decrease in membrane-associated Galpha(i3). Both effects were blocked by AGS3-SHORT. AGS3-SHORT also decreased the rate of Galpha(i3) decay. A mutant AGS3-SHORT incapable of binding G-protein was inactive. These data suggest that AGS3 and perhaps other G-protein regulatory motif-containing proteins increase the stability of Galpha(i) in the membrane, which influences the adaptation of the cell to prolonged activation of Galpha(i)-coupled receptors.