AGS3 and Signal Integration by Gαs- and Gαi-coupled Receptors

Abstract

AGS3-LONG and AGS3-SHORT contain G-protein regulatory motifs that interact with and stabilize the GDPbound conformation of G␣ i > G␣ 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 (G␣ s -mediated vasoactive intestinal peptide receptor) and inhibitory (G␣ i -mediated ␣ 2 -adrenergic receptor (␣ 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 ␣ 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 G␣ i -coupled receptor. Incubation of cells with the ␣ 2 -AR agonist UK14304 (1 M) for 18 h resulted in a ϳ1.8-fold increase in the vasoactive intestinal peptide-induced activation of adenylyl cyclase, and this was associated with a decrease in membrane-associated G␣ i3 . Both effects were blocked by AGS3-SHORT. AGS3-SHORT also decreased the rate of G␣ 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 G␣ i in the membrane, which influences the adaptation of the cell to prolonged activation of G␣ i -coupled receptors.The activation/deactivation cycle for heterotrimeric G-protein involves the exchange of GDP for GTP and the subsequent hydrolysis of GTP by G␣ subunits. These events are generally initiated by a G-protein-coupled receptor at the cell surface. However, increasing evidence indicates the existence of unexpected regulatory mechanisms for the G-protein activation/ deactivation cycle and a role for G-protein subunits in signaling events within the cell that do not involve a cell-surface receptor (1). These unexpected regulatory mechanisms include accessory proteins that accelerate GTPase activity, serve as novel binding partners for G␣ and G␤␥ subunits, and/or regulate guanine nucleotide exchange independent of a G-protein coupled receptor (1-6).

A Point Mutation to Gαi Selectively Blocks GoLoco Motif Binding

“…A particularly important ancillary use of the GoLoco-insensitivity mutation will be to delineate a potential role for GoLoco motif proteins in 7TMR-mediated signal transduction. Gain-of-function studies suggest that GPSM1/AGS3 can modulate the cellular levels of G␣ subunits and thus indirectly affect 7TMR signal transduction (75); other studies have suggested that 7TMR signaling in vivo may be modulated by GPSM1 and GPSM2 function (76 -78). Application of the GLi mutant to such studies will surely provide biochemical and structural insights into the biological function of this important class of G␣ regulatory proteins.…”

Identification of Transcription Factor E3 (TFE3) as a Receptor-independent Activator of Gα16

“…COS7 or HEK293 cells were cultured and transfected as described previously (17). In brief, cells were suspended at 0.5-1.0 ϫ 10 5 cells/ml, and 1.0 (12-well plate), 2.0 (35-mm dish), or 10 ml (100-mm dish) was plated.…”

“…The GST-TFE3 fusion protein was expressed in bacteria (Escherichia coli BL21; Amersham Biosciences) and purified on a glutathione affinity matrix. The GST fusion protein was eluted from the resin, and glutathione was removed by desalting to allow a solution-phase interaction assay (17). Protein interaction assays and immunoblotting were performed as described previously (17,29).…”

“…The transfection efficiency was 60 -80%. Cell lysis and fractionation were performed as described previously (17,30).…”

“…Another RGS protein, RGS7, interacts with G␤ 5 and migrates into the nucleus as an RGS7-G␤ 5 complex (16). Furthermore, signal alteration by G-protein and their various types of regulators is involved in adaptation of cells to maintain homeostasis under pathologic conditions (17)(18)(19)(20)(21). In fact, the expression of such regulatory proteins is altered with the development of cardiac hypertrophy in hypertension or in response to pressure overload stress (22,23).…”