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Abstract
Dexras1 is a novel GTP-binding protein (G protein) that was recently discovered on the basis of rapid mRNA up-regulation by glucocorticoids in murine AtT-20 corticotroph cells and in several primary tissues. The human homologue of Dexras1, termed activator of G protein signaling-1 (AGS-1), has been reported to stimulate signaling by G(i) heterotrimeric G proteins independently of receptor activation. The effects of Dexras1/AGS-1 on receptor-initiated signaling by G(i) have not been examined. Here we report that Dexras1 inhibits ligand-dependent signaling by the G(i)-coupled N-formyl peptide receptor (FPR). Dexras1 and FPR were transiently co-expressed in both COS-7 and HEK-293 cells. Activation of FPR by ligand (N-formyl-methionine-leucine-phenylalanine (f-MLF)) caused phosphorylation of endogenous Erk-1/2 that was reduced by co-expression of Dexras1. Direct effects of Dexras1 on the activity of co-expressed, epitope-tagged Erk-2 (hemagglutinin (HA)-Erk-2) were measured by immune complex in vitro kinase assay. Expression of Dexras1 alone resulted in a 1.9- to 4.9-fold increase in HA-Erk-2 activity; expression of the unliganded FPR alone resulted in a 6.2- to 8.1-fold increase in HA-Erk-2 activity. Stimulation of FPR by f-MLF produced a further 8- to 10-fold increase in HA-Erk-2 activity over the basal (non-ligand-stimulated) state, and this ligand-dependent activity was attenuated at the time points of maximal activity by co-expression of Dexras1 (reduced 31 +/- 6.8% in COS-7 at 10 min and 86 +/- 9.2% in HEK-293 at 5 min, p < 0.01 for each). Expression of Dexras1 did not influence protein expression of FPR or Erk, suggesting that the inhibitory effects of Dexras1 reflect a functional alteration in the signaling cascade from FPR to Erk. Expression of Dexras1 had no effect on expression of G(i)alpha species, but significantly impaired pertussis toxin-catalyzed ADP-ribosylation of membrane-associated G(i)alpha. Expression of Dexras1 also significantly decreased in vitro binding of GTPgammaS in f-MLF-stimulated membranes of cells co-transfected with FPR. These data suggest that Dexras1 inhibits signal transduction from FPR to Erk-1/2 through an effect that is very proximal to receptor-G(i) coupling. While Dexras1 weakly activates Erk in the resting state, more potent effects are evident in the modulation of ligand-stimulated receptor signal transduction, where Dexras1 functions as an inhibitor rather than activator of the Erk mitogen-activated protein kinase signaling cascade.
Highlights
Dexras1 is the prototypic member of a recently defined group of Ras-related intermediate molecular weight, basic GTP-binding proteins (G proteins)
The group, which includes Dexras1, the activator of G protein signaling 1 (AGS-1) (1), the Ras homologue enriched in striatum (2), tumor endothelial marker 2 (TEM-2) (3), and Drosophila Dexras, is characterized by highly basic net isoelectric points and molecular weights intermediate between those of other Ras family members and the heterotrimeric G protein ␣ subunits (4)
The yeast pheromone pathway (8) is analogous to the mammalian Erk-1/2 MAP1 kinase pathway that is initiated by activation of many Gi-coupled receptors, including the N-formyl peptide receptor (FPR), by their cognate ligand (9, 10)
Summary
Dexras is the prototypic member of a recently defined group of Ras-related intermediate molecular weight, basic GTP-binding proteins (G proteins). The yeast pheromone pathway (8) is analogous to the mammalian Erk-1/2 MAP1 kinase pathway that is initiated by activation of many Gi-coupled receptors, including the N-formyl peptide receptor (FPR), by their cognate ligand (9, 10). Direct effects of Dexras1/AGS-1 on the phosphorylation state or kinase activity of Erk have not been demonstrated in mammalian cells It is not known whether Dexras1/AGS-1 play a role in ligand-dependent signaling by Gi-coupled receptors. We hypothesized that guanyl nucleotidebound Dexras (Dexras11⁄7GTP or Dexras11⁄7GDP) interacts with Gi␣ in a ligand-independent fashion to effect catalytic activities similar to those associated with ligand-dependent, receptorinitiated activation of Gi␣ According to this hypothesis, the release of G␥ from the Gi heterotrimer initiated by Dexras would lead to ligand-independent activation of the Erk-1/2 MAP kinase pathway. We report that expression of Dexras increases Erk activity in COS-7 and HEK-293 cells but simultaneously attenuates further activation of Erk by ligand-stimulated FPR, acting through a mechanism that is proximal to receptorϪGi␣ coupling
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