Novel Activity of RGS14 on Goα and Giα Nucleotide Binding and Hydrolysis Distinct from Its RGS Domain and GDI Activity

Abstract

The bifunctional protein RGS14 is both a GTPase activating protein (GAP) for Gialpha and Goalphaand a guanine nucleotide dissociation inhibitor (GDI) for Gialpha. This GDI activity is isolated to a region of the protein distinct from the RGS domain that contains an additional G protein-binding domain (RBD/GL). Here, we report that RGS14 missing its RGS domain (R14-RBD/GL) binds directly to Go and Gi to modulate nucleotide binding and hydrolysis by mechanisms distinct from its defined GDI activity. In brain pull-down assays, full-length RGS14 and R14-RBD/GL (but not the isolated RGS domain of RGS14) bind Goalpha-GDP, Gialpha-GDP, and also Gbetagamma. When reconstituted with M2 muscarinic receptors (M2) plus either Gi or Go, RGS4 (which has no RBD/GL domain) and full-length RGS14 each markedly stimulates the steady-state GTPase activities of both G proteins, whereas R14-RBD/GL has little or no effect. R14-RBD/GL potentiates RGS4 GAP activity in membrane-based assays by increasing the apparent affinity of RGS4 for Gialpha and Goalpha, suggesting a cooperative interaction between the RBD/GL domain, RGS4, and Galpha. This activity of R14-RBD/GL on RGS4 is not apparent in single-turnover solution GAP assays with purified Gialpha or Goalpha, suggesting that membranes and/or receptors are required for this activity. When these findings are taken together, they indicate that regions of RGS14 outside of the RGS domain can bind inactive forms of Go and Gi to confer previously unappreciated activities that influence Galphanucleotide binding and/or hydrolysis by mechanisms distinct from its RGS domain and established GDI activity.

G Protein-coupled Receptors and Resistance to Inhibitors of Cholinesterase-8A (Ric-8A) Both Regulate the Regulator of G Protein Signaling 14 (RGS14)·Gαi1 Complex in Live Cells

“…The fact that the BRET signal was never completely abolished in the presence of the RGS14 and G␣ i1 double mutants that ablate G␣ binding to both the GPR and RGS domains (Fig. 2, B and C) is consistent with the existence of a third G protein binding site on RGS14, as has been postulated (51).…”

Regulators of G-Protein-Signaling Proteins: Negative Modulators of G-Protein-Coupled Receptor Signaling

“…RGS4-GFP was most abundant in the striatum and amygdala of the subcortex. In situ hybridization using RGS4 riboprobes revealed greater amounts of RGS4 in several regions of the frontal cortex with lower levels in the thalamus and striatum (Erdely et al, 2004;Ha et al, 2013;Heraud-Farlow et al, 2013;Kim et al, 2013;Lener et al, 2013;Li et al, 2013a;Steiner et al, 2014;Stratinaki et al, 2013;Wamsteeker Cusulin et al, 2013).…”

“…Further studies demonstrated that RGS14 interacts with and modulates the GTPSase activity of subunits of heterotrimeric proteins from G i/o families (Cho et al, 2000;Hepler et al, 2005;Traver et al, 2000). Actually, it is well known that RGS14 is implicated in a number of cellular processes, including, lymphocyte functions, centrosome formation and nuclear functions, and stress-induced signaling pathways (Cho and Kehrl, 2007;Cho et al, 2005Cho et al, , 2000Lin et al, 2011;Martin-McCaffrey et al, 2004a;Martin-McCaffrey et al, 2004b;MartinMcCaffrey et al, 2005b;Shu et al, 2007).…”

The impact of RGS and other G-protein regulatory proteins on Gαi-mediated signaling in immunity

“…Mouse RGS14 is a 547 amino acid proteins that has a N-terminal RGS domain, two Ras binding domains, and a GoLoco motif. RGS14 can simultaneously bind GDP-Gα i via it GoLoco motif and act as a GAP for GTP bound Gα i (108,118–120). RGS14 can interacts with the monomeric G proteins Rap1, Rap2, and H-Ras (121).…”