Activation of the Regulator of G Protein Signaling 14−Gαi1-GDP Signaling Complex Is Regulated by Resistance to Inhibitors of Cholinesterase-8A

Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322, United States.
Biochemistry (Impact Factor: 3.02). 02/2011; 50(5):752-62. DOI: 10.1021/bi101910n
Source: PubMed


RGS14 is a brain scaffolding protein that integrates G protein and MAP kinase signaling pathways. Like other RGS proteins, RGS14 is a GTPase activating protein (GAP) that terminates Gαi/o signaling. Unlike other RGS proteins, RGS14 also contains a G protein regulatory (also known as GoLoco) domain that binds Gαi1/3-GDP in cells and in vitro. Here we report that Ric-8A, a nonreceptor guanine nucleotide exchange factor (GEF), functionally interacts with the RGS14-Gαi1-GDP signaling complex to regulate its activation state. RGS14 and Ric-8A are recruited from the cytosol to the plasma membrane in the presence of coexpressed Gαi1 in cells, suggesting formation of a functional protein complex with Gαi1. Consistent with this idea, Ric-8A stimulates dissociation of the RGS14-Gαi1-GDP complex in cells and in vitro using purified proteins. Purified Ric-8A stimulates dissociation of the RGS14-Gαi1-GDP complex to form a stable Ric-8A-Gαi complex in the absence of GTP. In the presence of an activating nucleotide, Ric-8A interacts with the RGS14-Gαi1-GDP complex to stimulate both the steady-state GTPase activity of Gαi1 and binding of GTP to Gαi1. However, sufficiently high concentrations of RGS14 competitively reverse these stimulatory effects of Ric-8A on Gαi1 nucleotide binding and GTPase activity. This observation correlates with findings that show RGS14 and Ric-8A share an overlapping binding region within the last 11 amino acids of Gαi1. As further evidence that these proteins are functionally linked, native RGS14 and Ric-8A coexist within the same hippocampal neurons. These findings demonstrate that RGS14 is a newly appreciated integrator of unconventional Ric-8A and Gαi1 signaling.

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    • "We finally tested the role of the C-terminal region of Ga proteins in Ric-8A-mediated stabilization. It has been shown that the C-terminus of Gai is required for direct interaction with Ric- 8A, on the basis of assays using purified proteins [22] [34]. Here we tested the role of the C-terminal region in the interaction of Ric-8 with Gai2 and Gaq at the cellular level. "
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    ABSTRACT: The cytosolic protein Ric-8A acts as a guanine nucleotide exchange factor for Gα subunits of the Gi, Gq, and G12/13 classes of heterotrimeric G protein in vitro, and also increases the amounts of these Gα proteins in vivo. The mechanism whereby Ric-8 regulates Gα content has not been fully understood. Here we show that Ric-8A appears to stabilize Gαi2 and Gαq by preventing their ubiquitination. Ric-8A interacts with and stabilizes Gαi2, Gαq, Gα12, but not Gαs, when expressed in COS-7 cells. The protein levels of Gαi2 and Gαq appear to be controlled via the ubiquitin-proteasome degradation pathway, because these Gα subunits undergo polyubiquitination and are stabilized with the proteasome inhibitor MG132. The ubiquitination of Gαi2 and Gαq is suppressed by expression of Ric-8A. The suppression appears to require Ric-8A interaction with these Gα proteins, because the C-terminal truncation of Gαq and Gαi2 completely abrogates their interaction with Ric-8A, their stabilization by Ric-8A, and Ric-8A-mediated inhibition of Gα ubiquitination.
    Preview · Article · May 2013 · Biochemical and Biophysical Research Communications
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    • "Finally, substitution of the C-terminal twelve residues of Gαi1 with the corresponding residues of Gαs, a Gα protein that does not bind to Ric-8A [4], abrogated susceptibility to the GEF activity of Ric-8A (Figure 2) but did not impair GTP binding activity. These results are in accord with recent findings that pertussis toxin-catalyzed ADP ribosylation at the C-terminus of Gαi1 [8] and that truncation of the twelve Gαi1 C-terminal residues [25] blocks Ric-8A binding and GEF activity. On the other hand, truncation of 25 residues from the N-terminus of Gαi1 did not affect its susceptibility to the GEF activity of Ric-8A (Figure 2). "
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    ABSTRACT: Heterotrimeric G protein α subunits are activated upon exchange of GDP for GTP at the nucleotide binding site of Gα, catalyzed by guanine nucleotide exchange factors (GEFs). In addition to transmembrane G protein-coupled receptors (GPCRs), which act on G protein heterotrimers, members of the family cytosolic proteins typified by mammalian Ric-8A are GEFs for Gi/q/12/13-class Gα subunits. Ric-8A binds to Gα•GDP, resulting in the release of GDP. The Ric-8A complex with nucleotide-free Gαi1 is stable, but dissociates upon binding of GTP to Gαi1. To gain insight into the mechanism of Ric-8A-catalyzed GDP release from Gαi1, experiments were conducted to characterize the physical state of nucleotide-free Gαi1 (hereafter referred to as Gαi1[ ]) in solution, both as a monomeric species, and in the complex with Ric-8A. We found that Ric-8A-bound, nucleotide-free Gαi1 is more accessible to trypsinolysis than Gαi1•GDP, but less so than Gαi1[ ] alone. The TROSY-HSQC spectrum of [(15)N]Gαi1[ ] bound to Ric-8A shows considerable loss of peak intensity relative to that of [(15)N]Gαi1•GDP. Hydrogen-deuterium exchange in Gαi1[ ] bound to Ric-8A is 1.5-fold more extensive than in Gαi1•GDP. Differential scanning calorimetry shows that both Ric-8A and Gαi1•GDP undergo cooperative, irreversible unfolding transitions at 47° and 52°, respectively, while nucleotide-free Gαi1 shows a broad, weak transition near 35°. The unfolding transition for Ric-8A:Gαi1[ ] is complex, with a broad transition that peaks at 50°, suggesting that both Ric-8A and Gαi1[ ] are stabilized within the complex, relative to their respective free states. The C-terminus of Gαi1 is shown to be a critical binding element for Ric-8A, as is also the case for GPCRs, suggesting that the two types of GEF might promote nucleotide exchange by similar mechanisms, by acting as chaperones for the unstable and dynamic nucleotide-free state of Gα.
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    ABSTRACT: Beyond the core triad of receptor, Gαβγ and effector, there are multiple accessory proteins that provide alternative modes of signal input and regulatory adaptability to G-protein signalling systems. Such accessory proteins may segregate a signalling complex to microdomains of the cell, regulate the basal activity, efficiency and specificity of signal propagation and/or serve as alternative binding partners for Gα or Gβγ independent of the classical heterotrimeric Gαβγ complex. The latter concept led to the postulate that Gα and Gβγ regulate intracellular events distinct from their role as transducers for cell surface seven-transmembrane span receptors. One general class of such accessory proteins is defined by AGS proteins or activators of G-protein signalling that refer to mammalian cDNAs identified in a specific yeast-based functional screen. The discovery of AGS proteins and related entities revealed a number of unexpected mechanisms for regulation of G-protein signalling systems and expanded functional roles for this important signalling system.
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