Integration of G Protein Alpha (Gα) Signaling by the Regulator of G Protein Signaling 14

Abstract

RGS14 contains distinct binding sites for both active (GTP bound) and inactive (GDP bound) forms of Gα proteins. The N‐terminal RGS domain binds active Gαi/o‐GTP, whereas the C‐terminal GPR motif binds inactive Gαi1/3‐GDP. The molecular basis for how RGS14 binds different activation states of Gα to integrate G protein signaling is unknown. Here we explored the effects of G protein binding on the GPR motif and the RGS domain, and examined whether RGS14 can functionally interact with two forms of Gα simultaneously. Using cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gαi1‐GDP at the plasma membrane (PM), and that cytosolic RGS14 is recruited to the PM by activated Gαo‐AlF4‐. Bioluminescence resonance energy transfer (BRET) studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen deuterium exchange mass spectrometry (HDX‐MS) revealed RGS14 is a dynamic protein that undergoes allosteric conformational changes when inactive Gαi1‐GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gαo‐AlF4‐ and an AlF4‐‐insensitive mutant (G42R) of Gαi1‐GDP as observed by size exclusion chromatography and differential HDX‐MS. Finally, a preformed RGS14:Gαi1‐GDP complex exhibits full capacity to stimulate the GTPase activity of Gαo‐GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals.

Mechanisms of mGluR‐dependent plasticity in hippocampal area CA2

“…RGS14 has a number of functional domains/motifs that are distinct from its RGS domain, so RGS14 actions on mGluR-LTD could be independent of its GAP activity. Consistent with this idea, RGS14 contains a GPR motif that is required for plasma membrane anchoring (Brown et al, 2015), and this motif was found to be essential for RGS14 regulation of synaptic plasticity in hippocampal neurons independent of the RGS domain function (Squires et al, 2021). Additionally, RGS14 binds active H-Ras to inhibit ERK signaling (Shu et al, 2010).…”

RGS14 expression in CA2 hippocampus, amygdala, and basal ganglia: Implications for human brain physiology and disease

“…The G protein regulatory (GPR)/GoLoco motif binds inactive Gαi1/3-GDP (Hollinger et al, 2001;Kimple et al, 2001;Mittal & Linder, 2004). Binding of RGS14 to inactive Gαi1-GDP or Gαi3-GDP serves to anchor RGS14 at the plasma membrane thereby allowing for the RGS domain to bind and GAP a second active Gαi/o (Figure 1) (Brown et al, 2015;Brown et al, 2016).…”