Integration of G Protein α (Gα) Signaling by the Regulator of G Protein Signaling 14 (RGS14)

RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of G alpha subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active G alpha(i/o)-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive G alpha(i1/3)-GDP. The molecular basis for how RGS14 binds different activation states of G alpha proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of G alpha subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive G alpha(i1)-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated G alpha(o)-AlF4-. Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to G alpha in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive G alpha(i1)-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with G alpha(o)-AlF4- and an AlF4--insensitive mutant (G42R) of G alpha(i1)-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14 center dot G alpha(i1)-GDP complex exhibits full capacity to stimulate the GTPase activity of G alpha(o)-GTP, demonstrating that RGS14 can functionally engage two distinct forms of G alpha subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity.