Integration of G protein ? (G?) signaling by the regulator of G protein signaling 14 (RGS14).

ABSTRACT: RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of G? subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active G?i/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive G?i1/3-GDP. The molecular basis for how RGS14 binds different activation states of G? 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? subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive G?i1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated G?o-AlF4(-). Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to G? 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?i1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with G?o-AlF4(-) and an AlF4(-)-insensitive mutant (G42R) of G?i1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·G?i1-GDP complex exhibits full capacity to stimulate the GTPase activity of G?o-GTP, demonstrating that RGS14 can functionally engage two distinct forms of G? 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.

SUBMITTER: Brown NE 

PROVIDER: S-EPMC4423691 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature