Abstract
RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gαi/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gαi1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα 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 Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gαi1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gαo-AlF4(-). Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very 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 hydrogen/deuterium exchange. 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 in host CA2 hippocampal neurons to modulate synaptic plasticity.
Highlights
RGS14 binds distinct forms of active and inactive G␣ proteins through its regulator of G protein signaling (RGS) domain and G protein regulatory (GPR) motif
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
These results indicate that binding of G␣i1-GDP to RGS14 at the GPR motif does not alter the capacity of the RGS domain to bind G␣o-GTP and serve as a GTPase-activating protein (GAP)
Summary
RGS14 binds distinct forms of active and inactive G␣ proteins through its RGS domain and GPR motif. A preformed RGS141⁄7G␣i1GDP 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. Using a variety of complementary cellular and biochemical approaches, we show that binding of active or inactive G␣ subunit differentially affects RGS14 protein conformation and that RGS14 can bind both an inactive G␣-GDP and an active G␣-GDP-AlF4Ϫ subunit simultaneously to form a ternary signaling complex at the plasma membrane Based on these findings, we propose and discuss a working model for RGS14 regulation and integration of G protein signaling at postsynaptic spines of its natural host cell, CA2 hippocampal neurons
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