Premium
Integration of G Protein Alpha (Gα) Signaling by the Regulator of G Protein Signaling 14
Author(s) -
Brown Nicole,
Goswami Devrishi,
Branch Mary Rose,
Ramineni Suneela,
Ortlund Eric,
Griffin Patrick,
Hepler John
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.lb513
Subject(s) - allosteric regulation , gtp' , chemistry , g protein , regulator of g protein signaling , gtpase activating protein , cytosol , biochemistry , biophysics , microbiology and biotechnology , signal transduction , biology , enzyme
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‐AlF 4 ‐ . 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‐AlF 4 ‐ and an AlF 4 ‐ ‐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.