Year: 2025
Type: article
Abstract: G⍺ o , the alpha subunit of the most abundant heterotrimeric G protein in the brain, mediates signaling by opioids and by many neuromodulators to inhibit neural function. An open question is whether activated G⍺ o -GTP directly binds to and regulates effector molecules, like all other animal G⍺ proteins, or if it signals solely by releasing Gβ γ subunits. Using mouse brain lysates as native source of G⍺ o and its potential effectors, we analyzed immunopurified G⍺ o protein complexes by mass spectrometry. Pre-activating G⍺ o in the lysates with GTP γ S resulted in a ∼6 fold increase in the amount of the small G protein GTPase activators RASA3 and RASA2 in the purified complexes, the largest increase among all G⍺ o -associated proteins, making RASA2/3 candidate G⍺ o effectors. Using purified recombinant proteins, we found that RASA3 binds directly to G⍺ o -GTP γ S more strongly than it does to G⍺ o -GDP. We also found that the addition of Ca 2+ , a second messenger produced by the G⍺ q pathway that opposes G⍺ o signaling, strengthened binding of RASA3 to G⍺ o -GDP . A C-terminal fragment of RASA3 containing a predicted Ca 2+ site was sufficient to bind G⍺ o , albeit more weakly and without a preference for the activated state of G⍺ o . We present a model in which RASA3 could mediate G⍺ o signaling using two distinct G⍺ o -binding sites: one on full-length RASA3 that preferentially binds active G⍺ o -GTP, and a second on the RASA3 C-terminus that binds inactive G⍺ o in the presence of Ca 2+ .
Source: Journal of Biological Chemistry
Authors: Halie A.S. Bell, Andrew C. Olson, Juliana E. Gentile, TuKiet T. Lam, Michael R. Koelle
Institution: Yale University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,430
Awards: G1064903623, G1263806256, G2399585601, An Ultra-Performance Liquid Chromatography System to Support Metabolomics at Yale University, High Resolution Tandem Mass Spectrometer to Meet Growing Demand for Proteomics at Yale, G6936211521, G7169517480, G7229188214, 6500 QTrap Mass Spectrometer for Yale University, G882930799, G9000025815, Yale/NIDA Neuroproteomics Center
OpenAlex ID: W4416958456