PXD069453

PXD069453 is an original dataset announced via ProteomeXchange.

Title	RASA3 identified as a potential effector of the brain G protein G⍺o by mass spectrometry
Description	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 Gβ𝛾 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 GTP𝛾S 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-GTP𝛾S more strongly than it does to G⍺o-GDP. We also found that the addition of Ca2+, a second messenger produced by the G⍺q pathway that opposes G⍺o signaling, strengthened RASA3-G⍺o binding. A C-terminal fragment of RASA3 containing a predicted Ca2+ site was sufficient to bind G⍺o. Binding to G⍺q by this RASA3 fragment was strengthened by Ca2+ but, unlike full-length RASA3, showed a preference for binding G⍺o-GDP instead of G⍺o-GTP𝛾S. 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 via the G⍺o switch regions, and a second on the RASA3 C-terminus that preferentially binds G⍺o-GDP in the presence of Ca2+.
HostingRepository	PRIDE
AnnounceDate	2026-01-23
AnnouncementXML	Submission_2026-01-23_01:31:00.645.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	TuKiet Lam
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: NEWT:10090;
ModificationList	monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Q Exactive Plus

Revision	Datetime	Status	ChangeLog Entry
0	2025-10-14 12:13:59	ID requested
⏵ 1	2026-01-23 01:31:01	announced

10.1016/j.jbc.2025.110999;

Bell HAS, Olson AC, Gentile JE, Lam TT, Koelle MR, . J Biol Chem, 302(1):110999(2026) [pubmed]

submitter keyword: GTPase activating protein (GAP), proteomics, neurotransmitter signaling,Heterotrimeric G Protein, protein-protein interaction, RASA3, G⍺o

TuKiet Lam
contact affiliation	Keck MS & Proteomics Resource Yale School of Medicine
contact email	tukiet.lam@yale.edu
lab head
TuKiet Lam
contact affiliation	Yale University
contact email	tukiet.lam@yale.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD069453
     1. Label: PRIDE project
     2. Name: RASA3 identified as a potential effector of the brain G protein G⍺o by mass spectrometry