Date of Award

Fall 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Biophysics and Biochemistry

First Advisor

Koelle, Michael

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�� subunits. Using mouse brain lysates as native source of G⍺o and its potential effectors, I analyzed immunopurified G⍺o protein complexes by mass spectrometry. I identified several proteins that interact preferentially with inactive or active G⍺o. I focused my analysis on proteins that are significantly upregulated in activated G⍺o protein complexes as would be expected for an effector protein. Pre-activating G⍺o in the lysates with GTP��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-GTP��S-associated proteins, making RASA2/3 candidate G⍺o effectors. Using purified recombinant proteins, I found that RASA3 binds directly to G⍺o-GTP��S more strongly than it does to G⍺o-GDP. I also found that the addition of Ca2+, a second messenger produced by the G⍺q pathway that opposes G⍺o signaling, enhanced RASA3-G⍺o binding in the inactive GDP condition. A C-terminal fragment of RASA3 containing a predicted Ca2+ site was sufficient to bind G⍺o-GDP in the presence of Ca2+. Finally, I 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 that preferentially binds G⍺o-GDP in the presence of Ca2+.

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