# Structural and kinetic investigation of the guanine nucleotide exchange factor Ric-8A and its interaction with G proteins (Gαq/Gαi)

> **NIH NIH F32** · UNIVERSITY OF MONTANA · 2020 · $62,712

## Abstract

PROJECT SUMMARY/ABSTRACT
 Heterotrimeric G proteins are involved in the regulation of many physiological processes and are essential
to multiple cell signaling pathways. G protein heterotrimers are formed through association of α, β, and γ subunits
with guanidine diphosphate (GDP) bound to the Gα subunit. The canonical signaling mechanism involves
displacement of GDP from the Gα subunit by guanidine triphosphate (GTP) and subsequent disassociation of
the Gβγ dimeric complex from Gα-GTP; the Gβγ and Gα-GTP complexes then act to regulate downstream
effectors in the cell. G protein coupled receptors (GPCRs) represent the most well-studied class of proteins
responsible for catalyzing the exchange of GDP for GTP on the Gα subunit. However, other nonreceptor guanine
exchange factor (GEF) proteins also exist and have been shown to be crucial to regulation of G protein signaling.
Resistance to Inhibitors of Cholinesterase-8A (Ric-8A) is a protein that has been shown to act as a GEF and
molecular chaperone for Gα subunits of Gi, Gq and G12/13 families. Although Ric-8A acts as a GEF to activate
both Gαq/Gαi, previous research has demonstrated that the affinity of Ric-8A is different for Gαq in comparison
to Gαi, and that the kinetics of the Ric-8A-catalyzed guanine nucleotide reaction are unique in relation to these
two subtypes. Further, Ric-8A contains several phosphorylation sites and the phosphorylated form of Ric-8A has
enhanced GEF activity toward Gα subunits. The effect of phosphorylation of Ric-8A has been shown to be
different for Gαq relative to Gαi. Aim 1 of this work is to systematically measure the kinetics of GEF activity of
Ric-8A for Gαq/Gαi using stopped-flow fluorescence spectroscopy to allow for a direct comparison, and to
determine the effect of Ric-8A phosphorylation. Aim 2 is to elucidate the structure of Ric-8A:Gαq complexes in
the GDP-free and GDP-bound forms by X-ray crystallography and cryo-electron microscopy. These studies will
provide new insight into the distinct mechanism of Ric-8A GEF activity for Gαq/Gαi, and provide, for the first
time, an atomic-resolution model of the Ric-8A:Gαq complex. This work will enhance our understanding of the
role of Ric-8A in G protein signaling and have broad implications in human health and disease by laying the
foundation for development of future therapeutics that target cytoplasmic G protein activation.

## Key facts

- **NIH application ID:** 9909050
- **Project number:** 1F32GM136014-01
- **Recipient organization:** UNIVERSITY OF MONTANA
- **Principal Investigator:** Sascha Stump
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $62,712
- **Award type:** 1
- **Project period:** 2020-01-01 → 2020-12-18

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9909050

## Citation

> US National Institutes of Health, RePORTER application 9909050, Structural and kinetic investigation of the guanine nucleotide exchange factor Ric-8A and its interaction with G proteins (Gαq/Gαi) (1F32GM136014-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9909050. Licensed CC0.

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