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Structural and kinetic investigation of the guanine nucleotide exchange factor Ric-8A and its interaction with G proteins (G?q/G?i)

Award Number: F32GM136014
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS

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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 a, ß, and ¿ subunits with guanidine diphosphate (GDP) bound to the Ga subunit. The canonical signaling mechanism involves displacement of GDP from the Ga subunit by guanidine triphosphate (GTP) and subsequent disassociation of the Gß¿ dimeric complex from Ga-GTP; the Gß¿ and Ga-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 Ga 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 Ga subunits of Gi, Gq and G12/13 families. Although Ric-8A acts as a GEF to activate both Gaq/Gai, previous research has demonstrated that the affinity of Ric-8A is different for Gaq in comparison to Gai, 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 Ga subunits. The effect of phosphorylation of Ric-8A has been shown to be different for Gaq relative to Gai. Aim 1 of this work is to systematically measure the kinetics of GEF activity of Ric-8A for Gaq/Gai 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:Gaq 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 Gaq/Gai, and provide, for the first time, an atomic-resolution model of the Ric-8A:Gaq 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.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2022 ( Subtotal = -$5,158 )
2022	2021	UNIVERSITY OF MONTANA	32 CAMPUS DR	MISSOULA	MT	59812	MISSOULA	USA	Biomedical Research and Research Training	000	2	4/22/2022	NON-COMPETING CONTINUATION	$0
2022	2020	UNIVERSITY OF MONTANA	32 CAMPUS DR	MISSOULA	MT	59812	MISSOULA	USA	Biomedical Research and Research Training	001	1	4/22/2022	NEW	-$5,158
														Subtotal = -$5,158

Issue Date FY: 2021 ( Subtotal = -$1,842 )
2021	2021	UNIVERSITY OF MONTANA	32 CAMPUS DR MAIN HALL	MISSOULA	MT	59812	MISSOULA	USA	Biomedical Research and Research Training	001	2	1/13/2021	NON-COMPETING CONTINUATION	-$64,926
2021	2021	UNIVERSITY OF MONTANA	32 CAMPUS DR MAIN HALL	MISSOULA	MT	59812	MISSOULA	USA	Biomedical Research and Research Training	000	2	12/16/2020	NON-COMPETING CONTINUATION	$64,926
2021	2020	UNIVERSITY OF MONTANA	32 CAMPUS DR MAIN HALL	MISSOULA	MT	59812	MISSOULA	USA	Biomedical Research and Research Training	002	1	2/1/2021	NEW	-$1,842
														Subtotal = -$1,842

Issue Date FY: 2020 ( Subtotal = $64,554 )
2020	2020	UNIVERSITY OF MONTANA	32 CAMPUS DR MAIN HALL	MISSOULA	MT	59812	MISSOULA	USA	Biomedical Research and Research Training	000	1	12/30/2019	NEW	$65,354
2020	2020	UNIVERSITY OF MONTANA	32 CAMPUS DR MAIN HALL	MISSOULA	MT	59812	MISSOULA	USA	Biomedical Research and Research Training	002	1	1/16/2020	NEW	$0
2020	2020	UNIVERSITY OF MONTANA	32 CAMPUS DR MAIN HALL	MISSOULA	MT	59812	MISSOULA	USA	Biomedical Research and Research Training	001	1	1/16/2020	NEW	-$4,500
2020	2020	UNIVERSITY OF MONTANA	32 CAMPUS DR MAIN HALL	MISSOULA	MT	59812	MISSOULA	USA	Biomedical Research and Research Training	003	1	2/27/2020	NEW	$3,700
														Subtotal = $64,554

Grand Total All Awards = $57,554

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Structural and kinetic investigation of the guanine nucleotide exchange factor Ric-8A and its interaction with G proteins (G?q/G?i)

Award Number: F32GM136014

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS

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