# Inter-Domain Regulation of p120RasGAP

> **NIH NIH F31** · YALE UNIVERSITY · 2023 · $47,694

## Abstract

PROJECT SUMMARY
GAPs (GTPase activating proteins) play an essential role in the GTPase cycle, allowing for the regulation of
complex processes such as signal transduction; consequently, their dysfunction has significant ramifications. In
the case of p120RasGAP (RasGAP, p120; RASA1), the first GAP described, dysfunctions have been linked to
vascular diseases such as capillary malformation-arteriovenous malformation syndromes (CM-AVM) and vein of
Galen malformations (VOGM). The protein consists of an N-terminal Src homology 2 (SH2)-SH3-SH2 cassette,
followed by pleckstrin homology (PH), C2, and GAP domains. Despite its importance and relatively long history
as a target of study, p120RasGAP has not been adequately analyzed at the structural and biochemical levels.
In particular, although full-length p120RasGAP has been shown to have greater activity than the GAP domain
alone, the contributions of the PH and C2 domains have not specifically been isolated. I hypothesize that the
GAP activity of p120RasGAP is controlled through conformational changes induced by the protein’s
regulatory domains, which are in turn affected by lipid head groups and/or other binding proteins. Specifically,
my preliminary data demonstrate that the C2 domain may play a role in GAP regulation. I will test these
mechanisms in two aims. In Aim 1, I can already express and purify constructs of the GAP, C2, and PH domains
of p120RasGAP, and preliminary enzymatic analysis suggests that the C2 domain accelerates enzymatic
activity. I will conduct detailed enzymatic studies in vitro using GAP assays to assess the roles of these domains.
I will then assess the effect of disease-causing mutations on enzyme activity. Additionally, I will quantify the
affinities of the PH and C2 domains for different lipid head groups and assess C2 calcium sensitivity. These
data will then be used to conduct in vitro GAP assays using vesicle-bound Ras to test the role of membrane
association in PH and C2 regulatory behavior. In Aim 2, I have already obtained and am refining the first crystal
structure of the C2-GAP region of p120RasGAP. This crystal structure demonstrates that the C2 and GAP
domains are connected by a flexible linker and illustrates that the C2 domain is ideally positioned to interact with
the allosteric lobes of Ras in an “opposable thumb” formation. I will also obtain a crystal structure of the C2-
GAP region in complex with HRas to observe this binding directly. Based on analysis of these structures, I will
validate interactions made by the C2 domain via site-directed point mutagenesis and GAP assays. Taken
together, these aims will provide a comprehensive analysis of the roles of the PH and C2 domains in regulation
of p120RasGAP and reveal interactions responsible for the observed changes in activity. These studies will
elucidate molecular bases for vascular diseases including CM-AVM and VOGM.

## Key facts

- **NIH application ID:** 10605754
- **Project number:** 1F31HL167578-01
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Maxum Paul
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $47,694
- **Award type:** 1
- **Project period:** 2023-02-01 → 2026-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10605754

## Citation

> US National Institutes of Health, RePORTER application 10605754, Inter-Domain Regulation of p120RasGAP (1F31HL167578-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10605754. Licensed CC0.

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