# CLIC function in GPCR-mediated Rho/Rac signaling

> **NIH NIH R01** · UNIVERSITY OF ILLINOIS AT CHICAGO · 2021 · $397,295

## Abstract

G-protein-coupled receptor (GPCR)-mediated regulation of the GTPases Rho and Rac is a conserved signaling
module required for vital cell behaviors. However, the mechanisms that connect GPCRs, and their heterotrimeric
G-protein (Gα/β/γ) partners, to Rho/Rac are not fully delineated. Our studies in C. elegans and human endothelial
cells have revealed a new conserved player required for Rho/Rac signaling: the Chloride Intracellular Channel
(CLIC) family of proteins. Our data shows that CLICs are required in two important endothelial GPCR pathways
(S1P/S1P Receptor and thrombin/PAR) that function through Gα12/13 and Gαi to activate Rho and Rac. This
CLIC function is evolutionarily conserved, because we found that in C. elegans the CLIC ortholog exc-4
genetically interacts with the Gα12 ortholog gpa-12 and with the Rac orthologs ced-10 and mig-2. The molecular
function of CLICs has long remained a mystery. Based on sequence and structural similarities, they have been
proposed to function as chloride channels and/or as glutathione S-transferases (GST). Previous work has shown
that EXC-4 membrane localization is mediated by an N-terminal domain and this localization is critical for function.
We have now found that CLIC membrane localization is also critical for its role in GPCR-mediated Rho/Rac
activation in endothelial cells, and that replacement of the membrane-targeting N-terminus with a myristoylation
signal is sufficient to restore this function. Since the channel and GST activities of CLICs require an intact N-
terminus we have discovered a novel activity for CLICs. We hypothesize that CLICs are membrane-localized
regulators of Rho and Rac that respond to GPCR-Gα signaling. In Aim 1 we will determine how CLICs couple
GPCR-Gα (Gα12/13 and Gαi) signaling to Rho and Rac. We will survey the requirement for CLICs in different cell
and signaling contexts to define key GPCR-Gα combinations that utilize CLICs to regulate of Rho and Rac. We
will use cutting-edge bio-sensors and genetic tools to measure and modulate signaling to determine which step
in the GPCR-Gαβγ-Rho/Rac cascade requires CLICs. Finally, we will test whether CLICs physically interact with
Rho/Rac to modulate signaling. In Aim 2 we will define the determinants by which CLICs regulate Rho/Rac in
human cells and in C. elegans by performing structure-function analyses, focused on the EXC-4/CLIC C-
terminus. Critical domains defined in this Aim will be tested for their ability to interact with Rho/Rac (as defined
in Aim 1). In Aim 3 we will carry out unbiased genetic and proteomic screens in C. elegans to find conserved
players that genetically and physically interact with EXC-4/CLIC to further elucidate how CLICs regulate Rho/Rac
signaling. We will then test whether human orthologs of genes identified in these screens influence Rho/Rac
signaling in mammalian cells. By defining new mechanisms of action for CLICs in GPCR-Gαβγ-Rho/Rac signaling
we will significantly increase our knowledge of how...

## Key facts

- **NIH application ID:** 10135123
- **Project number:** 5R01GM134032-02
- **Recipient organization:** UNIVERSITY OF ILLINOIS AT CHICAGO
- **Principal Investigator:** Jan K. Kitajewski
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $397,295
- **Award type:** 5
- **Project period:** 2020-04-01 → 2024-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10135123

## Citation

> US National Institutes of Health, RePORTER application 10135123, CLIC function in GPCR-mediated Rho/Rac signaling (5R01GM134032-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10135123. Licensed CC0.

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