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GTPases and their Regulation

GTPases and their Regulation

Guanine nucleotide-binding proteins (G-proteins), also known as GTPases, are a superfamily of proteins that regulate many cellular processes, such as cell signaling, vesicular transport, and the regulation of cell shape and motility. Mutation or dysfunction of these proteins can lead to disease. There are around 40,000 known G-proteins that can broadly be classified into two groups ‒ small G-proteins consisting of a single domain and large multi-domain G-proteins.
Large G-proteins,...

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Related Experiment Video

Updated: Jun 13, 2025

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay

Published on: November 11, 2018

Quantifying small GTPase activation status using a novel fluorescence HPLC-based assay.

Makoto Araki¹, Yukika Kasuya¹, Kaho Yoshimoto¹

¹Department of Biochemistry, Meiji Pharmaceutical University, Tokyo, Japan.

The Journal of Biological Chemistry

|April 26, 2025

Summary

This summary is machine-generated.

A new Fluor-HPLC assay precisely quantifies small GTPase activation states, crucial for understanding cancer and cellular signaling. This method advances research into GTPase function and disease pathogenesis.

Keywords:

HRAS KRAS RHEB anticancer drug high-performance liquid chromatography (HPLC)small GTPase

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Published on: March 31, 2012

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Last Updated: Jun 13, 2025

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Published on: November 11, 2018

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

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Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein GST-RhoAG17A from Epithelial Cell Lysates

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein GST-RhoAG17A from Epithelial Cell Lysates

Published on: March 31, 2012

Area of Science:

Biochemistry
Cell Biology
Molecular Biology

Background:

Small GTPases are key regulators of cellular signaling pathways.
Their activation states (GDP-bound inactive vs. GTP-bound active) are critical for function.
Dysregulation of GTPase states, like in RAS oncogenes, contributes to diseases such as cancer.

Purpose of the Study:

To develop a highly sensitive and versatile method for quantifying small GTPase activation states.
To overcome limitations of existing techniques in small GTPase analysis.
To enable precise measurement of GTPase nucleotide-binding states in cellular and in vivo models.

Main Methods:

Development of a highly sensitive High-Performance Liquid Chromatography (HPLC)-based assay with fluorescence detection (Fluor-HPLC).
Quantification of guanine nucleotide-binding states of small GTPases at endogenous expression levels.
Application of Fluor-HPLC in cellular studies and syngeneic mouse models.

Main Results:

Fluor-HPLC successfully quantified small GTPase guanine nucleotide-binding states with high sensitivity.
The method elucidated RHEB and HRAS activation in response to extracellular stimuli.
KRAS activation dynamics in tumor tissues were investigated, and therapeutic efficacy was evaluated in mouse models.

Conclusions:

Fluor-HPLC is a versatile and sensitive method for precise quantification of small GTPase activation states.
This technique facilitates the study of GTPase roles in cellular processes and disease.
The method has potential to accelerate understanding of GTPase-related pathogenesis and therapeutic strategies.