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Small GTPases - Ras and Rho

Small GTPases - Ras and Rho

Ras and Rho are small monomeric GTPases that act downstream of receptor tyrosine kinase (RTK) and regulate various cellular processes. These GTPases switch between active and inactive states by binding to guanine nucleotides.
Three regulatory proteins control their activity:

Activation and Inactivation of G Proteins

Activation and Inactivation of G Proteins

Heterotrimeric G proteins are guanine nucleotide-binding proteins. As the name suggests, heterotrimeric G proteins are composed of three subunits: alpha, beta, and gamma. They remain GDP-bound or GTP-bound inside the cells and switch between inactive/active states. The Gα subunit possesses the nucleotide-binding pocket that binds guanine nucleotides and switches between GDP or GTP-bound states. In contrast, the Gꞵ and Gγ subunits are always bound together with high affinity and are together...

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, also known...

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, also known...

Rab Proteins

Rab Proteins

Rab proteins constitute the largest family of monomeric GTPases, of which 70 members are present in humans. Rab proteins and their effectors regulate consecutive stages of vesicle transport such as vesicle transport, docking, and fusion to the correct recipient membrane.
Rab proteins switch between a cytosolic, GDP-bound inactive state and a membrane-anchored, GTP-bound active state. By themselves, Rabs show slow rates of GDP/GTP exchange and GTP hydrolysis. Thus, Rab proteins are considered...

GPCRs Regulate Adenylyl Cylase Activity

GPCRs Regulate Adenylyl Cylase Activity

Some GPCRs transmit signals through adenylyl cyclase (AC), a transmembrane enzyme. AC helps synthesize second messenger cyclic adenosine monophosphate (cAMP). AC catalyzes cyclization reaction and converts ATP to cAMP by releasing a pyrophosphate. The pyrophosphate is further hydrolyzed to phosphate by the enzyme pyrophosphatase, which drives cAMP synthesis to completion. However, cAMP is rapidly degraded to 5′ AMP by the enzymes phosphodiesterase (PDE), preventing overstimulation of cells.
Two...

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Updated: Jul 6, 2026

RhoC GTPase Activation Assay

RhoC GTPase Activation Assay

Published on: August 22, 2010

Rho GTPase activation assays.

Stéphanie Pellegrin¹, Harry Mellor

¹Mammalian Cell Biology Laboratory, Department of Biochemistry, School of Medical Sciences, University of Bristol, United Kingdom.

Current Protocols in Cell Biology

|March 25, 2008

Summary

This study details pull-down assays to measure Rho GTPase activation, a key process in cell signaling. The methods enable quantification of RhoA, Rac, and Cdc42 activation levels in cell extracts.

Area of Science:

Cellular Biology
Molecular Biology
Biochemistry

Background:

Rho GTPases are crucial regulators of dynamic cellular processes.
Understanding Rho GTPase activation is vital for studying cell signaling pathways.

Purpose of the Study:

To describe standardized techniques for Rho GTPase activation assays.
To provide practical guidance for performing pull-down assays for RhoA, Rac, and Cdc42.

Main Methods:

Utilizes pull-down assays with specific binding proteins to capture activated Rho GTPases.
Employs Western blotting with specific antibodies for quantification of captured proteins.
Details reagent preparation for RhoA, Rac, and Cdc42 assays.

Main Results:

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Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

Published on: March 31, 2012

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Last Updated: Jul 6, 2026

RhoC GTPase Activation Assay

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Published on: August 22, 2010

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

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Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

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Published on: March 31, 2012

The described methods allow for the investigation and quantification of Rho GTPase activation.
Practical tips are provided for successful assay application in various experimental contexts.

Conclusions:

Standardized pull-down assay protocols facilitate the study of Rho GTPase signaling.
These techniques are essential for researchers investigating cellular dynamics and signaling pathways.