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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, also known...

Directionality of Nuclear Transport

Directionality of Nuclear Transport

Ras-related nuclear protein or Ran is a small G protein that cycles between its GTP and GDP bound states. Ran specific regulators, a Ran GTPase Activating Protein or RanGAP present in the cytosol and a Ran guanine nucleotide exchange factor or RanGEF present inside the nucleus regulate GTP/GDP exchange. A high concentration of GTP inside the cells, in addition to this asymmetric distribution of Ran-specific regulators, leads to a higher RanGTP concentration inside the nucleus. This...

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...

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...

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...

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Related Experiment Video

Updated: Jul 16, 2026

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

Molecular basis for Rac1 recognition by guanine nucleotide exchange factors.

A E Karnoub¹, D K Worthylake, K L Rossman

¹Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina 27599, USA.

Nature Structural Biology

|October 31, 2001

Summary

Researchers identified key Rac1 protein regions essential for recognition by guanine nucleotide exchange factors (GEFs). A specific mutation altered Rac1

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

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Last Updated: Jul 16, 2026

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

Comparing the Affinity of GTPase-binding Proteins using Competition Assays

Comparing the Affinity of GTPase-binding Proteins using Competition Assays

Published on: October 8, 2015

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

Area of Science:

Molecular Biology
Structural Biology
Biochemistry

Background:

Rho GTPases are crucial regulators of cellular processes.
Guanine nucleotide exchange factors (GEFs), particularly Dbl family proteins, activate Rho GTPases.
The structural mechanisms underlying GEF-GTPase specificity are not fully understood.

Purpose of the Study:

To elucidate the structural basis for the specific interaction between Rac1 and its GEF, Tiam1.
To identify the structural determinants within Rac1 that govern GEF recognition and specificity.

Main Methods:

X-ray crystallography of Tiam1 DH/PH domains complexed with Rac1.
Site-directed mutagenesis of Rac1 to probe interaction interfaces.
Functional assays to assess GEF activity and downstream signaling.

Main Results:

Specific residues within the Rac1 beta2-beta3 region are critical for Tiam1 binding.
A single point mutation (W56F) in Rac1 abrogated Tiam1 sensitivity.
This mutation enabled recognition by a different GEF (Intersectin) without affecting Rac1's downstream functions.

Conclusions:

Identified novel GEF specificity determinants within Rac1.
Provided structural insights into the DH/PH domain-mediated selection of GTPase targets.
Demonstrated the potential to engineer GEF-GTPase interaction specificity.