Related Experiment Video

Updated: Aug 11, 2026

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates
11:28

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

Identification of the guanine nucleotide dissociation stimulator for Ral as a putative effector molecule of R-ras,

M Spaargaren1, J R Bischoff

  • 1ONYX Pharmaceuticals, Richmond, CA 94806.

Proceedings of the National Academy of Sciences of the United States of America
|December 20, 1994
PubMed

Insights

Researchers identified Ral guanine nucleotide dissociation stimulator (RalGDS) as a binding protein for Ras-related protein R-ras. RalGDS interacts with active Ras-like GTPases, suggesting it acts as an effector molecule.

Area of Science:

  • Molecular biology
  • Cell signaling
  • Protein-protein interactions

Background:

  • Ras GTPases are key regulators of cellular signaling pathways.
  • Identifying effector proteins is crucial for understanding Ras-mediated signaling.
  • R-ras is a member of the Ras superfamily with less understood functions.

Purpose of the Study:

  • To identify proteins that interact with the Ras-related protein R-ras.
  • To characterize the binding domain and specificity of identified interacting proteins.
  • To determine if Ral guanine nucleotide dissociation stimulator (RalGDS) functions as an effector for Ras-like GTPases.

Main Methods:

  • Yeast two-hybrid cDNA library screening to identify R-ras binding partners.
  • In vitro binding assays using purified proteins to confirm direct interactions.
  • Competition assays to assess binding site overlap with other Ras-binding proteins.

Main Results:

  • The C-terminal region of Ral guanine nucleotide dissociation stimulator (RalGDS) was identified as an R-ras binding partner.
  • The R-ras-binding domain of RalGDS (RalGDS-RBD) specifically binds to active forms of H-ras, K-ras, and Rap.
  • Direct, GTP-dependent interactions were confirmed in vitro between Ras-like GTPases and RalGDS.
  • RalGDS-RBD competes with Raf-1 for binding to Ras-like GTPases, indicating shared binding sites.

Conclusions:

  • Ral guanine nucleotide dissociation stimulator (RalGDS) is a direct binding partner for R-ras.
  • RalGDS acts as a putative effector molecule for multiple Ras-like GTPases, including R-ras, H-ras, K-ras, and Rap.
  • These findings elucidate a novel interaction in Ras signaling pathways and highlight RalGDS's role as a downstream effector.

More Related Videos

Comparing the Affinity of GTPase-binding Proteins using Competition Assays10:37

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 Assay13:51

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

Published on: November 11, 2018

Related Concept Videos

GTPases and their Regulation02:14

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 Proteins01:14

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...
The Ras Gene02:38

The Ras Gene

The Ras-gene-encoded proteins are regulators of signaling pathways controlling cell proliferation, differentiation, or cell survival. The Ras-gene family in humans constitutes three primary members—the HRas, NRas, and KRas. These genes code for four functionally distinct yet closely related proteins—the HRas, NRas, KRas4A, and KRas4B. The involvement of mutant Ras genes in human cancer was first discovered in 1982 and is among the most common causes of human tumorigenesis.
Ras is a superfamily...
Rab Cascades01:25

Rab Cascades

Rab GTPases act in a regulated cascade during membrane fusion, helping the lipid bilayers mix. The Rab family of proteins are active when bound to GTP, and inactive when bound to GDP. Hence, they act as guanine nucleotide-dependent molecular switches. Rab-GTP recognizes and binds to long or short-range tethering proteins to capture the target vesicle. These tethers coordinate with SNAREs on the vesicle and the target membrane to assemble the trans SNARE complex that locks the mixing bilayers.
Small GTPases - Ras and Rho01:24

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 Proteins01:22

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...