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Related Concept Videos

Small GTPases - Ras and Rho01:24

Small GTPases - Ras and Rho

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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:
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Cell Polarization by Rho Proteins01:21

Cell Polarization by Rho Proteins

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Cell polarity is the asymmetric distribution of cellular and membrane components, making one side of the cell different from the other. This polarity is essential to many processes such as embryogenesis, axon migration, glucose transport across epithelial cells, and directional cell migration. A migrating cell responds to intracellular or extracellular signals via molecular cascades that reorganize the actin cytoskeleton to establish this polarity. In these cells, the Rho family proteins Cdc42,...
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MAPK Signaling Cascades01:07

MAPK Signaling Cascades

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Mitogen-activated protein kinase, or MAPK pathway, activates three sequential kinases to regulate cellular responses such as proliferation, differentiation, survival, and apoptosis. The canonical MAPK pathway starts with a mitogen or growth factor binding to an RTK. The activated RTKs stimulate Ras, which recruits Raf or MAP3 Kinase (MAPKKK), the first kinase of the MAPK signaling cascade. Raf further phosphorylates and activates MEK or MAP2 Kinases (MAPKK), which in turn phosphorylates MAP...
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Amplifying Signals via Enzymatic Cascade01:22

Amplifying Signals via Enzymatic Cascade

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When a ligand binds to a cell-surface receptor, the receptor's intracellular domain changes shape, which may either activate its enzyme function or allow its binding to other molecules. The initial signal is amplified by most signal transduction pathways. This means that a single ligand molecule can activate multiple molecules of a downstream target. Proteins that relay a signal are most commonly phosphorylated at one or more sites, activating or inactivating the protein. Kinases catalyze...
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Interactions Between Signaling Pathways01:19

Interactions Between Signaling Pathways

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Signaling cascades usually lack linearity. Multiple pathways interact and regulate one another, allowing cells to integrate and respond to diverse environmental stimuli.
Convergence and divergence, and cross-talk between signaling pathways
Two distinct signaling pathways can converge on a single functional unit, which may either be a single protein or a complex of proteins. The response is either functionally distinct or synergistic between the two pathways but different from the response...
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Receptor Tyrosine Kinases01:26

Receptor Tyrosine Kinases

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Receptor tyrosine kinases or RTKs are membrane-bound receptors that phosphorylate specific tyrosine on protein substrates. RTKs regulate cellular growth, differentiation, survival, and migration. They contain an extracellular ligand binding domain, a transmembrane domain, and a cytosolic tail with intrinsic kinase activity. Several extracellular signaling molecules activate RTKs in one or more ways and relay the signal downstream. Ligands such as platelet-derived growth factor (PDGF) or...
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RhoC GTPase Activation Assay
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RhoC GTPase Activation Assay

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Quantitation of RhoA activation: differential binding to downstream effectors.

Yu-Wen Zhang1, Holly M Torsilieri2, James E Casanova2

  • 1Department of Cell Biology, University of Virginia, School of Medicine, Charlottesville, VA 22908 USA.

Small Gtpases
|August 11, 2022
PubMed
Summary
This summary is machine-generated.

RhoA activation is not solely determined by Rhotekin binding. Different RhoA forms and stimuli show effector-specific binding, indicating differential pathway regulation.

Keywords:
BRETGST pulldownROCK2Rho-binding domainRhoARhoA activation assayRhotekinmDia1

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

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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-RhoAG17A from Epithelial Cell Lysates
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Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay
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Area of Science:

  • Cellular biology
  • Molecular signaling
  • GTPase biology

Background:

  • The small GTPase RhoA regulates crucial cellular functions via downstream effectors.
  • RhoA effectors typically possess a Rho-binding domain (RBD) that interacts with active RhoA.
  • GST-RBD fusion proteins, particularly Rhotekin RBD, are standard tools for measuring RhoA activation.

Purpose of the Study:

  • To investigate whether all active RhoA forms interact with the Rhotekin RBD.
  • To explore the specificity of RhoA interactions with different effectors.
  • To determine if RhoA activation is effector-specific.

Main Methods:

  • GST pulldown assays to assess RhoA-RBD interactions.
  • BRET (Bioluminescence Resonance Energy Transfer) assay to study RhoA binding in a cellular context.
  • Utilizing constitutively active RhoA mutants (e.g., RhoA-G14V) and RhoA activation by cellular stimuli.

Main Results:

  • A constitutively active RhoA-G14V mutant failed to bind the Rhotekin RBD, despite interacting with ROCK2 and mDIA1 RBDs.
  • Active RhoA induced by various stimuli exhibited differential binding preferences for its effectors.
  • The study identified limitations in using Rhotekin RBD as the sole indicator of RhoA activation.

Conclusions:

  • RhoA activation is effector-specific, leading to differential regulation of downstream pathways.
  • Rhotekin RBD interaction does not encompass all forms of active RhoA.
  • RhoA activation status should be assessed considering its interaction with diverse effectors, not just Rhotekin.