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Related Concept Videos

Phosphoinositides and PIPs01:42

Phosphoinositides and PIPs

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Phosphoinositides are a group of phospholipids containing a glycerol backbone with two fatty acid chains and a phosphate attached to a myoinositol sugar ring. The inositol head group extends into the cytoplasm, where it is modified by adding phosphate groups to form phosphatidylinositol phosphates or PIPs.
Different phosphoinositides are synthesized and recruited on the cytosolic face of the plasma membrane. The localization of specific phosphoinositides concentrated in separate membrane...
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IP3/DAG Signaling Pathway01:11

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Membrane lipids such as phosphatidylinositol (PI) are precursors for several membrane-bound and soluble second messengers. Specific kinases phosphorylate PI and produce phosphorylated inositol phospholipids. One such inositol phospholipids are the  phosphatidylinositol-4,5 bisphosphate [PI(4,5)P2], present in the inner half of the lipid bilayer. Upon ligand binding, GPCR stimulates Gq proteins to turn on phospholipase Cꞵ. Activated phospholipase Cꞵ cleaves PI(4,5)P2 and...
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GTPases and their Regulation02:14

GTPases and their Regulation

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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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Amplifying Signals via Second Messengers01:15

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Many receptor binding ligands are hydrophilic; they do not cross the cell membrane but bind to cell-surface receptors. Thus, their message must be relayed by second messengers present in the cell cytoplasm. There are several second messenger pathways, each with its own way of relaying information. For example, the G protein-coupled receptors can activate both phosphoinositol and cyclic AMP (cAMP) second messenger pathways. The phosphoinositol pathway is active when the receptor induces...
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Related Experiment Video

Updated: Nov 20, 2025

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

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Liposome-Based Methods to Study GTPase Activation by Phosphoinositides.

Julien Viaud1, Laurie Ceccato1, Bernard Payrastre1,2

  • 1INSERM U1048 and Université Toulouse 3, Toulouse Cedex, France.

Methods in Molecular Biology (Clifton, N.J.)
|January 22, 2021
PubMed
Summary

Phosphoinositides (PIPs) regulate cell processes by interacting with proteins. A new assay quantifies GTPase activation by measuring PIPs, GEFs, and GTPases together, reflecting in vivo conditions.

Keywords:
GTPase exchange factorsGTPase–liposome interactionLiposomePhosphoinositide

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Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation
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PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions
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Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation
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PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions
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PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

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Area of Science:

  • Cell Biology
  • Lipid Signaling
  • Biochemistry

Background:

  • Phosphoinositides (PIPs) are crucial lipid messengers regulating diverse cellular functions.
  • PIPs control processes like cytoskeleton dynamics and membrane trafficking via protein domain interactions.
  • PIPs modulate small GTPase activity by activating Guanyl-nucleotide Exchange Factors (GEFs).

Purpose of the Study:

  • To address limitations in current in vitro assays for GTPase activation.
  • To develop a more physiologically relevant assay for studying PIP-mediated GTPase regulation.
  • To quantify GTPase activation in the presence of relevant membrane and protein co-activators.

Main Methods:

  • Utilized classical protein-lipid overlay and liposome sedimentation assays.
  • Developed a novel three-component assay system.
  • Incorporated liposomes mimicking target membranes, specific GEF domains, and recombinant GTPases.

Main Results:

  • The developed assay enables precise quantification of GTPase activation.
  • The assay incorporates essential co-activators like membranes and protein partners.
  • This method provides a more accurate assessment of PIP-mediated GTPase activity compared to traditional assays.

Conclusions:

  • The novel assay accurately quantifies GTPase activation by phosphoinositides.
  • This method better reflects in vivo conditions by including membrane and protein co-activators.
  • The assay is valuable for studying the role of PIPs in regulating GTPase function.