Related Experiment Video
Updated: Aug 10, 2026
11:49
Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay
Published on: July 28, 2014
Characteristics of GTP-mediated microsomal Ca2+ release
S K Joseph1, H L Rice, C V Nicchitta
1Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia 19104.
Biochimica Et Biophysica Acta
|November 22, 1988
Summary
Guanosine triphosphate (GTP) reversibly alters liver microsomal membrane permeability, affecting calcium (Ca2+) release and enhancing responses to inositol trisphosphate (IP3). This suggests GTP modulates a transmembrane pore, influencing intracellular Ca2+ signaling pathways.
Area of Science:
- Cellular Biology
- Biochemistry
- Membrane Transport
Background:
- Guanosine triphosphate (GTP) is known to release Ca2+ from liver microsomes.
- Polyethylene glycol (PEG) enhances GTP-mediated Ca2+ release and responses to D-myo-inositol 1,4,5-trisphosphate (IP3).
Purpose of the Study:
- To investigate the mechanism behind GTP-mediated Ca2+ release and its interaction with IP3 responses in liver microsomes.
- To determine if GTP affects microsomal membrane permeability.
Main Methods:
- Incubation of liver microsomes with GTP and PEG.
- Use of GTP gamma S as a GTP antagonist.
- Measurement of Ca2+ fluxes.
- Assay of intraluminal esterase leakage.
- Determination of mannose 6-phosphate hydrolysis latency.
- Electron microscopy to assess vesicle fusion.
- Microsomal density analysis.
Main Results:
- GTP gamma S did not promote Ca2+ re-uptake after GTP-mediated release.
- GTP's effects were reversible by washing or dilution.
- PEG induced microsome aggregation, but not fusion.
- GTP, in the presence of PEG, increased esterase leakage, reduced vesicle density, and decreased mannose 6-phosphate hydrolysis latency.
- These permeability changes developed slowly, contrasting with rapid Ca2+ flux effects.
- Mannose 6-phosphate permeability was restored upon GTP removal.
Conclusions:
- GTP induces non-specific permeability changes in microsomal membranes.
- These permeability alterations may underlie GTP's effects on Ca2+ release.
- GTP can reversibly modulate a transmembrane pore in microsomes, affecting ion and macromolecule passage.
- This mechanism might link IP3-sensitive vesicles with other Ca2+-containing compartments.
Related Concept Videos
02:14GTPases 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...
Large G-proteins, also known...
01:24Small 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:
Three regulatory proteins control their activity:
01:22Activation 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...
01:11IP3/DAG Signaling Pathway
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 produces two-second...
01:27Feedback Regulation of Calcium Concentration
Calcium is an essential signaling molecule required for various cellular functions. Calcium pumps and ion channels on cell and organellar membranes, such as those on the endoplasmic reticulum (ER), regulate calcium concentrations inside the cell. They remain closed, keeping the cytosolic calcium levels low at a resting state.
Various transmembrane receptors, such as G protein-coupled receptors (GPCRs), elicit a response to extracellular signals by increasing cytosolic calcium. Activated GPCRs...
Various transmembrane receptors, such as G protein-coupled receptors (GPCRs), elicit a response to extracellular signals by increasing cytosolic calcium. Activated GPCRs...
01:16Calmodulin-dependent Signaling
Calmodulin (CaM) is a calcium-binding protein in eukaryotes that controls various calcium-regulated cellular processes. It has four calcium-binding sites that bind calcium to form the calcium-calmodulin ( Ca2+-CaM) complex. GPCR stimulation increases the calcium levels in the cells that bind to CaM and induces a conformational change.
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...