Related Experiment Video
Updated: Jun 14, 2025
11:49
Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay
Published on: July 28, 2014
40.1K
Direct detection of endogenous Gαi activity in cells with a sensitive conformational biosensor.
Alex Luebbers1, Remi Janicot1, Jingyi Zhao1
1Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine, Boston University, Boston, MA 02118, USA.
Biorxiv : the Preprint Server for Biology
|September 4, 2024
Summary
This study introduces a novel optical biosensor for robustly detecting endogenous G-protein activation by G-protein-coupled receptors (GPCRs). The biosensor overcomes limitations of existing methods, enabling sensitive and interference-free analysis of native signaling pathways.
Area of Science:
- Cellular signaling and molecular biology.
- Biophysics and biosensor development.
- Pharmacology and drug discovery.
Background:
- G-protein-coupled receptors (GPCRs) mediate crucial cellular signals via heterotrimeric G-proteins (Gαβγ).
- Existing methods for measuring G-protein activity, such as optical biosensors, often require exogenous proteins or interfere with native signaling.
- Limitations include compromised readout fidelity and narrow dynamic range, hindering broad applicability.
Purpose of the Study:
- To develop a novel optical biosensor for sensitive detection of endogenous Gαi-GTP, the active species of Gαi subunits.
- To overcome limitations of current biosensors by ensuring minimal interference with endogenous G-protein signaling pathways.
- To validate the biosensor's performance in diverse experimental systems utilizing endogenous GPCRs.
Main Methods:
- Development of a bystander Bioluminescence Resonance Energy Transfer (BRET)-based optical biosensor.
- Leveraging the Gαi-binding protein GINIP as a high-affinity detector for GTP-bound Gαi.
- Optimization to prevent interference with Gi-dependent cyclic AMP (cAMP) inhibition.
- Implementation in primary astroglial cells and cell lines expressing endogenous GPCRs (neurotransmitter and opioid receptors).
Main Results:
- The developed biosensor robustly detects endogenous Gαi-GTP upon stimulation of endogenous GPCRs.
- The biosensor design prevents interference with Gi-dependent signaling pathways.
- Application in primary astroglial and cell line models revealed distinct opioid neuropeptide-mediated activation profiles.
- Demonstrated sensitive and direct detection of endogenous G-protein activation without disrupting downstream signaling.
Conclusions:
- Introduced a novel, sensitive optical biosensor for endogenous G-protein activation by GPCRs.
- The biosensor offers improved fidelity and minimal interference compared to existing technologies.
- Enables robust investigation of native GPCR-G-protein signaling in various cellular contexts.
- Provides a valuable tool for studying drug mechanisms and cellular responses involving endogenous GPCRs.
More Related Videos
09:12G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay
Published on: September 10, 2016
9.6K
08:46A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors
Published on: December 2, 2022
2.2K
Related Concept Videos
01:22Activation and Inactivation of G Proteins
6.9K
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...
6.9K
01:15G Protein-coupled Receptors
11.6K
G Protein-Coupled Receptors or GPCRs are membrane-bound receptors that transiently associate with heterotrimeric G proteins and induce an appropriate response to sensory stimuli such as light, odors, hormones, cytokines, or neurotransmitters.
GPCRs are also called heptahelical, 7TM, or serpentine receptors, and consist of seven (H1-H7) transmembrane alpha-helices that span the bilayer to form a cylindrical core. The transmembrane helices are connected by three extracellular loops and three...
GPCRs are also called heptahelical, 7TM, or serpentine receptors, and consist of seven (H1-H7) transmembrane alpha-helices that span the bilayer to form a cylindrical core. The transmembrane helices are connected by three extracellular loops and three...
11.6K
01:21G-protein Coupled Receptors
117.1K
G-protein coupled receptors are ligand binding receptors that indirectly affect changes in the cell. The actual receptor is a single polypeptide that transverses the cell membrane seven times creating intracellular and extracellular loops. The extracellular loops create a ligand specific pocket which binds to neurotransmitters or hormones. The intracellular loops holds onto the G-protein.
117.1K
01:09GPCRs Regulate Adenylyl Cylase Activity
5.4K
Some GPCRs transmit signals through adenylyl cyclase (AC), a transmembrane enzyme. AC helps synthesize second messenger cyclic adenosine monophosphate (cAMP). AC catalyzes cyclization reaction and converts ATP to cAMP by releasing a pyrophosphate. The pyrophosphate is further hydrolyzed to phosphate by the enzyme pyrophosphatase, which drives cAMP synthesis to completion. However, cAMP is rapidly degraded to 5′ AMP by the enzymes phosphodiesterase (PDE), preventing overstimulation of...
5.4K
01:30Transducer Mechanism: G Protein–Coupled Receptors
1.9K
G Protein–Coupled Receptors (GPCRs) are membrane-bound receptors that transiently associate with heterotrimeric G proteins and induce an appropriate response to various stimuli. GPCRs regulate critical physiological pathways and are excellent drug targets for treating diseases such as diabetes, cancer, obesity, depression, or Alzheimer's. Nearly 35% of approved drugs implement their therapeutic effects by selectively interacting with specific GPCRs.
GPCRs are also called heptahelical,...
GPCRs are also called heptahelical,...
1.9K
01:11IP3/DAG Signaling Pathway
11.9K
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...
11.9K