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Related Concept Videos

G-protein Coupled Receptors01:21

G-protein Coupled Receptors

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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.
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G Protein-coupled Receptors01:15

G Protein-coupled Receptors

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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...
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Transducer Mechanism: G Protein–Coupled Receptors01:30

Transducer Mechanism: G Protein–Coupled Receptors

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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,...
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Ligand Binding and Linkage00:49

Ligand Binding and Linkage

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Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked.  In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence...
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Ligand Binding Sites02:40

Ligand Binding Sites

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Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...
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Internal Receptors01:31

Internal Receptors

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Many cellular signals are hydrophilic and therefore cannot pass through the plasma membrane. However, small or hydrophobic signaling molecules can cross the hydrophobic core of the plasma membrane and bind to internal, or intracellular, receptors that reside within the cell. Many mammalian steroid hormones use this mechanism of cell signaling, as does nitric oxide (NO) gas.
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Related Experiment Video

Updated: Jan 27, 2026

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors
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A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

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High-Throughput Fluorescence Polarization Assay to Identify Ligands Using Purified G Protein-Coupled Receptor.

P Heine1, G Witt2, A Gilardi2

  • 1Department of Biochemistry, University of Zurich, Zurich, Switzerland.

SLAS Discovery : Advancing Life Sciences R & D
|March 31, 2019
PubMed
Summary

Researchers developed a cell-free assay to find new drugs for G protein-coupled receptors (GPCRs). This method successfully identified potential drug compounds targeting the neurotensin receptor type 1 (NTS1), aiding GPCR drug discovery.

Keywords:
G protein-coupled receptorfluorescence polarizationhigh-throughput screeningneurotensinsurface plasmon resonance

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Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay
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Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

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A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators
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A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

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Related Experiment Videos

Last Updated: Jan 27, 2026

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors
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A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

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Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay
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Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

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A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators
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A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

Published on: February 20, 2018

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Area of Science:

  • Biochemistry
  • Pharmacology
  • Molecular Biology

Background:

  • High-throughput (HT) screening for G protein-coupled receptor (GPCR) drug discovery faces challenges with cell-free methods.
  • Mutational strategies stabilize purified GPCRs, overcoming limitations of low receptor density and micelle instability for in vitro screening.

Purpose of the Study:

  • To develop a cell-free, high-throughput fluorescence polarization (FP) assay for screening G protein-coupled receptor (GPCR) ligands.
  • To identify novel small molecules targeting the neurotensin receptor type 1 (NTS1) using the developed assay.

Main Methods:

  • Development of a 384-well, DMSO-tolerant, high-throughput fluorescence polarization (FP) assay for the neurotensin receptor type 1 (NTS1).
  • Screening of a 1272-compound library.
  • Validation of primary hits using orthogonal assays, including surface plasmon resonance (SPR).

Main Results:

  • The FP assay identified 12 primary hits (~1%) from 1272 compounds.
  • SPR validation confirmed binding for seven compounds (0.6%).
  • One validated compound exhibited submicromolar affinity for the orthosteric binding pocket; another bound to a nonorthosteric site and showed specific biological activity.

Conclusions:

  • A robust cell-free HT FP assay for NTS1 was successfully developed and validated.
  • The assay facilitates the identification of GPCR ligands, expanding the potential for discovering novel antagonistic or agonistic pharmaceuticals.
  • The findings demonstrate the utility of cell-free assays in advancing GPCR drug discovery pipelines.