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Related Concept Videos

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

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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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Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay
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Automated Plate Reader-Based Assays of Light-Activated GPCRs.

Elliot J Gerrard1,2,3, Alexandra-Madelaine Tichy2,3, Harald Janovjak2,3,4

  • 1Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.

Methods in Molecular Biology (Clifton, N.J.)
|December 26, 2024
PubMed
Summary
This summary is machine-generated.

This study presents a novel method for measuring light-sensitive G-protein coupled receptor (GPCR) activity in mammalian cells using standard plate readers. The protocol enables optogenetic control and assaying without specialized optical equipment.

Keywords:
AutomationCell signalingOpsinOptoXROptogeneticsSynthetic biology

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Area of Science:

  • Optogenetics
  • Molecular Biology
  • Cell Signaling

Background:

  • Light-sensitive G-protein coupled receptors (GPCRs) offer precise temporal control over cellular signaling pathways.
  • Standard assay practices are often incompatible with light-sensitive molecular tools like opsins and chimeric OptoXRs.
  • Quantifying the activity of these tools in mammalian cells presents a significant challenge.

Purpose of the Study:

  • To develop and present a method for quantifying opsin activity in automated plate reader-based assays.
  • To demonstrate the utility of internal plate reader optics for activating and detecting light-sensitive molecular tools.
  • To provide guidance for optimizing assays involving light-sensitive molecular tools.

Main Methods:

  • Utilizing internal optical components of common plate readers for both activation and detection.
  • Applying the protocol to assess cyclic adenosine monophosphate (cAMP) levels downstream of a chimeric OptoXR.
  • Adapting the method for other opsins and second messengers like calcium (Ca2+) mobilization.

Main Results:

  • Successful quantification of opsin activity without external optical hardware.
  • Demonstration of kinetic and dose-response relationship detection using standard plate readers.
  • Validation of the protocol for cAMP level assessment.

Conclusions:

  • A practical method is established for assaying light-sensitive GPCRs in mammalian cells using readily available plate readers.
  • This approach simplifies optogenetic experiments by eliminating the need for specialized light sources.
  • The protocol is versatile and can be extended to various opsins and signaling pathways.