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Updated: Aug 8, 2026

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators
07:41

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

Published on: February 20, 2018

A novel cell-based assay for G-protein-coupled receptor-mediated cyclic adenosine monophosphate response element

Julie V Selkirk1, Lisa M Nottebaum, Ian C Ford

  • 1Department of Neuroscience, Neurocrine Biosciences Inc., San Diego, CA 92130, USA. jselkirk@neurocrine.com

Journal of Biomolecular Screening
|June 6, 2006
PubMed

Insights

Researchers developed a high-throughput method to measure cyclic adenosine monophosphate response element binding protein (CREB) phosphorylation, offering a faster way to assess G-protein-coupled receptor (GPCR) activity for drug discovery.

Area of Science:

  • Pharmacology
  • Molecular Biology
  • Biochemistry

Background:

  • Gs/olf-coupled receptor activity is commonly assessed by measuring cyclic adenosine monophosphate (cAMP) accumulation.
  • Downstream phosphorylation of cAMP response element binding protein (CREB) indicates gene transcription, a key G-protein-coupled receptor (GPCR) signaling outcome.
  • Existing CREB phosphorylation assays lack the high throughput required for efficient drug discovery.

Purpose of the Study:

  • To develop a novel, high-throughput method for quantifying CREB phosphorylation.
  • To validate this new method for assessing the functional activity of GPCRs.
  • To demonstrate its utility in drug discovery applications.

Main Methods:

  • Utilized the Odyssey infrared imaging system for a novel, higher throughput CREB phosphorylation assay.
  • Validated the method using both endogenous (adenosine 2A receptor) and heterologous (human melanocortin 4 receptor) expression systems.
  • Correlated functional potencies with radioligand binding affinities.

Main Results:

  • The novel method demonstrated high throughput for CREB phosphorylation measurement.
  • Functional potencies obtained via CREB phosphorylation correlated well with radioligand binding affinities.
  • The antagonist ZM241385 showed consistent affinity and functional potency values across cAMP and CREB assays.

Conclusions:

  • The developed Odyssey-based CREB phosphorylation assay is a valuable tool for assessing GPCR activity in whole cells.
  • This method offers improved throughput compared to existing techniques, making it suitable for drug discovery.
  • The assay provides reliable functional data that correlates with binding affinities, aiding in the characterization of receptor modulators.

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