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Luciferase-based GloSensor™ cAMP assay: Temperature optimization and application to cell-based kinetic studies.

Fang I Wang1, Gucci Ding1, Garmen S Ng1

  • 1Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Canada.

Methods (San Diego, Calif.)
|November 5, 2021
PubMed
Summary

The GloSensor™ cAMP assay effectively monitors G protein-coupled receptor (GPCR) signaling. Room temperature is optimal for assay performance, and the GloSensor-22F version offers the best signal, with temperature effects being reversible.

Keywords:
Drug discoveryGPCRsGloSensorHigh-throughput screeningOptimization

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Area of Science:

  • Biochemistry
  • Cellular Biology
  • Pharmacology

Background:

  • G protein-coupled receptors (GPCRs) are crucial drug targets involved in numerous signaling pathways.
  • Cyclic adenosine monophosphate (cAMP) is a key second messenger in GPCR signaling.
  • Luminescence-based biosensors, like GloSensor™, offer real-time monitoring of intracellular cAMP levels.

Purpose of the Study:

  • To investigate the temperature sensitivity and reversibility of the GloSensor™ cAMP assay.
  • To identify the optimal GloSensor™ version for measuring cytosolic cAMP.
  • To provide a detailed protocol for monitoring cAMP in live cells expressing GPCRs.

Main Methods:

  • HEK293H cells were transfected with adenosine receptor A2a and GloSensor™ plasmids (pGloSensor-20F or -22F).
  • Temperature optimization studies involved preincubation and luminescence readings at various temperatures.
  • Comparison of GloSensor™-20F and GloSensor™-22F for signal-to-background ratio.

Main Results:

  • Optimal assay performance, including preincubation and luminescence reading, was achieved at room temperature.
  • The GloSensor™-22F biosensor demonstrated a superior signal-to-background ratio compared to GloSensor™-20F.
  • The impact of temperature on biosensor activity was found to be reversible.

Conclusions:

  • Room temperature is optimal for the GloSensor™ cAMP assay, with reversible temperature effects on biosensor activity.
  • The GloSensor™-22F variant is recommended for enhanced cytosolic cAMP measurement.
  • The assay is a valuable tool for GPCR signaling analysis in drug discovery, though thermal instability may limit in vivo applications.