Technical note | Measurement of intracellular cAMP using a BRET biosensor

Abstract

G protein-coupled receptors (GPCRs) are the targets of some chemical warfare agents, and many medical countermeasures to these agents. Techniques for measuring GPCR-mediated cell signalling are essential for chemical warfare agent research and medical countermeasure development. However, commercially available kits for measuring GPCR signalling are typically very expensive. This report describes a simple cost-effective method of measuring the activity of GPCRs coupled to the cAMP signalling pathway. The assay utilises a commonly used cAMP bioluminescence resonance energy transfer (BRET) biosensor to detect changes in cAMP levels in live cells. This method was used to measure activation, antagonism and allosteric modulation of a muscarinic acetylcholine receptor in a cultured mammalian cell line.

Executive Summary

The Agent-based Genomics and Cell Biology Science and Technology Capability (AG&CB STC) is developing a capability to investigate the in vitro pharmacology of G protein coupled receptors (GPCRs). GPCRs are the target of 35-40% of currently available pharmaceuticals and are major targets for new drug development. These receptors are also the target of some traditional chemical warfare agents, many emerging threat agents and their medical countermeasures (MCMs). Thus, a specific focus on GPCR pharmacology will enable the AG&CB STC to contribute to the collaborative development of new MCM drugs as well as the biological hazard assessment of chemical agents.

Methods to quantitatively measure the activation or inhibition of GPCRs by compounds of interest are essential for the AG&CB STC’s in vitro pharmacology program. Many GPCRs, including opioid, muscarinic, dopaminergic and adrenergic receptors, are coupled to the cyclic adenosine monophosphate (cAMP) signalling pathway. A simple cost-effective method for in vitro measurement of intracellular cAMP levels in live cultured cells involves transfection of cells to express a bioluminescence resonance energy transfer (BRET) biosensor for cAMP.

This technical note describes the successful implementation of this method to measure cAMP levels in a mammalian cell line stably overexpressing muscarinic acetylcholine receptor 2 (M2). Stimulation, antagonism and allosteric modulation of the M2 receptor are demonstrated.

This method will be a core technique in the suite of in vitro pharmacology assays for the AG&CB STC and will be applied to both the hazard assessment of chemical agents as well as the assessment of novel MCMs currently under development. For a more comprehensive analysis of compounds of interest, this method can be easily adapted for the measurement of other GPCR signalling pathways using BRET-based biosensors described in the open literature.