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Journal of Biomolecular Screening
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A Novel Cell-Based Assay for G-Protein-Coupled Receptor-Mediated Cyclic Adenosine Monophosphate Response Element Binding Protein Phosphorylation

Julie V. Selkirk

Department of Neuroscience, Neurocrine Biosciences Inc., San Diego, CA

Lisa M. Nottebaum

Department of Neuroscience, Neurocrine Biosciences Inc., San Diego, CA

Ian C. Ford

Department of Neuroscience, Neurocrine Biosciences Inc., San Diego, CA

Mark Santos

Department of Pharmacology, Neurocrine Biosciences Inc., San Diego, CA

Siobhan Malany

Department of Pharmacology, Neurocrine Biosciences Inc., San Diego, CA

Alan C. Foster

Department of Neuroscience, Neurocrine Biosciences Inc., San Diego, CA

Sandra M. Lechner

Department of Neuroscience, Neurocrine Biosciences Inc., San Diego, CA

Currently, the most popular means of assessing functional activity of Gs/olf-coupled receptors is via the measurement of intracellular cyclic adenosine monophosphate (cAMP) accumulation. An additional readout is the downstream phosphorylation of cAMP response element binding protein (CREB), which gives an indication of gene transcription, the ultimate response of many G-protein-coupled receptor (GPCR) signals. Current methods of quantifying CREB phosphorylation are low throughput, and so we have designed a novel higher throughput method using the OdysseyTM infrared imaging system. Functional potencies of both agonists and antagonists correlate well with radioligand binding affinities determined using examples of both an endogenous (adenosine2A receptor in PC-12 cells) and a heterologous (human melanocortin 4 receptor in HEK-293 cells) expression system. For example, the antagonist ZM241385 demonstrates 0.23 ± 0.03 nM affinity for the A2A receptor and has a functional potency of 0.26 ± 0.04 nM determined using cAMP and 0.15 ± 0.06 nM using CREB phosphorylation. These data demonstrate that this novel approach for the measurement of CREB phosphorylation is a useful tool for the assessment of GPCR activity in whole cells and is more amenable to the throughput required for the purposes of drug discovery.

Key Words: CREB • in-cell Western • adenosine2A receptor • OdysseyTM • phosphorylation

This version was published on June 1, 2006

Journal of Biomolecular Screening, Vol. 11, No. 4, 351-358 (2006)
DOI: 10.1177/1087057106286608
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