Journal
ANALYTICAL BIOCHEMISTRY
Volume 393, Issue 1, Pages 95-104Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2009.06.018
Keywords
ERK; GPCR; MAPK; 5-HT1A; Serotonin; HTS; BacMam; GFP; Immunoassay
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The activation of G-protein-coupled receptors (GPCRs) can result in the stimulation Of numerous signaling networks that extend beyond canonical secondary messenger-dependent pathways. It is well-established that many of these diverse networks converge on the MAPK pathway, resulting in the activation of extracellular-signal regulated kinase 1/2 (ERK). Since the link between GPCRs and ERK can be modulated via both G-protein-dependent and -independent mechanisms, measurement of ERK phosphorylation may serve as an ideal surrogate for GPCR activation. We have combined BacMam-mediated gene delivery of the GFP-ERK2 with a time-resolved Foerster resonance energy transfer (TR-FRET) immunoassay for the measurement of intracellular phospho-ERK2 levels. Together these technologies enable a flexible platform for measuring GPCR and MAPK activation in the cell line of interest. This technology has been applied to the measurement of activation of the serotonin 5-hydroxytryptamine-1A (5-HT1A) receptor expressed in CHO-K1 cells. In addition to demonstrating the flexibility of this assay platform, we provide the first reported profile for 5-HT1A receptor-mediated ERK activation using a panel of known Parkinson's disease drugs. Our results demonstrate the value of using ERK activation as a downstream sensor for GPCR function, providing an attractive complement to upstream endpoints such as ligand occupancy and binding of GTP gamma S. (C) 2009 Elsevier Inc. All rights reserved.
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