title: Characterization of serotonin 5-hydroxytryptamine-1A receptor activation using a phospho-extracellular-signal regulated kinase 2 sensor.
abstract: 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-HT(1A)) receptor expressed in CHO-K1 cells. In addition to demonstrating the flexibility of this assay platform, we provide the first reported profile for 5-HT(1A) 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 GTPgammaS.
authors.affiliation: Life Technologies, 501 Charmany Drive, Madison, WI 53719, USA.
authors.forename: Kristin G
authors.initials: KG
authors.lastname: Huwiler
authors.forename: Thomas
authors.initials: T
authors.lastname: Machleidt
authors.forename: Lucas
authors.initials: L
authors.lastname: Chase
authors.forename: Bonnie
authors.initials: B
authors.lastname: Hanson
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Robers
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002460:Cell Line; D006224:Cricetinae; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D044282:Receptor, Serotonin, 5-HT1A / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*
chemical_list: D010750:Phosphoproteins; D044282:Receptor, Serotonin, 5-HT1A; D019950:Mitogen-Activated Protein Kinase 1