Characterization of serotonin 5-hydroxytryptamine-1A receptor activation using a phospho-extracellular-signal regulated kinase 2 sensor.
Anal Biochem
; 393(1): 95-104, 2009 Oct 01.
Article
en En
| MEDLINE
| ID: mdl-19539597
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Fosfoproteínas
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Técnicas Biosensibles
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Proteína Quinasa 1 Activada por Mitógenos
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Receptor de Serotonina 5-HT1A
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Anal Biochem
Año:
2009
Tipo del documento:
Article
País de afiliación:
Estados Unidos