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Abstract for A Homogenous, High-Throughput Luminescent cAMP
Assay to Monitor Modulation of Gs and Gi Protein-Coupled Receptors
Said A. Goueli1,2, Meera Kumar1, Kevin Hsiao1, Jolanta
Vidugiriene1, and Bob Bulleit1
1Cell Signaling Group, Cellular Analysis Platform, Dept. R&D,
Promega Corporation, 2800 Woods Hollow Road, Madison WI 53711, USA
2Dept. of Pathology and Laboratory Medicine, University of Wisconsin
School of Medicine and Public Health, Madison, WI
G Protein-Coupled Receptors (GPCRs) represent a large validated
target for drug discovery research. They are classified into three
main groups based on the G protein associated with the receptor. The
Gs group is coupled to activation of adenylate cyclase, and the Gi
group is coupled to inhibition of adenylate cyclase, while Gq is
coupled to activation of phospholipase b. There are two main
strategies to monitor the activation of GPCRs, reporter-based assays
and cAMP accumulation-based assays. We report here on a new assay
for monitoring modulation of GPCRs that are linked to activation or
inhibition of adenylate cyclase (Gs or Gi). The assay can be used to
monitor cAMP accumulation or depletion in the cell upon treatment
with agonists or antagonists of Gs- or Gi-coupled receptors. The
assay is homogenous and amenable to high-throughput screening of
modulators of GPCRs. A Z′ value higher than 0.7 attests to the
robustness of the assay and is carried out in 96- and 384-well
plates and potentially adaptable to 1536-well format. The assay is
based on luminescence, and thus it does not suffer from fluorescence
interference by library compounds. We have successfully generated
EC50 values for agonists and IC50 values for antagonists of
Gs-coupled receptors that are similar to those reported in the
literature. The assay is easy to use, can be carried out in less
than 60 minutes and does not require antibodies or expensive
instrumentation for signal detection. The signal output is
relatively stable for several hours and thus can be used for
screening large numbers of plates. This luminescent assay is fast,
homogenous, and reliable, as indicated by high Z′ values, making it
an attractive screening tool for identifying agonist or antagonists
that modulate Gs- and Gi-coupled receptors. |
