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A Homogenous bioluminescent, and fast assay for monitoring alteration in cAMP cellular and tissue cAMP and monitoring the Modulation of Gs and Gi Protein Coupled Receptors
Author(s) -
Goueli Said,
Hsiao Kevin
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.666.3
Subject(s) - g protein coupled receptor , adenylate kinase , receptor , second messenger system , cyclase , bioluminescence , g protein , phospholipase c , chemistry , signal transduction , microbiology and biotechnology , gi alpha subunit , biology , biochemistry
G Protein Coupled Receptors represent a large validated target for Drug discovery research. The G s is coupled to activation of adenylate cyclase and G i is coupled to inhibition of adenylate cyclase while G q is coupled to activation of phospholipase β. Cellular concentrations of second messengers such as cAMP and cGMP have been known to play major roles in smooth muscle relaxation, contractile activity of cardiovascular system, and central nervous system related pathologies. We report here on a new assay for determining cAMP concentrations in cellular and tissue extract and for monitoring modulation of GPCR that are linked to activation or inhibition of adenylate cyclase (Gs or G i ). The assay is homogenous, fast, HTS formatted with high Z′, and adaptable to multi plate formats. We have successfully generated EC 50 values for agonists and IC 50 values for antagonists of G s coupled receptors that are similar to those reported in the literature. The assay is easy to use, and can be carried out in less than 60 minutes and does not require antibodies or expensive instrumentation for signal detection and the signal output is relatively stable for few hours.