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Gq‐coupled Receptor signaling – A kinetic analysis in living cells
Author(s) -
Hoffmann Carsten,
Hein Peter,
Zabel Ulrike,
Ziegler Nicole,
Berlot Cathrine,
Lohse Martin J,
Bünemann Moritz
Publication year - 2008
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.22.1_supplement.722.1
Subject(s) - heterotrimeric g protein , förster resonance energy transfer , g protein coupled receptor , gq alpha subunit , g protein , receptor , chemistry , biophysics , carbachol , muscarinic acetylcholine receptor m5 , acetylcholine receptor , agonist , muscarinic acetylcholine receptor , microbiology and biotechnology , yellow fluorescent protein , muscarinic acetylcholine receptor m3 , g beta gamma complex , signal transduction , biochemistry , biology , fluorescence , physics , quantum mechanics , gene
The activation of G protein‐coupled receptors (GPCRs) is the initial step in the signaling cascade which is triggered by binding of an agonist to a GPCR. Here we report the kinetics of the initial steps in the signaling cascade of a Gq‐coupled receptor. We employed the M 3 ‐subtype of the muscarinic acetylcholine receptor (M 3 ‐AChR) as a model receptor to study Gq‐signaling. Using a fluorescence resonance energy transfer (FRET) approach based on the FlAsH‐tetracysteine tag technology in combination with CFP we generated a M 3 ‐receptor construct that reports conformational changes during receptor activation by changes in the FRET‐signal. Acetylcholine or carbachol both rapidly activated receptors on a millisecond timescale. FRET based detection of M 3 ‐AChR – Gq‐Protein interaction revealed kinetics that closely resembled receptor activation kinetics. Furthermore G‐Protein activation was measured using YFP‐ tagged Gαq and an N‐terminally tagged CFP‐γ 2 subunit of the heterotrimeric G‐protein. Activation of the Gq‐Protein was significantly slower than receptor activation and undistinguishable for acetylcholine or carbachol.