Comparison of the activation kinetics of the M3 acetylcholine receptor and a constitutively active mutant receptor in living cells.

Molecular Pharmacology
C HoffmannMartin J Lohse

Abstract

Activation of G-protein-coupled receptors is the first step of the signaling cascade triggered by binding of an agonist. Here we compare the activation kinetics of the G(q)-coupled M(3) acetylcholine receptor (M(3)-AChR) with that of a constitutively active mutant receptor (M(3)-AChR-N514Y) using M(3)-AChR constructs that report receptor activation by changes in the fluorescence resonance energy transfer (FRET) signal. We observed a leftward shift in the concentration-dependent FRET response for acetylcholine and carbachol with M(3)-AChR-N514Y. Consistent with this result, at submaximal agonist concentrations, the activation kinetics of M(3)-AChR-N514Y were significantly faster, whereas at maximal agonist concentrations the kinetics of receptor activation were identical. Receptor deactivation was significantly faster with carbachol than with acetylcholine and was significantly delayed by the N514Y mutation. Receptor-G-protein interaction was measured by FRET between M(3)-AChR-yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)-Gγ(2). Agonist-induced receptor-G-protein coupling was of a time scale similar to that of receptor activation. As observed for receptor deactivation, receptor-G-protein dissociation was sl...Continue Reading

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Citations

Nov 13, 2015·British Journal of Pharmacology·A D Stumpf, C Hoffmann
Jul 23, 2015·Journal of Molecular Medicine : Official Organ of the Gesellschaft Deutscher Naturforscher Und Ärzte·Martin J Lohse
Jul 1, 2015·Trends in Pharmacological Sciences·Frederick J Ehlert
Sep 24, 2013·Biochimica Et Biophysica Acta·Rajashri SridharanMark E Dumont
Jul 18, 2015·Molecular Pharmacology·Martin J Lohse, Klaus Peter Hofmann
Jun 1, 2020·Molecular Pharmacology·Cristina Perpiñá-VicianoCarsten Hoffmann
Dec 21, 2019·Nature Communications·Serap ErdogmusMichael Mederos Y Schnitzler
Apr 25, 2018·Proceedings of the National Academy of Sciences of the United States of America·Olga S IlyaskinaMoritz Bünemann
Jan 25, 2021·Biosensors & Bioelectronics·Hannes SchihadaGunnar Schulte
May 29, 2021·Communications Biology·Volker JelinekMoritz Bünemann

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