PT - JOURNAL ARTICLE AU - Carsten Hoffmann AU - Susanne Nuber AU - Ulrike Zabel AU - Nicole Ziegler AU - Christiane Winkler AU - Peter Hein AU - Catherine H. Berlot AU - Moritz Bünemann AU - Martin J. Lohse TI - Comparison of the Activation Kinetics of the M<sub>3</sub> Acetylcholine Receptor and a Constitutively Active Mutant Receptor in Living Cells AID - 10.1124/mol.112.077578 DP - 2012 Aug 01 TA - Molecular Pharmacology PG - 236--245 VI - 82 IP - 2 4099 - http://molpharm.aspetjournals.org/content/82/2/236.short 4100 - http://molpharm.aspetjournals.org/content/82/2/236.full SO - Mol Pharmacol2012 Aug 01; 82 AB - 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 Gq-coupled M3 acetylcholine receptor (M3-AChR) with that of a constitutively active mutant receptor (M3-AChR-N514Y) using M3-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 M3-AChR-N514Y. Consistent with this result, at submaximal agonist concentrations, the activation kinetics of M3-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 M3-AChR-yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)-Gγ2. 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 slower for acetylcholine than that for carbachol. Acetylcholine-stimulated increases in receptor-G-protein coupling of M3-AChR-N514Y reached only 12% of that of M3-AChR and thus cannot be kinetically analyzed. G-protein activation was measured using YFP-tagged Gαq and CFP-tagged Gγ2. Activation of Gq was significantly slower than receptor activation and indistinguishable for the two agonists. However, Gq deactivation was significantly prolonged for acetylcholine compared with that for carbachol. Consistent with decreased agonist-stimulated coupling to Gq, agonist-stimulated Gq activation by M3-AChR-N514Y was not detected. Taken together, these results indicate that the N514Y mutation produces constitutive activation of M3-AChR by decreasing the rate of receptor deactivation, while having minimal effect on receptor activation.