RT Journal Article
SR Electronic
T1 Comparison of the Activation Kinetics of the M3-ACh-receptor and a Constitutively Active Mutant Receptor in Living Cells
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP mol.112.077578
DO 10.1124/mol.112.077578
A1 Carsten Hoffmann
A1 Susanne Nuber
A1 Ulrike Zabel
A1 Nicole Ziegler
A1 Christiane Winkler
A1 Peter Hein
A1 Catherine H Berlot
A1 Moritz Bunemann
A1 Martin J Lohse
YR 2012
UL http://molpharm.aspetjournals.org/content/early/2012/05/07/mol.112.077578.abstract
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 FRET-signal. We observed a leftward shift in the concentration dependent FRET-response for acetylcholine and carbachol with M3-AChR-N514Y. Consistent with this, at sub-maximal agonist concentrations the activation kinetics of M3-AChR-N514Y were significantly faster, while at maximal agonist concentrations the kinetics of receptor activation were identical. Receptor deactivation was significantly faster with carbachol than acetylcholine and significantly delayed by the N514Y mutation. Receptor-G-protein interaction was measured by FRET between M3-AChR-YFP and CFP-Gγ2. Agonist induced receptor-G-protein coupling was of a similar time scale as receptor activation. As observed for receptor deactivation, receptor-G-protein dissociation was slower for acetylcholine compared to 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-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 to carbachol. Consistent with decreased agonist-stimulated coupling to Gq, agonist-stimulated Gq activation by M3-AChR-N514Y was not detected. Collectively, 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.