A fluorescence resonance energy transfer-based M2 muscarinic receptor
sensor reveals rapid kinetics of allosteric modulation
M. Maier-Peuschel, N. Frolich, C. Dees, L. Hommers, C. Hoffmann, V. Nikolaev, and M. Lohse. J Biol Chem, 285 (12):
8793-800(March 2010)Maier-Peuschel, Monika Frolich, Nadine Dees, Christian Hommers, Leif
G Hoffmann, Carsten Nikolaev, Viacheslav O Lohse, Martin J Research
Support, Non-U.S. Gov't United States The Journal of biological chemistry
J Biol Chem. 2010 Mar 19;285(12):8793-800. Epub 2010 Jan 18..
Abstract
Allosteric modulators have been identified for several G protein-coupled
receptors, most notably muscarinic receptors. To study their mechanism
of action, we made use of a recently developed technique to generate
fluorescence resonance energy transfer (FRET)-based sensors to monitor
G protein-coupled receptor activation. Cyan fluorescent protein was
fused to the C terminus of the M(2) muscarinic receptor, and a specific
binding sequence for the small fluorescent compound fluorescein arsenical
hairpin binder, FlAsH, was inserted into the third intracellular
loop; the latter site was labeled in intact cells by incubation with
FlAsH. We then measured FRET between the donor cyan fluorescent protein
and the acceptor FlAsH in intact cells and monitored its changes
in real time. Agonists such as acetylcholine and carbachol induced
rapid changes in FRET, indicative of agonist-induced conformational
changes. Removal of the agonists or addition of an antagonist caused
a reversal of this signal with rate constants between 400 and 1100
ms. The allosteric ligands gallamine and dimethyl-W84 caused no changes
in FRET when given alone, but increased FRET when given in the presence
of an agonist, compatible with an inactivation of the receptors.
The kinetics of these effects were very rapid, with rate constants
of 80-100 ms and approximately 200 ms for saturating concentrations
of gallamine and dimethyl-W84, respectively. Because these speeds
are significantly faster than the responses to antagonists, these
data indicate that gallamine and dimethyl-W84 are allosteric ligands
and actively induce a conformation of the M(2) receptor with a reduced
affinity for its agonists.
Maier-Peuschel, Monika Frolich, Nadine Dees, Christian Hommers, Leif
G Hoffmann, Carsten Nikolaev, Viacheslav O Lohse, Martin J Research
Support, Non-U.S. Gov't United States The Journal of biological chemistry
J Biol Chem. 2010 Mar 19;285(12):8793-800. Epub 2010 Jan 18.
%0 Journal Article
%1 Maier-Peuschel2010
%A Maier-Peuschel, M.
%A Frolich, N.
%A Dees, C.
%A Hommers, L. G.
%A Hoffmann, C.
%A Nikolaev, V. O.
%A Lohse, M. J.
%D 2010
%J J Biol Chem
%K 50 Acetylcholine/chemistry Allosteric Animals CHO Carbachol/chemistry Concentration Confocal/methods Cricetinae Cricetulus Energy Fluorescence Fluorescent Gallamine Green Humans Inhibitory Ligands M2/*chemistry Microscopy, Muscarinic Phthalimides/chemistry Protein Proteins/chemistry Resonance Site Structure, Tertiary Transfer/*methods Triethiodide/chemistry Receptor Cell
%N 12
%P 8793-800
%T A fluorescence resonance energy transfer-based M2 muscarinic receptor
sensor reveals rapid kinetics of allosteric modulation
%U http://www.ncbi.nlm.nih.gov/pubmed/20083608
%V 285
%X Allosteric modulators have been identified for several G protein-coupled
receptors, most notably muscarinic receptors. To study their mechanism
of action, we made use of a recently developed technique to generate
fluorescence resonance energy transfer (FRET)-based sensors to monitor
G protein-coupled receptor activation. Cyan fluorescent protein was
fused to the C terminus of the M(2) muscarinic receptor, and a specific
binding sequence for the small fluorescent compound fluorescein arsenical
hairpin binder, FlAsH, was inserted into the third intracellular
loop; the latter site was labeled in intact cells by incubation with
FlAsH. We then measured FRET between the donor cyan fluorescent protein
and the acceptor FlAsH in intact cells and monitored its changes
in real time. Agonists such as acetylcholine and carbachol induced
rapid changes in FRET, indicative of agonist-induced conformational
changes. Removal of the agonists or addition of an antagonist caused
a reversal of this signal with rate constants between 400 and 1100
ms. The allosteric ligands gallamine and dimethyl-W84 caused no changes
in FRET when given alone, but increased FRET when given in the presence
of an agonist, compatible with an inactivation of the receptors.
The kinetics of these effects were very rapid, with rate constants
of 80-100 ms and approximately 200 ms for saturating concentrations
of gallamine and dimethyl-W84, respectively. Because these speeds
are significantly faster than the responses to antagonists, these
data indicate that gallamine and dimethyl-W84 are allosteric ligands
and actively induce a conformation of the M(2) receptor with a reduced
affinity for its agonists.
@article{Maier-Peuschel2010,
abstract = {Allosteric modulators have been identified for several G protein-coupled
receptors, most notably muscarinic receptors. To study their mechanism
of action, we made use of a recently developed technique to generate
fluorescence resonance energy transfer (FRET)-based sensors to monitor
G protein-coupled receptor activation. Cyan fluorescent protein was
fused to the C terminus of the M(2) muscarinic receptor, and a specific
binding sequence for the small fluorescent compound fluorescein arsenical
hairpin binder, FlAsH, was inserted into the third intracellular
loop; the latter site was labeled in intact cells by incubation with
FlAsH. We then measured FRET between the donor cyan fluorescent protein
and the acceptor FlAsH in intact cells and monitored its changes
in real time. Agonists such as acetylcholine and carbachol induced
rapid changes in FRET, indicative of agonist-induced conformational
changes. Removal of the agonists or addition of an antagonist caused
a reversal of this signal with rate constants between 400 and 1100
ms. The allosteric ligands gallamine and dimethyl-W84 caused no changes
in FRET when given alone, but increased FRET when given in the presence
of an agonist, compatible with an inactivation of the receptors.
The kinetics of these effects were very rapid, with rate constants
of 80-100 ms and approximately 200 ms for saturating concentrations
of gallamine and dimethyl-W84, respectively. Because these speeds
are significantly faster than the responses to antagonists, these
data indicate that gallamine and dimethyl-W84 are allosteric ligands
and actively induce a conformation of the M(2) receptor with a reduced
affinity for its agonists.},
added-at = {2010-12-14T18:12:02.000+0100},
author = {Maier-Peuschel, M. and Frolich, N. and Dees, C. and Hommers, L. G. and Hoffmann, C. and Nikolaev, V. O. and Lohse, M. J.},
biburl = {https://www.bibsonomy.org/bibtex/2c771523817be6e24104923ab7b3c6b34/pharmawuerz},
comment = {2838301},
endnotereftype = {Journal Article},
groups = {private},
interhash = {4de76e0eda842d9cd5809d33c8987b9c},
intrahash = {c771523817be6e24104923ab7b3c6b34},
issn = {1083-351X (Electronic) 0021-9258 (Linking)},
journal = {J Biol Chem},
keywords = {50 Acetylcholine/chemistry Allosteric Animals CHO Carbachol/chemistry Concentration Confocal/methods Cricetinae Cricetulus Energy Fluorescence Fluorescent Gallamine Green Humans Inhibitory Ligands M2/*chemistry Microscopy, Muscarinic Phthalimides/chemistry Protein Proteins/chemistry Resonance Site Structure, Tertiary Transfer/*methods Triethiodide/chemistry Receptor Cell},
month = {Mar 19},
note = {Maier-Peuschel, Monika Frolich, Nadine Dees, Christian Hommers, Leif
G Hoffmann, Carsten Nikolaev, Viacheslav O Lohse, Martin J Research
Support, Non-U.S. Gov't United States The Journal of biological chemistry
J Biol Chem. 2010 Mar 19;285(12):8793-800. Epub 2010 Jan 18.},
number = 12,
pages = {8793-800},
shorttitle = {A fluorescence resonance energy transfer-based M2 muscarinic receptor
sensor reveals rapid kinetics of allosteric modulation},
timestamp = {2010-12-14T18:20:40.000+0100},
title = {A fluorescence resonance energy transfer-based M2 muscarinic receptor
sensor reveals rapid kinetics of allosteric modulation},
url = {http://www.ncbi.nlm.nih.gov/pubmed/20083608},
volume = 285,
year = 2010
}