Fluorescence resonance energy transfer analysis of alpha 2a-adrenergic
receptor activation reveals distinct agonist-specific conformational
changes
A. Zurn, U. Zabel, J. Vilardaga, H. Schindelin, M. Lohse, and C. Hoffmann. Mol Pharmacol, 75 (3):
534-41(March 2009)Zurn, A Zabel, U Vilardaga, J-P Schindelin, H Lohse, M J Hoffmann,
C Comparative Study Research Support, Non-U.S. Gov't United States
Molecular pharmacology Mol Pharmacol. 2009 Mar;75(3):534-41. Epub
2008 Dec 23..
Abstract
Several lines of evidence suggest that G-protein-coupled receptors
can adopt different active conformations, but their direct demonstration
in intact cells is still missing. Using a fluorescence resonance
energy transfer (FRET)-based approach we studied conformational changes
in alpha(2A)-adrenergic receptors in intact cells. The receptors
were C-terminally labeled with cyan fluorescent protein and with
fluorescein arsenical hairpin binder at different sites in the third
intracellular loop: N-terminally close to transmembrane domain V
(I3-N), in the middle of the loop (I3-M), or C-terminally close to
transmembrane domain VI (I3-C). All constructs retained normal ligand
binding and signaling properties. Changes in FRET between the labels
were determined in intact cells in response to different agonists.
The full agonist norepinephrine evoked similar FRET changes for all
three constructs. The strong partial agonists clonidine and dopamine
induced partial FRET changes for all constructs. However, the weak
partial agonists octopamine and norphenephrine only induced detectable
changes in the construct I3-C but no change in I3-M and I3-N. Dopamine-induced
FRET-signals were approximately 1.5-fold slower than those for norepinephrine
in I3-C and I3-M but >3-fold slower in I3-N. Our data indicate that
the different ligands induced conformational changes in the receptor
that were sensed differently in different positions of the third
intracellular loop. This agrees with X-ray receptor structures indicating
larger agonist-induced movements at the cytoplasmic ends of transmembrane
domain VI than V and suggests that partial agonism is linked to distinct
conformational changes within a G-protein-coupled receptor.
Zurn, A Zabel, U Vilardaga, J-P Schindelin, H Lohse, M J Hoffmann,
C Comparative Study Research Support, Non-U.S. Gov't United States
Molecular pharmacology Mol Pharmacol. 2009 Mar;75(3):534-41. Epub
2008 Dec 23.
%0 Journal Article
%1 Zurn2009
%A Zurn, A.
%A Zabel, U.
%A Vilardaga, J. P.
%A Schindelin, H.
%A Lohse, M. J.
%A Hoffmann, C.
%D 2009
%J Mol Pharmacol
%K Adrenergic Agonists/metabolism/*pharmacology Animals Binding/drug Cell Clonidine/metabolism Conformation/drug Dopamine/metabolism Energy Fluorescence Humans Ligands Line Mice Norepinephrine/metabolism Octopamine/metabolism Protein Resonance Transfer/methods alpha-2/*agonists/*chemistry/metabolism effects effects/physiology Receptor
%N 3
%P 534-41
%T Fluorescence resonance energy transfer analysis of alpha 2a-adrenergic
receptor activation reveals distinct agonist-specific conformational
changes
%U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19106230
%V 75
%X Several lines of evidence suggest that G-protein-coupled receptors
can adopt different active conformations, but their direct demonstration
in intact cells is still missing. Using a fluorescence resonance
energy transfer (FRET)-based approach we studied conformational changes
in alpha(2A)-adrenergic receptors in intact cells. The receptors
were C-terminally labeled with cyan fluorescent protein and with
fluorescein arsenical hairpin binder at different sites in the third
intracellular loop: N-terminally close to transmembrane domain V
(I3-N), in the middle of the loop (I3-M), or C-terminally close to
transmembrane domain VI (I3-C). All constructs retained normal ligand
binding and signaling properties. Changes in FRET between the labels
were determined in intact cells in response to different agonists.
The full agonist norepinephrine evoked similar FRET changes for all
three constructs. The strong partial agonists clonidine and dopamine
induced partial FRET changes for all constructs. However, the weak
partial agonists octopamine and norphenephrine only induced detectable
changes in the construct I3-C but no change in I3-M and I3-N. Dopamine-induced
FRET-signals were approximately 1.5-fold slower than those for norepinephrine
in I3-C and I3-M but >3-fold slower in I3-N. Our data indicate that
the different ligands induced conformational changes in the receptor
that were sensed differently in different positions of the third
intracellular loop. This agrees with X-ray receptor structures indicating
larger agonist-induced movements at the cytoplasmic ends of transmembrane
domain VI than V and suggests that partial agonism is linked to distinct
conformational changes within a G-protein-coupled receptor.
@article{Zurn2009,
abstract = {Several lines of evidence suggest that G-protein-coupled receptors
can adopt different active conformations, but their direct demonstration
in intact cells is still missing. Using a fluorescence resonance
energy transfer (FRET)-based approach we studied conformational changes
in alpha(2A)-adrenergic receptors in intact cells. The receptors
were C-terminally labeled with cyan fluorescent protein and with
fluorescein arsenical hairpin binder at different sites in the third
intracellular loop: N-terminally close to transmembrane domain V
(I3-N), in the middle of the loop (I3-M), or C-terminally close to
transmembrane domain VI (I3-C). All constructs retained normal ligand
binding and signaling properties. Changes in FRET between the labels
were determined in intact cells in response to different agonists.
The full agonist norepinephrine evoked similar FRET changes for all
three constructs. The strong partial agonists clonidine and dopamine
induced partial FRET changes for all constructs. However, the weak
partial agonists octopamine and norphenephrine only induced detectable
changes in the construct I3-C but no change in I3-M and I3-N. Dopamine-induced
FRET-signals were approximately 1.5-fold slower than those for norepinephrine
in I3-C and I3-M but >3-fold slower in I3-N. Our data indicate that
the different ligands induced conformational changes in the receptor
that were sensed differently in different positions of the third
intracellular loop. This agrees with X-ray receptor structures indicating
larger agonist-induced movements at the cytoplasmic ends of transmembrane
domain VI than V and suggests that partial agonism is linked to distinct
conformational changes within a G-protein-coupled receptor.},
added-at = {2010-12-14T18:12:02.000+0100},
author = {Zurn, A. and Zabel, U. and Vilardaga, J. P. and Schindelin, H. and Lohse, M. J. and Hoffmann, C.},
biburl = {https://www.bibsonomy.org/bibtex/23d1ab294c29a9b599cd277ddba6a2c22/pharmawuerz},
endnotereftype = {Journal Article},
interhash = {6cff8a941a7dbc9e54c3afecad460bca},
intrahash = {3d1ab294c29a9b599cd277ddba6a2c22},
issn = {1521-0111 (Electronic) 1521-0111 (Linking)},
journal = {Mol Pharmacol},
keywords = {Adrenergic Agonists/metabolism/*pharmacology Animals Binding/drug Cell Clonidine/metabolism Conformation/drug Dopamine/metabolism Energy Fluorescence Humans Ligands Line Mice Norepinephrine/metabolism Octopamine/metabolism Protein Resonance Transfer/methods alpha-2/*agonists/*chemistry/metabolism effects effects/physiology Receptor},
month = Mar,
note = {Zurn, A Zabel, U Vilardaga, J-P Schindelin, H Lohse, M J Hoffmann,
C Comparative Study Research Support, Non-U.S. Gov't United States
Molecular pharmacology Mol Pharmacol. 2009 Mar;75(3):534-41. Epub
2008 Dec 23.},
number = 3,
pages = {534-41},
shorttitle = {Fluorescence resonance energy transfer analysis of alpha 2a-adrenergic
receptor activation reveals distinct agonist-specific conformational
changes},
timestamp = {2010-12-14T18:22:43.000+0100},
title = {Fluorescence resonance energy transfer analysis of alpha 2a-adrenergic
receptor activation reveals distinct agonist-specific conformational
changes},
url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19106230},
volume = 75,
year = 2009
}