TY - JOUR
T1 - FRET-based monitoring of conformational change of the β2 adrenergic receptor in living cells
AU - Nakanishi, Jun
AU - Takarada, Tohru
AU - Yunoki, Shinya
AU - Kikuchi, Yukiko
AU - Maeda, Mizuo
N1 - Funding Information:
This work was supported in part by a Grant-in-Aid for Young Scientists (B) (to J.N.). J.N. thanks the Special Postdoctoral Researcher Program of RIKEN.
PY - 2006/5/19
Y1 - 2006/5/19
N2 - The β2 adrenergic receptor (β2AR) is a G protein-coupled receptor that is selective to epinephrine. We demonstrate herein monitoring of an agonist-induced conformational change of β2AR in living cells. The monitoring method is based on fluorescence resonance energy transfer from a cyan fluorescent protein (CFP) to a biarsenical fluorophore, FlAsH, attached to the C-terminus, and the third intracellular loop (ICL3), respectively. Recombinant β2ARs exhibited agonist-induced increases in the FlAsH/CFP emission ratio, indicating that the ICL3 approached the C-terminus upon activation. Since the emission ratio changes were on a time scale of seconds, the conformational change of β2AR in living cells was more rapid than that of purified β2AR measured in vitro. Interestingly, the direction of the emission ratio change of β2AR was opposite to that of the norepinephrine-responsive α2A adrenergic receptor reported recently. It was suggested that this discrepancy corresponds directly to the diametric biological functions, i.e., the activation or inactivation of adenylyl cyclase.
AB - The β2 adrenergic receptor (β2AR) is a G protein-coupled receptor that is selective to epinephrine. We demonstrate herein monitoring of an agonist-induced conformational change of β2AR in living cells. The monitoring method is based on fluorescence resonance energy transfer from a cyan fluorescent protein (CFP) to a biarsenical fluorophore, FlAsH, attached to the C-terminus, and the third intracellular loop (ICL3), respectively. Recombinant β2ARs exhibited agonist-induced increases in the FlAsH/CFP emission ratio, indicating that the ICL3 approached the C-terminus upon activation. Since the emission ratio changes were on a time scale of seconds, the conformational change of β2AR in living cells was more rapid than that of purified β2AR measured in vitro. Interestingly, the direction of the emission ratio change of β2AR was opposite to that of the norepinephrine-responsive α2A adrenergic receptor reported recently. It was suggested that this discrepancy corresponds directly to the diametric biological functions, i.e., the activation or inactivation of adenylyl cyclase.
KW - Agonist
KW - Conformational change
KW - Cyan fluorescent protein
KW - FlAsH
KW - Fluorescence imaging
KW - Fluorescence resonance energy transfer
KW - G protein-coupled receptor
KW - β Adrenergic receptor
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U2 - 10.1016/j.bbrc.2006.03.064
DO - 10.1016/j.bbrc.2006.03.064
M3 - Article
C2 - 16580633
AN - SCOPUS:33646052569
VL - 343
SP - 1191
EP - 1196
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 4
ER -