FRET-based monitoring of conformational change of the beta2 adrenergic receptor in living cells.
The beta(2) adrenergic receptor (beta(2)AR) is a G protein-coupled receptor that is selective to epinephrine. We demonstrate herein monitoring of an agonist-induced conformational change of beta(2)AR 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 beta(2)ARs 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 beta(2)AR in living cells was more rapid than that of purified beta(2)AR measured in vitro. Interestingly, the direction of the emission ratio change of beta(2)AR was opposite to that of the norepinephrine-responsive alpha(2A) 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.
Bioengineering Laboratory, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan.
Jun
J
Nakanishi
Tohru
T
Takarada
Shinya
S
Yunoki
Yukiko
Y
Kikuchi
Mizuo
M
Maeda
D058666:Adrenergic beta-2 Receptor Agonists; D058672:Adrenergic beta-2 Receptor Antagonists; D000595:Amino Acid Sequence; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism; D005452:Fluoresceins; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D008969:Molecular Sequence Data; D009942:Organometallic Compounds; D011412:Propanolamines / Q000494:pharmacology; D011487:Protein Conformation; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism
C113276:4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein; D058666:Adrenergic beta-2 Receptor Agonists; D058672:Adrenergic beta-2 Receptor Antagonists; C485182:Cyan Fluorescent Protein; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009942:Organometallic Compounds; D011412:Propanolamines; D018343:Receptors, Adrenergic, beta-2; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; C026777:ICI 118551; D003545:Cysteine; D007545:Isoproterenol