Agonists induce conformational changes in transmembrane domains III and VI of the beta2 adrenoceptor.
Agonist binding to G protein-coupled receptors is believed to promote a conformational change that leads to the formation of the active receptor state. However, the character of this conformational change which provides the important link between agonist binding and G protein coupling is not known. Here we report evidence that agonist binding to the beta2 adrenoceptor induces a conformational change around 125Cys in transmembrane domain (TM) III and around 285Cys in TM VI. A series of mutant beta2 adrenoceptors with a limited number of cysteines available for chemical derivatization were purified, site-selectively labeled with the conformationally sensitive, cysteine-reactive fluorophore IANBD and analyzed by fluorescence spectroscopy. Like the wild-type receptor, mutant receptors containing 125Cys and/or 285Cys showed an agonist-induced decrease in fluorescence, while no agonist-induced response was observed in a receptor where these two cysteines were mutated. These data suggest that IANBD bound to 125Cys and 285Cys are exposed to a more polar environment upon agonist binding, and indicate that movements of transmembrane segments III and VI are involved in activation of G protein-coupled receptors.
Gether
U
U
Howard Hughes Medical Institute, Stanford University Medical School, Stanford, CA 94305, USA. gether@mfi.ku.dk
Lin
S
S
Ghanouni
P
P
Ballesteros
J A
JA
Weinstein
H
H
Kobilka
B K
BK
D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000595:Amino Acid Sequence; D003198:Computer Simulation; D003545:Cysteine / Q000235:genetics; D004082:Dihydroalprenolol / Q000494:pharmacology; D005456:Fluorescent Dyes; D007545:Isoproterenol / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation / Q000187:drug effects*; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics; D013050:Spectrometry, Fluorescence
D000318:Adrenergic beta-Agonists; D005456:Fluorescent Dyes; D018343:Receptors, Adrenergic, beta-2; D004082:Dihydroalprenolol; D003545:Cysteine; D007545:Isoproterenol