Agonist-induced conformational changes in the G-protein-coupling domain of the beta 2 adrenergic receptor.
The majority of extracellular physiologic signaling molecules act by stimulating GTP-binding protein (G-protein)-coupled receptors (GPCRs). To monitor directly the formation of the active state of a prototypical GPCR, we devised a method to site specifically attach fluorescein to an endogenous cysteine (Cys-265) at the cytoplasmic end of transmembrane 6 (TM6) of the beta(2) adrenergic receptor (beta(2)AR), adjacent to the G-protein-coupling domain. We demonstrate that this tag reports agonist-induced conformational changes in the receptor, with agonists causing a decline in the fluorescence intensity of fluorescein-beta(2)AR that is proportional to the biological efficacy of the agonist. We also find that agonists alter the interaction between the fluorescein at Cys-265 and fluorescence-quenching reagents localized to different molecular environments of the receptor. These observations are consistent with a rotation and/or tilting of TM6 on agonist activation. Our studies, when compared with studies of activation in rhodopsin, indicate a general mechanism for GPCR activation; however, a notable difference is the relatively slow kinetics of the conformational changes in the beta(2)AR, which may reflect the different energetics of activation by diffusible ligands.
Ghanouni
P
P
Howard Hughes Medical Institute and Department of Biochemistry, Oregon Health Sciences University, Portland, OR 97201, USA.
Steenhuis
J J
JJ
Farrens
D L
DL
Kobilka
B K
BK
D000318:Adrenergic beta-Agonists / Q000737:chemistry*; D000420:Albuterol / Q000737:chemistry; D001665:Binding Sites; D003545:Cysteine; D003902:Detergents / Q000737:chemistry; D004280:Dobutamine / Q000737:chemistry; D004837:Epinephrine / Q000737:chemistry; D005452:Fluoresceins; D005453:Fluorescence; D005456:Fluorescent Dyes; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005960:Glucosides / Q000737:chemistry; D006801:Humans; D007545:Isoproterenol / Q000737:chemistry; D007700:Kinetics; D008239:Lysine; D008823:Micelles; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry* / Q000235:genetics; D013229:Stearic Acids / Q000737:chemistry
D000318:Adrenergic beta-Agonists; D003902:Detergents; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005960:Glucosides; D008823:Micelles; D018343:Receptors, Adrenergic, beta-2; D013229:Stearic Acids; D004280:Dobutamine; C031183:stearic acid; C040358:dodecyl maltoside; C037041:fluorescein 5-maleimide; D019204:GTP-Binding Proteins; D008239:Lysine; D003545:Cysteine; D007545:Isoproterenol; D000420:Albuterol; D004837:Epinephrine