期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 98, 期 11, 页码 5997-6002出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.101126198
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资金
- NIGMS NIH HHS [5T32GM07365, T32 GM007365] Funding Source: Medline
- NINDS NIH HHS [R01 NS028471, 5 RO1 NS28471] Funding Source: Medline
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.
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