Agonist-induced conformational changes in the G-protein-coupling domain of the β2 adrenergic receptor

Pejman Ghanouni, Jacqueline J. Steenhuis, David Farrens, Brian K. Kobilka

Research output: Contribution to journalArticle

295 Citations (Scopus)

Abstract

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 β2 adrenergic receptor (β2AR), 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-β2AR 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 β2AR, which may reflect the different energetics of activation by diffusible ligands.

Original languageEnglish (US)
Pages (from-to)5997-6002
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume98
Issue number11
DOIs
StatePublished - May 22 2001

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Fluorescein
GTP-Binding Proteins
Adrenergic Receptors
G-Protein-Coupled Receptors
Fluorescence
Rhodopsin
Cysteine
Ligands
Protein Domains

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Agonist-induced conformational changes in the G-protein-coupling domain of the β2 adrenergic receptor. / Ghanouni, Pejman; Steenhuis, Jacqueline J.; Farrens, David; Kobilka, Brian K.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 98, No. 11, 22.05.2001, p. 5997-6002.

Research output: Contribution to journalArticle

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