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FRET-Based Measurement of GPCR Conformational Changes

Authors :
Sébastien Granier
Charles Parnot
Samuel Kim
Juan Jose Fung
Michael P. Bokoch
Source :
Methods in Molecular Biology ISBN: 9781603273169
Publication Year :
2009
Publisher :
Humana Press, 2009.

Abstract

The C-termini of G protein-coupled receptors (GPCRs) interact with specific kinases and arrestins in an agonist-dependent manner suggesting that conformational changes induced by ligand binding within the transmembrane domains are transmitted to the C-terminus. Forster resonance energy transfer (FRET) can be used to monitor changes in distance between two protein domains if each site can be specifically and efficiently labeled with a donor or acceptor fluorophore. In order to probe GPCR conformational changes, we have developed a FRET technique that uses site-specific donor and acceptor fluorophores introduced by two orthogonal labeling chemistries. Using this strategy, we examined ligand-induced changes in the distance between two labeled sites in the beta(2) adrenoceptor (beta(2)-AR), a well-characterized GPCR model system. The donor fluorophore, LumioGreen, is chelated by a CCPGCC motif [Fluorescein Arsenical Helix or Hairpin binder (FlAsH) site] introduced through mutagenesis. The acceptor fluorophore, Alexa Fluor 568, is attached to a single reactive cysteine (C265). FRET analyses revealed that the average distance between the intracellular end of transmembrane helix (TM) six and the C-terminus of the beta(2)-AR is 62 A. This relatively large distance suggests that the C-terminus is extended and unstructured. Nevertheless, ligand-specific conformational changes were observed (1). The results provide new insight into the structure of the beta(2)-AR C-terminus and ligand-induced conformational changes that may be relevant to arrestin interactions. The FRET labeling technique described herein can be applied to many GPCRs (and other membrane proteins) and is suitable for conformational studies of domains other than the C-terminus.

Details

ISBN :
978-1-60327-316-9
ISBNs :
9781603273169
Database :
OpenAIRE
Journal :
Methods in Molecular Biology ISBN: 9781603273169
Accession number :
edsair.doi...........acd20179cea3274bad8ff2ef774d8a5d
Full Text :
https://doi.org/10.1007/978-1-60327-317-6_18