Functional assay for T4 lysozyme-engineered G protein-coupled receptors with an ion channel reporter.

Katarzyna Niescierowicz; Lydia Caro; Vadim Cherezov; Michel Vivaudou; Christophe J Moreau

doi:10.1016/j.str.2013.10.002

Functional assay for T4 lysozyme-engineered G protein-coupled receptors with an ion channel reporter.

Katarzyna Niescierowicz ¹ Lydia Caro ² Vadim Cherezov Michel Vivaudou ¹ Christophe J Moreau ³

1 IBS - UMR 5075 - Institut de biologie structurale

2 ECAM - ECAM Rennes - Louis de Broglie

3 Foie, métabolismes et cancer

Abstract : Structural studies of G protein-coupled receptors (GPCRs) extensively use the insertion of globular soluble protein domains to facilitate their crystallization. However, when inserted in the third intracellular loop (i3 loop), the soluble protein domain disrupts their coupling to G proteins and impedes the GPCRs functional characterization by standard G protein-based assays. Therefore, activity tests of crystallization-optimized GPCRs are essentially limited to their ligand binding properties using radioligand binding assays. Functional characterization of additional thermostabilizing mutations requires the insertion of similar mutations in the wild-type receptor to allow G protein-activation tests. We demonstrate that ion channel-coupled receptor technology is a complementary approach for a comprehensive functional characterization of crystallization-optimized GPCRs and potentially of any engineered GPCR. Ligand-induced conformational changes of the GPCRs are translated into electrical signal and detected by simple current recordings, even though binding of G proteins is sterically blocked by the added soluble protein domain.

Type de document :

Article dans une revue

Structure (London, England : 1993), 2014, 22 (1), pp.149-55. 〈10.1016/j.str.2013.10.002〉

Domaine :

Sciences du Vivant [q-bio] / Biochimie, Biologie Moléculaire / Biologie structurale [q-bio.BM]

Liste complète des métadonnées

http://hal.univ-grenoble-alpes.fr/hal-01130648
Contributeur : Frank Thomas <>
Soumis le : jeudi 12 mars 2015 - 10:28:44
Dernière modification le : lundi 11 février 2019 - 16:51:06

Functional assay for T4 lysozyme-engineered G protein-coupled receptors with an ion channel reporter.

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Functional assay for T4 lysozyme-engineered G protein-coupled receptors with an ion channel reporter.

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