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

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.
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Structure (London, England : 1993), 2014, 22 (1), pp.149-55. 〈10.1016/j.str.2013.10.002〉
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http://hal.univ-grenoble-alpes.fr/hal-01130648
Contributeur : Frank Thomas <>
Soumis le : jeudi 12 mars 2015 - 10:28:44
Dernière modification le : lundi 26 mars 2018 - 14:51:15

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Katarzyna Niescierowicz, Lydia Caro, Vadim Cherezov, Michel Vivaudou, Christophe J Moreau. Functional assay for T4 lysozyme-engineered G protein-coupled receptors with an ion channel reporter.. Structure (London, England : 1993), 2014, 22 (1), pp.149-55. 〈10.1016/j.str.2013.10.002〉. 〈hal-01130648〉

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