title: 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.
authors.affiliation: University Grenoble Alpes, Institut de Biologie Structurale (IBS), F-38027 Grenoble, France; Le Centre National de la Recherche Scientifique (CNRS), IBS, LabEx ICST, F-38027 Grenoble, France; Direction des Sciences du Vivant du Comissariat à l'Energie Atomique (CEA), F-38027 Grenoble, France.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Niescierowicz
authors.affiliation: University Grenoble Alpes, Institut de Biologie Structurale (IBS), F-38027 Grenoble, France; Le Centre National de la Recherche Scientifique (CNRS), IBS, LabEx ICST, F-38027 Grenoble, France; Direction des Sciences du Vivant du Comissariat à l'Energie Atomique (CEA), F-38027 Grenoble, France.
authors.forename: Lydia
authors.initials: L
authors.lastname: Caro
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Vadim
authors.initials: V
authors.lastname: Cherezov
authors.affiliation: University Grenoble Alpes, Institut de Biologie Structurale (IBS), F-38027 Grenoble, France; Le Centre National de la Recherche Scientifique (CNRS), IBS, LabEx ICST, F-38027 Grenoble, France; Direction des Sciences du Vivant du Comissariat à l'Energie Atomique (CEA), F-38027 Grenoble, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Vivaudou
authors.affiliation: University Grenoble Alpes, Institut de Biologie Structurale (IBS), F-38027 Grenoble, France; Le Centre National de la Recherche Scientifique (CNRS), IBS, LabEx ICST, F-38027 Grenoble, France; Direction des Sciences du Vivant du Comissariat à l'Energie Atomique (CEA), F-38027 Grenoble, France. Electronic address: christophe.moreau@ibs.fr.
authors.forename: Christophe J
authors.initials: CJ
authors.lastname: Moreau
mesh_terms: D000818:Animals; D017122:Bacteriophage T4 / Q000737:chemistry / Q000201:enzymology; D001681:Biological Assay; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D009113:Muramidase / Q000235:genetics / Q000378:metabolism; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D043585:Receptor, Muscarinic M2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis; D000099161:Kcnj11 Channel
chemical_list: D009113:Muramidase; D024661:Potassium Channels, Inwardly Rectifying; D021122:Protein Subunits; D043585:Receptor, Muscarinic M2; D011993:Recombinant Fusion Proteins; D000099161:Kcnj11 Channel