Spirohexene-Tetrazine Ligation Enables Bioorthogonal Labeling of Class B G Protein-Coupled Receptors in Live Cells.
J Am Chem Soc
; 139(38): 13376-13386, 2017 09 27.
Article
en En
| MEDLINE
| ID: mdl-28876923
ABSTRACT
A new bioorthogonal reactant pair, spiro[2.3]hex-1-ene (Sph) and 3,6-di(2-pyridyl)-s-tetrazine (DpTz), for the strain-promoted inverse electron-demand Diels-Alder cycloaddition, that is, tetrazine ligation, is reported. As compared to the previously reported strained alkenes such as trans-cyclooctene (TCO) and 1,3-disubstituted cyclopropene, Sph exhibits balanced reactivity and stability in tetrazine ligation with the protein substrates. A lysine derivative of Sph, SphK, was site-selectively incorporated into the extracellular loop regions (ECLs) of GCGR and GLP-1R, two members of class B G protein-coupled receptors (GPCRs) in mammalian cells with the incorporation efficiency dependent on the location. Subsequent bioorthogonal reactions with the fluorophore-conjugated DpTz reagents afforded the fluorescently labeled GCGR and GLP-1R ECL mutants with labeling yield as high as 68%. A multitude of functional assays were performed with these GPCR mutants, including ligand binding, ligand-induced receptor internalization, and ligand-stimulated intracellular cAMP accumulation. Several positions in the ECL3s of GCGR and GLP-1R were identified that tolerate SphK mutagenesis and subsequent bioorthogonal labeling. The generation of functional, fluorescently labeled ECL3 mutants of GCGR and GLP-1R should allow biophysical studies of conformation dynamics of this important class of GPCRs in their native environment in live cells.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Compuestos de Espiro
/
Coloración y Etiquetado
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Receptores Acoplados a Proteínas G
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Alquenos
Límite:
Animals
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Humans
Idioma:
En
Revista:
J Am Chem Soc
Año:
2017
Tipo del documento:
Article
País de afiliación:
Estados Unidos