Spirohexene-Tetrazine Ligation Enables Bioorthogonal Labeling of Class B G Protein-Coupled Receptors in Live Cells

Carlo P. Ramil, Maoqing Dong, Peng An, Tracey M. Lewandowski, Zhipeng Yu, Laurence J Miller, Qing Lin

Research output: Contribution to journalArticle

22 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)13376-13386
Number of pages11
JournalJournal of the American Chemical Society
Volume139
Issue number38
DOIs
StatePublished - Sep 27 2017

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G-Protein-Coupled Receptors
Labeling
Ligation
Emitter coupled logic circuits
Ligands
Proteins
Mutagenesis
Cycloaddition
Fluorophores
Cycloaddition Reaction
Alkenes
Lysine
Conformations
Assays
Cells
Electrons
Olefins
Derivatives
Substrates

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Spirohexene-Tetrazine Ligation Enables Bioorthogonal Labeling of Class B G Protein-Coupled Receptors in Live Cells. / Ramil, Carlo P.; Dong, Maoqing; An, Peng; Lewandowski, Tracey M.; Yu, Zhipeng; Miller, Laurence J; Lin, Qing.

In: Journal of the American Chemical Society, Vol. 139, No. 38, 27.09.2017, p. 13376-13386.

Research output: Contribution to journalArticle

Ramil, Carlo P. ; Dong, Maoqing ; An, Peng ; Lewandowski, Tracey M. ; Yu, Zhipeng ; Miller, Laurence J ; Lin, Qing. / Spirohexene-Tetrazine Ligation Enables Bioorthogonal Labeling of Class B G Protein-Coupled Receptors in Live Cells. In: Journal of the American Chemical Society. 2017 ; Vol. 139, No. 38. pp. 13376-13386.
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