Selective covalent labeling of tag-fused GPCR proteins on live cell surface with a synthetic probe for their functional analysis

Hiroshi Nonaka, Sho Hei Fujishima, Shohei Uchinomiya, Akio Ojida, Itaru Hamachi

Research output: Contribution to journalArticle

64 Citations (Scopus)

Abstract

Selective protein labeling with a small molecular probe is a versatile method for elucidating protein functions in living cells. In this paper, we report a covalent labeling method of tag-fused G-protein coupled receptor (GPCR) proteins expressing on cell surfaces utilizing small functional molecules. This method employs the selective and rapid reaction of a peptide tag and a molecular probe, which comprises the cysteine-containing short CA6D4×2 tag (CAAAAAADDDDGDDDD) and a tetranuclear Zn(II)-DpaTyr probe containing a reactive α-chloroacetyl moiety. The covalent labeling of tag-fused GPCRs such as bradykinin receptor (B2R) and acetylcholine receptor (m1AchR) selectively proceeded under physiological conditions during short incubation (10-30 min) with Zn(II)-DpaTyr probes bearing various functional groups. Labeling with fluorophore-appended Zn(II)-DpaTyr probes enabled visualization of the GPCRs on the surface of HEK293 cells by fluorescence. Labeling with the biotin-appended probe allowed introduction of a biotin unit into the GPCRs. This biotin label was utilized for fluorescence bioimaging studies and postlabeling blotting analysis of the labeled GPCRs by use of the specific biotin-streptavidin interaction. The utility of this labeling method was demonstrated in several function analyses of GPCRs, such as fluorescence visualization of the stimuli-responsive internalization of GPCRs and pH change in endosomes containing the internalized GPCRs.

Original languageEnglish
Pages (from-to)9301-9309
Number of pages9
JournalJournal of the American Chemical Society
Volume132
Issue number27
DOIs
Publication statusPublished - Jul 14 2010
Externally publishedYes

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Functional analysis
Biotin
G-Protein-Coupled Receptors
Labeling
Proteins
Molecular Probes
Fluorescence
Bradykinin Receptors
Streptavidin
HEK293 Cells
Endosomes
Cholinergic Receptors
Bearings (structural)
Visualization
Cysteine
Fluorophores
Peptides
Functional groups
Labels
Cells

All Science Journal Classification (ASJC) codes

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

Cite this

Selective covalent labeling of tag-fused GPCR proteins on live cell surface with a synthetic probe for their functional analysis. / Nonaka, Hiroshi; Fujishima, Sho Hei; Uchinomiya, Shohei; Ojida, Akio; Hamachi, Itaru.

In: Journal of the American Chemical Society, Vol. 132, No. 27, 14.07.2010, p. 9301-9309.

Research output: Contribution to journalArticle

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