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 journalArticlepeer-review

71 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

All Science Journal Classification (ASJC) codes

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

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