Selective covalent labeling of tag-fused GPCR proteins on live cell surface with a synthetic probe for their functional analysis.
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 CA6D4x2 tag (CAAAAAADDDDGDDDD) and a tetranuclear Zn(II)-DpaTyr probe containing a reactive alpha-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.
Nonaka
Hiroshi
H
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura Campus, Nishikyo-ku, Kyoto 615-8510, Japan.
Fujishima
Sho-hei
SH
Uchinomiya
Sho-hei
SH
Ojida
Akio
A
Hamachi
Itaru
I
D001710:Biotin; D002460:Cell Line; D003545:Cysteine; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005452:Fluoresceins; D006801:Humans; D015336:Molecular Probe Techniques; D015335:Molecular Probes / Q000737:chemistry*; D009842:Oligopeptides; D009942:Organometallic Compounds; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000378:metabolism; D013194:Staining and Labeling
D005452:Fluoresceins; D015335:Molecular Probes; D009842:Oligopeptides; D009942:Organometallic Compounds; D043562:Receptors, G-Protein-Coupled; C521584:Zn(II)-DpaTyr; D001710:Biotin; D003545:Cysteine