Selective Covalent Labeling of Tag-Fused GPCR Proteins on Live Cell Surface with a Synthetic Probe for Their Functional Analysis

• Published in: Journal of the American Chemical Society 2010-07, Vol.132 (27), p.9301-9309
• Main Authors: Nonaka, Hiroshi, Fujishima, Sho-hei, Uchinomiya, Sho-hei, Ojida, Akio, Hamachi, Itaru
• Format: Article
• Language: English
• Subjects: Biotin; Cell Line; Cysteine; Endocytosis; Endosomes - metabolism; Fluoresceins; Humans; Molecular Probe Techniques; Molecular Probes - chemistry; Oligopeptides; Organometallic Compounds; Receptors, G-Protein-Coupled - chemistry; Receptors, G-Protein-Coupled - metabolism; Staining and Labeling
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja910703v

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 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.