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

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 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.

Chemical labelling for visualizing native AMPA receptors in live neurons

“…Receptors located on the cell surface are then enzymatically and covalently labelled with fluorescent probes such as fluorescein and Alexa dyes78. To reduce the size of the protein tags (20–33 kDa), a complementary recognition pairs comprising a short peptide tag (1–3 kDa) and a small molecular probe are also being developed910111213. A combination of bio-orthogonal chemistry and genetic incorporation of a non-naturally occurring amino acid is also claimed to effectively label cell-surface receptors with minimal structural disturbance14.…”

Real-Time Monitoring of ATP-Responsive Drug Release Using Mesoporous-Silica-Coated Multicolor Upconversion Nanoparticles

“…53–55 To develop the ATP-responsive molecular valve in our DDS, we synthesized a compact block polypeptide containing oligo-aspartate side chains using N -carboxyl anhydride (NCA) based ring opening metathesis polymerization (ROMP) technique with two-generation polymerization. 63–65 This technique allows for extensive control over the polymerization process, thereby leading to the production of highly monodisperse synthetic polypeptides, while avoiding any unfavorable side reactions.…”

“…The high binding affinity of the oligo-aspartate moieties in polypeptide side chains to TDPA-Zn 2+ moieties on the surface of the UCNP@MSN allows the loaded drugs to remain entrapped inside the mesopores. 53–55 It is expected that, because of the overlap of broad absorption peaks of loaded model drugs [ e.g. , doxorubicin (DOX) and camptothecin (CPT)] with the multiple sharp emission peaks of UCNP, as well as their close proximity, luminescence resonance energy transfer (LRET) would occur from UCNP (donor) to drug moieties (acceptor), which will result in quenching of the UV–vis emission of UCNPs.…”

Labeling and Single-Molecule Methods To Monitor G Protein-Coupled Receptor Dynamics

“…The four Zn(II) atoms coordinate with the oligo-aspartate tag fused to the N-terminus of the receptor, facilitating the formation of a thioester bond with the N-terminal cysteine. 271 (e) Template-directed labeling based on a coiled-coil motif. 272 Similarly to FRET, BRET has been used to examine receptor oligomerization.…”