Affinity-Driven Aryl Diazonium Labeling of Peptide Receptors on Living Cells

Peptide–receptor interactions play critical roles
in a wide
variety of physiological processes. Methods to link bioactive peptides
covalently to unmodified receptors on the surfaces of living cells
are valuable for studying receptor signaling, dynamics, and trafficking
and for identifying novel peptide–receptor interactions. Here,
we utilize peptide analogues bearing deactivated aryl diazonium groups
for the affinity-driven labeling of unmodified receptors. We demonstrate
that aryl diazonium-bearing peptide analogues can covalently label
receptors on the surface of living cells using both the neurotensin
and the glucagon-like peptide 1 receptor systems. Receptor labeling
occurs in the complex environment of the cell surface in a sequence-specific
manner. We further demonstrate the utility of this covalent labeling
approach for the visualization of peptide receptors by confocal fluorescence
microscopy and for the enrichment and identification of labeled receptors
by mass spectrometry-based proteomics. Aryl diazonium-based affinity-driven
receptor labeling is attractive due to the high abundance of tyrosine
and histidine residues susceptible to azo coupling in the peptide
binding sites of receptors, the ease of incorporation of aryl diazonium
groups into peptides, and the relatively small size of the aryl diazonium
group. This approach should prove to be a powerful and relatively
general method to study peptide–receptor interactions in cellular
contexts.