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.

Evaluating functional ligand-GPCR interactions in cell-based assays.

G protein-coupled receptors (GPCRs) are a family of transmembrane proteins that act as major mediators of cellular signaling, and are the primary targets for a large portion of clinical therapeutics. Despite their critical role in biology and medicine, a large number of GPCRs are poorly understood, lacking validated ligands or potent synthetic modulators. Ligand-induced GPCR activation can be measured in cell-based assays to test hypotheses about ligand-receptor interactions or to evaluate efficacy of synthetic agonists or antagonists. However, the techniques necessary to develop and implement a cell-based assay to study a given receptor of interest are not commonplace in all laboratories. This chapter outlines methods to develop a cell-based assay to evaluate agonist-induced activation for a GPCR of interest, which can be useful to evaluate the effectiveness of predicted ligands. Examples of sample preparation protocols and data analysis are provided to help researchers from interdisciplinary fields, especially those in fields with relatively little molecular biology or cell culture experience.