Probing antibody binding sites on g protein-coupled receptors using genetically encoded photo-activatable cross-linkers

Abstract

We describe a methodology to map epitopes of monoclonal antibodies that bind to G protein-coupled receptors (GPCRs). The method relies on an amber codon suppression strategy to genetically encode photo-activatable cross-linkers, such as p-azido-l-phenylalanine (azF) or p-benzoly-l-phenylalanine (BzF), in GPCRs expressed in mammalian cells in culture. Individual receptor variants that harbor a site-specific photo-crosslinker residue can be assayed for functional activity in standard cell-based assays. The interaction sites between the receptor variants and an antibody can be mapped by determining which of the azF or BzF residues cross-link to the antibody upon UV irradiation. A whole cell enzyme-linked immunosorbent assay (ELISA) is used to quantiate cross-linking efficiency. A binding “footprint” of the antibody of the surface of the receptor is obtained by comparing the sites of amino acid replacements that cause loss of antibody binding with those that create colvalent cross-links with bound antibody. The precision of the receptor-antibody binding-site map is determined by the number of mutants tested and whether or not high resolution crystal structures or homology models are available. The targeted photo-cross-linking method is complementary to loss-of-function mutagenesis and is especially useful to study anti-receptor antibodies with discontinuous epitopes.