Probing Antibody Binding Sites on G Protein-Coupled Receptors Using Genetically Encoded Photo-Activatable Cross-Linkers.

We describe a methodology to map epitopes of monoclonal antibodies (mAbs) that bind to G protein-coupled receptors (GPCRs). The method involves using genetic code expansion technology to introduce a non-canonical amino acid (ncAA) residue into an expressed GPCR that can serve as a photo-activatable cross-linkers in mammalian cells in culture. Interaction sites between the engineered receptor variants and the cognate mAb are mapped by determining which of the p-azido-L-phenylalanine (azF) or p-benzoyl-L-phenylalanine (BzF) residues cross-link to the mAb upon ultraviolet (UV) irradiation. These sites are compared with the sites of amino acid replacements that cause loss of mAb binding to create a surface binding map of the mAb epitope. The precision of the GPCR-mAb binding-site map is dependent on the number of receptor mutants studied and the availability of a high-resolution three-dimensional structural models. One advantage of the method is that anti-receptor mAbs with discontinuous epitopes may also be elucidated. In addition, the method is also applicable to map the cell-surface epitopes of mAbs targeting proteins other than GPCRs.