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 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.

Development of a CCK1R-membrane nanoparticle as a fish-out tool for bioactive peptides.

The cholecystokinin receptor type 1 (CCK1R) is a G protein-coupled receptor (GPCR) that is involved in several biological processes including the regulation of the secretion of digestive enzymes. The peptide hormone cholecystokinin (CCK) binds to CCK1R, which is an important pharmacological target for several diseases, including obesity. Interestingly, nutritional dietary peptides also appear to activate CCK1R, and may play a role in CCK1R signaling in the gut. In this study, a novel technique to screen for CCK1R ligands based on affinity-selection is described. Functional expressed CCK1R is reconstituted into membrane nanoparticles called NABBs (nanoscale apo-lipoprotein bound bilayers). NABBs are native-like bilayer membrane systems for incorporation of GPCRs. CCK1R-NABBs were characterized using a fluorescently labeled CCK analog and can be used as a cutting-edge technology to screen for CCK1R ligands using affinity-selection mass spectrometry.

Antibody epitopes on g protein-coupled receptors mapped with genetically encoded photoactivatable cross-linkers.

We developed a strategy for creating epitope maps of monoclonal antibodies (mAbs) that bind to G protein-coupled receptors (GPCRs) containing photo-cross-linkers. Using human CXC chemokine receptor 4 (CXCR4) as a model system, we genetically incorporated the photolabile unnatural amino acid p-azido-l-phenylalanine (azF) at various positions within extracellular loop 2 (EC2). We then mapped the interactions of the azF-CXCR4 variants with mAb 12G5 using targeted loss-of-function studies and photo-cross-linking in whole cells in a microplate-based format. We used a novel variation of a whole cell enzyme-linked immunosorbent assay to quantitate cross-linking efficiency. 12G5 cross-linked primarily to residues 184, 178, and 189 in EC2 of CXCR4. Mapping of the data to the crystal structure of CXCR4 showed a distinct mAb epitope footprint with the photo-cross-linked residues clustered around the loss-of-function sites. We also used the targeted photo-cross-linking approach to study the interaction of human CC chemokine receptor 5 (CCR5) with PRO 140, a humanized mAb that inhibits human immunodeficiency virus-1 cellular entry, and 2D7. The mAbs produced distinct cross-linking patterns on EC2 of CCR5. PRO 140 cross-linked primarily to residues 174 and 175 at the amino-terminal end of EC2, and 2D7 cross-linked mainly to residues 170, 176, and 184. These results were mapped to the recent crystal structure of CCR5 in complex with maraviroc, showing cross-linked residues at the tip of the maraviroc binding crevice formed by EC2. As a strategy for mapping mAb epitopes on GPCRs, our targeted photo-cross-linking method is complementary to loss-of-function mutagenesis results and should be especially useful for studying mAbs with discontinuous epitopes.