Furan Cross-Linking Technology for Investigating GPCR–Ligand Interactions
Interactions between G protein-coupled receptors and their ligands hold extensive potential for drug discovery. Studying these interactions poses technical problems due to their transient nature and the inherent difficulties when working with G protein-coupled receptors (GPCR) that are only functional in a membrane setting. Here, we describe the use of a furan-based chemical cross-linking methodology to achieve selective covalent coupling between a furan-modified peptide ligand and its native GPCR present on the surface of living cells under normal cell culture conditions. This methodology relies on the oxidation of the furan moiety, which can be achieved by either addition of an external oxidation signal or by the reactive oxygen species produced by the cell. The cross-linked ligand–GPCR complex is subsequently detected by Western blotting based on the biotin label that is incorporated in the peptide ligand.
Key wordsFuran Receptor GPCR Chemical cross-linking Peptide Solid phase peptide synthesis Western blot Singlet oxygen Reactive oxygen species
- 1.Huang W, Manglik A, Venkatakrishnan AJ, Laeremans T, Feinberg EN, Sanborn AL, Kato HE, Livingston KE, Thorsen TS, Kling RC, Granier S, Gmeiner P, Husbands SM, Traynor JR, Weis WI, Steyaert J, Dror RO, Kobilka BK (2015) Structural insights into μ-opioid receptor activation. Nature 524:315–321CrossRefGoogle Scholar
- 15.https://www.licor.com/bio/products/reagents/irdye/streptavidin/. Accessed 19 May 2018
- 16.García-Martin F, Albericio F (2008) Solid supports for the synthesis of peptides–from the first resin used to the most sophisticated in the market. Chem Today 26:29–34Google Scholar
- 23.Alegria-Schaffer A (2014) Western blotting using chemiluminescent substrates. Methods Enzymol 541:251–259. https://doi.org/10.1016/B978-0-12-420119-4.00019-7CrossRefPubMedGoogle Scholar