Advertisement

Furan Cross-Linking Technology for Investigating GPCR–Ligand Interactions

  • Marleen Van Troys
  • Willem Vannecke
  • Christophe Ampe
  • Annemieke MadderEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1947)

Abstract

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 words

Furan Receptor GPCR Chemical cross-linking Peptide Solid phase peptide synthesis Western blot Singlet oxygen Reactive oxygen species 

References

  1. 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
  2. 2.
    Stevens RC, Cherezov V, Katritch V, Abagyan R, Kuhn P, Rosen H, Wüthrich K (2013) The GPCR network: a large-scale collaboration to determine human GPCR structure and function. Nat Rev Drug Discov 12(1):25–34.  https://doi.org/10.1038/nrd3859CrossRefPubMedGoogle Scholar
  3. 3.
    Fredriksson R, Lagerström MC, Lundin LG, Schiöth HB (2003) The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol Pharmacol 63(6):1256–1272.  https://doi.org/10.1124/mol.63.6.1256CrossRefPubMedGoogle Scholar
  4. 4.
    Shemesh R, Toporik A, Levine Z, Hecht I, Rotman G, Wool A, Dahary D, Gofer E, Kliger Y, Soffer MA, Rosenberg A, Eshel D, Cohen Y (2008) Discovery and validation of novel peptide agonists for G-protein-coupled receptors. J Biol Chem 283:34643–34649CrossRefGoogle Scholar
  5. 5.
    Sriram K, Insel PA (2018) GPCRs as targets for approved drugs: How many targets and how many drugs? Mol Pharmacol.  https://doi.org/10.1124/mol.117.111062CrossRefGoogle Scholar
  6. 6.
    Hauser AS, Chavali S, Masuho I, Jahn LJ, Martemyanov KA, Gloriam DE, Babu MM (2018) Pharmacogenomics of GPCR drug targets. Cell 172(1–2):41–54.e19.  https://doi.org/10.1016/j.cell.2017.11.033CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Corgiat BA, Nordman JC, Kabbani N (2014) Chemical crosslinkers enhance detection of receptor interactomes. Front Pharmacol 4:171.  https://doi.org/10.3389/fphar.2013.00171CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Grunbeck A, Sakmar TP (2013) Probing G protein-coupled receptor-ligand interactions with targeted photoactivatable cross-linkers. Biochemistry 52(48):8625–8632.  https://doi.org/10.1021/bi401300yCrossRefPubMedGoogle Scholar
  9. 9.
    Scalabrin M, Dixit SM, Makshood MM, Krzemien CE, Fabris D (2018) Bifunctional cross-linking approaches for mass spectrometry-based investigation of nucleic acids and protein-nucleic acid assemblies. Methods 144:64–78.  https://doi.org/10.1016/j.ymeth.2018.05.001CrossRefPubMedGoogle Scholar
  10. 10.
    Carrette LLG, GysselsE D, Laet N, Madder A (2016) Furan oxidation based cross-linking: a new approach for the study and targeting of nucleic acid and protein interactions. Chem Commun 52:1539–1554.  https://doi.org/10.1039/c5cc08766jCrossRefGoogle Scholar
  11. 11.
    Op de Beeck M, Madder A (2012) Sequence specific furan based DNA crosslinking with visual light. J Am Chem Soc 134(26):10737–10740CrossRefGoogle Scholar
  12. 12.
    Stevens K, Madder A (2009) Furan-modified oligonucleotides for fast, high-yielding and site-selective DNA inter-strand cross-linking with non-modified complements. Nucleic Acids Res 37(5):1555–1565CrossRefGoogle Scholar
  13. 13.
    Halila S, Velasco T, De Clercq P, Madder A (2005) Fine-tuning furan toxicity: fast and quantitative DNA interchain cross-link formation upon selective oxidation of a furan containing oligonucleotide. Chem Commun 7:936–938CrossRefGoogle Scholar
  14. 14.
    Vannecke W, Ampe C, Van Troys M, Beltramo M, Madder A (2017) Cross-linking furan-modified kisspeptin-10 to the KISS receptor. ACS Chem Biol 12(8):2191–2200.  https://doi.org/10.1021/acschembio.7b00396CrossRefPubMedGoogle Scholar
  15. 15.
  16. 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
  17. 17.
    Redmond RW, Gamlin JN (1999) A compilation of singlet oxygen yields from biologically relevant molecules. Photochem Photobiol 70:391–475CrossRefGoogle Scholar
  18. 18.
    Kochevar IE, Lambert CR, Lynch MC, Tedesco AC (1996) Comparison of photosensitized plasma membrane damage caused by singlet oxygen and free radicals. Biochim Biophys Acta 1280:223–230CrossRefGoogle Scholar
  19. 19.
    Antonatou E, Hoogewijs K, Kalaitzakis D, Baudot A, Vassilikogiannakis G (2016) Madder a singlet oxygen-induced furan oxidation for site-specific and chemoselective peptide ligation. Chem Eur J 22(25):8457–8461.  https://doi.org/10.1002/chem.201601113CrossRefPubMedGoogle Scholar
  20. 20.
    Rabilloud T, Adessi C, Giraudel A, Lunardi J (1997) Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 18(3–4):307–316CrossRefGoogle Scholar
  21. 21.
    He F (2011) Laemmli-SDS-PAGE. Bio Protocol Bio101:e80.  https://doi.org/10.21769/BioProtoc.80CrossRefGoogle Scholar
  22. 22.
    Misu R, Oishi S, Setsuda S, Noguchi T, Kaneda M, Ohno H, Evans B, Navenot JM, Peiper SC, Fujii N (2013) Characterization of the receptor binding residues of kisspeptins by positional scanning using peptide photoaffinity probes. Bioorg Med Chem Lett 23:2628–2631CrossRefGoogle Scholar
  23. 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
  24. 24.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Marleen Van Troys
    • 1
    • 2
  • Willem Vannecke
    • 3
  • Christophe Ampe
    • 1
    • 2
  • Annemieke Madder
    • 3
    Email author
  1. 1.Department of Biochemistry, Faculty of Medicine and Health SciencesGhent UniversityGhentBelgium
  2. 2.Department of Biomolecular Medicine, Faculty of Medicine and Health SciencesGhent UniversityGhentBelgium
  3. 3.Organic and Biomimetic Chemistry Research Group, Department of Organic and Macromolecular ChemistryGhent UniversityGhentBelgium

Personalised recommendations