Advertisement

Detection and Characterization of Receptor Interactions with PDZ Domains

  • Stefanie L. Ritter
  • Randy A. HallEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 756)

Abstract

Many transmembrane receptors are regulated by associations with scaffold proteins containing PDZ domains, which interact with receptor carboxyl-termini to control receptor signaling, trafficking, and localization. We describe here approaches for detecting and characterizing interactions between receptors and PDZ scaffolds. These approaches include the construction and screening of proteomic arrays, blot overlays using fusion proteins, and co-immunoprecipitation studies to assess interactions in cells.

Key words

G protein-coupled receptor PDZ domain Scaffold Affinity Growth factor Tyrosine kinase 

Notes

Acknowledgments

The authors’ research is funded by the National Institutes of Health, USA.

References

  1. 1.
    Overington, J. P., Al-Lazikani, B., and Hopkins, A. L. (2006) How many drug targets are there? Nat Rev Drug Discov 5, 993–6.PubMedCrossRefGoogle Scholar
  2. 2.
    Ritter, S. L., and Hall, R. A. (2009) Fine-tuning of GPCR activity by receptor-interacting proteins. Nat Rev Mol Cell Biol 10, 819–30.PubMedCrossRefGoogle Scholar
  3. 3.
    Sheng, M., and Sala, C. (2001) PDZ domains and the organization of supramolecular complexes. Annu Rev Neurosci 24, 1–29.PubMedCrossRefGoogle Scholar
  4. 4.
    Hillier, B. J., Christopherson, K. S., Prehoda, K. E., Bredt, D. S., and Lim, W. A. (1999) Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS-syntrophin complex. Science 284, 812–5.PubMedCrossRefGoogle Scholar
  5. 5.
    Stiffler, M. A., Chen, J. R., Grantcharova, V. P., Lei, Y., Fuchs, D., Allen, J. E., Zaslavskaia, L. A., and MacBeath, G. (2007) PDZ domain binding selectivity is optimized across the mouse proteome. Science 317, 364–9.PubMedCrossRefGoogle Scholar
  6. 6.
    Fields, S., and Song, O. (1989) A novel genetic system to detect protein-protein interactions. Nature 340, 245–6.PubMedCrossRefGoogle Scholar
  7. 7.
    Bair, C. L., Oppenheim, A., Trostel, A., Prag, G., and Adhya, S. (2008) A phage display system designed to detect and study protein-protein interactions. Mol Microbiol 67, 719–28.PubMedCrossRefGoogle Scholar
  8. 8.
    Park, S. H., and Raines, R. T. (2004) Fluorescence polarization assay to quantify protein-protein interactions. Methods Mol Biol 261, 161–6.PubMedGoogle Scholar
  9. 9.
    Brymora, A., Valova, V. A., and Robinson, P. J. (2004) Protein-protein interactions identified by pull-down experiments and mass spectrometry. Curr Protoc Cell Biol Chapter 17, Unit 17 15.Google Scholar
  10. 10.
    Fam, S. R., Paquet, M., Castleberry, A. M., Oller, H., Lee, C. J., Traynelis, S. F., Smith, Y., Yun, C. C., and Hall, R. A. (2005) P2Y1 receptor signaling is controlled by interaction with the PDZ scaffold NHERF-2. Proc Natl Acad Sci U S A 102, 8042–7.PubMedCrossRefGoogle Scholar
  11. 11.
    He, J., Bellini, M., Inuzuka, H., Xu, J., Xiong, Y., Yang, X., Castleberry, A. M., and Hall, R. A. (2006) Proteomic analysis of beta1-adrenergic receptor interactions with PDZ scaffold proteins. J Biol Chem 281, 2820–7.PubMedCrossRefGoogle Scholar
  12. 12.
    Ciruela, F. (2008) Fluorescence-based methods in the study of protein-protein interactions in living cells. Curr Opin Biotechnol 19, 338–43.PubMedCrossRefGoogle Scholar
  13. 13.
    Vikis, H. G., and Guan, K. L. (2004) Glutathione-S-transferase-fusion based assays for studying protein-protein interactions, Methods Mol Biol 261, 175–186.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of PharmacologyEmory University School of MedicineAtlantaUSA

Personalised recommendations