The Interaction of GTP Cyclohydrolase I and GTP Cyclohydrolase Feedback Regulatory Protein Can be Detected using the Yeast Two-Hybrid System

  • Lance Swick
  • Kei Hirayama
  • Gregory Kapatos


The enzyme GTP cyclohydrolase I (GTPCH) catalyzes the first and rate-limiting step in the synthesis of tetrahydrobiopterin (BH4) (1), the required cofactor for tyrosine and tryptophan hydroxylases (2). All members of the family of nitric oxide synthases also require BH4 (3). Intracellular concentrations of BH4 are normally subsaturating for these enzymes (4) and alterations in BH4 levels brought about by changes in GTPCH activity can modify the synthesis of monoamine and nitric oxide neurotransmitters. GTPCH has a regulatory subunit known as GTP cyclohydrolase feedback regulatory protein (GFRP) (5). In the presence of GFRP BH4 inhibits GTPCH activity thereby inhibiting BH4 synthesis. The interaction between GTPCH and GFRP is not completely understood, but it is thought that two pentameric molecules of GFRP bind to the outer surface of the GTPCH homodecamer (6). We have begun to use the yeast two-hybrid system to study the protein domains required for the association of GTPCH and GFRP. Our results indicate that the two-hybrid system can detect a strong interaction between GTPCH and GFRP. More detailed analysis shows that deletion of the N-terminal domain of GTPCH eliminates the interaction between GTPCH and GFRP. We propose that the association of GFRP and GTPCH involves the N-terminal, proline-rich domain of GTPCH and possibly SH3-like regions in GFRP.


Blue Coloni Prey Plasmid Aromatic Amino Acid Hydroxylase Galactosidase Assay Competent Yeast Cell 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Nichol C.A., Smith G.K., Duch D.S. Biosynthesis and metabolism of tetrahydrobiopterin and molybdoptein. Annu. Rev. Biochem. 54: 729–764, 1985.PubMedCrossRefGoogle Scholar
  2. 2.
    Kaufman S. “Properties of the pterin-dependent aromatic amino acid hydroxylases.” In CIBA Foundation Symposium, Vol. 22: Aromatic Amino Acids in the Brain, Elsevier, New York, pp. 85–115, 1974.Google Scholar
  3. 3.
    Kwon N.S., Nathan C.F., Struehr D.J. Reduced biopterin as a cofactor in the generation of nitrogen oxides by murine macrophages. J. Biol. Chem. 264: 20496–20501, 1989.PubMedGoogle Scholar
  4. 4.
    Kettler R., Bartholini G., Pletscher A. In vivo enhancement of tyrosine hydroxylation in rat striatum by tetrahydrobiopterin. Nature 249: 476–478, 1974.PubMedCrossRefGoogle Scholar
  5. 5.
    Harada T., Kagamiyama H., Hatakeyama K. Feedback regulation mechanisms for the control of GTP cyclohydrolase I activity. Science 260: 1507–1510, 1993.PubMedCrossRefGoogle Scholar
  6. 6.
    Yoneyama T., Brewer J.M., Hatakeyama K. GTP cyclohydrolase I feedback regulatory protein is a pentamer of identical subunits. Purification, cDNA cloning, and bacterial expression. J. Biol. Chem. 272: 9690–9696, 1997.PubMedCrossRefGoogle Scholar
  7. 7.
    Togari A., Ichinose H., Matsumoto S., Fujita K., Nagatsu T. Multiple mRNA forms of human GTP cyclohydrolase I. Biochem. Biophys. Res. Commun. 187: 359–365, 1992.PubMedCrossRefGoogle Scholar
  8. 8.
    Milstien S., Jaffe H., Kowlessur D., Bonner T.I. Purification and cloning of the GTP cyclohydrolase I feedback regulatory protein, GFRP. J. Biol. Chem. 271: 19743–19751, 1996.PubMedCrossRefGoogle Scholar
  9. 9.
    Auerbach G., Herrmann A., Bracher A., Bader G., Gutlich M., Fischer M., Neukamm M., Garrido-Franco M., Richardson J., Nar H., Huber R., Bacher A. Zinc plays a key role in human and bacterial GTP cyclohydrolase I. PNAS 97: 13567–13572, 2000.PubMedCrossRefGoogle Scholar
  10. 10.
    Nar H., Huber R., Meining W., Schmid C., Weinkauf S., Bacher A. Atomic Structure of GTP cyclohydrolase I. Structure 3: 459–456, 1995.PubMedCrossRefGoogle Scholar
  11. 11.
    Cohen G.B., Ren R., Baltimore D. Modular binding domains in signal transduction proteins. Cell 80: 237–248, 1995.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2002

Authors and Affiliations

  • Lance Swick
    • 1
  • Kei Hirayama
    • 1
  • Gregory Kapatos
    • 1
  1. 1.Department of Psychiatry and Behavioral Neuroscience and Center for Molecular Medicine and GeneticsWayne State University School of MedicineDetroitUSA

Personalised recommendations