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Author Notes:

Corresponding author: David G. Harrison, Division of Cardiology, Emory University School of Medicine, 1639 Pierce Drive WMBR 319, Atlanta, GA 30322, dharr02@emory.edu, Phone: (404) 727 3710, Fax: (404) 727 3585

Disclosures: None

Subjects:

Research Funding:

This work was supported by NIH Grants P01HL58000, R01HL39006, P01HL58000 and a Department of Veterans Affairs merit grant (to D.G.H.) and an American Heart Association Predoctoral Grant (to L.L.).

Keywords:

  • shear stress
  • eNOS uncoupling
  • electron spin resonance
  • high performance liquid chromatography
  • co-immunoprecipitation
  • transfection
  • site directed mutagenesis

GTP Cyclohydrolase I Phosphorylation and Interaction with GTP Cyclohydrolase Feedback Regulatory Protein Provide Novel Regulation of Endothelial Tetrahydrobiopterin and Nitric Oxide

Tools:

Journal Title:

Circulation Research

Volume:

Volume 106, Number 2

Publisher:

, Pages 328-328

Type of Work:

Article | Post-print: After Peer Review

Abstract:

Rationale GTP cyclohydrolase I (GTPCH-1) is the rate-limiting enzyme involved in de novo biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthases and aromatic amino acid hydroxylases. GTPCH-1 undergoes negative feedback regulation by its end-product BH4 via interaction with the GTP cyclohydrolase feedback regulatory protein (GFRP). Such a negative feedback mechanism should maintain cellular BH4 levels within a very narrow range; however, we recently identified a phosphorylation site (S81) on human GTPCH-1 that markedly increases BH4 production in response to laminar shear. Objective To define how S81 phosphorylation alters GTPCH-1 enzyme activity and how this is modulated by GFRP. Methods and Results Using prokaryotically expressed proteins, we found that the GTPCH-1 phospho-mimetic mutant (S81D) has increased enzyme activity, reduced binding to GFRP and resistance to inhibition by GFRP compared to wild-type GTPCH-1. Using siRNA or overexpressing plasmids, GFRP was shown to modulate phosphorylation of GTPCH-1, BH4 levels and nitric oxide (NO) production in human endothelial cells. Laminar, but not oscillatory shear stress caused dissociation of GTPCH-1 and GFRP, promoting GTPCH-1 phosphorylation. We also found that both GTPCH-1 phosphorylation and GFRP down-regulation prevents eNOS uncoupling in response to oscillatory shear. Finally oscillatory shear was associated with impaired GTPCH-1 phosphorylation and reduced BH4 levels in vivo. Conclusion These studies provide a new mechanism for regulation of endothelial GTPCH-1 by its phosphorylation and interplay with GFRP. This mechanism allows for escape from GFRP negative feedback and permits large amounts of BH4 to be produced in response to laminar shear stress.

Copyright information:

© 2010 American Heart Association, Inc.

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