Published online before print November 19, 2009, doi: 10.1161/CIRCRESAHA.109.210658
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Submitted on June 23, 2009
Revised on November 8, 2009
Accepted on November 10, 2009
GTP Cyclohydrolase I Phosphorylation and Interaction With GTP Cyclohydrolase Feedback Regulatory Protein Provide Novel Regulation of Endothelial Tetrahydrobiopterin and Nitric Oxide
Li Li ;
From the Division of Cardiology (L.L., A.R., A.H., K.K., H.J., D.G.H., W.C.), Department of Medicine; and Graduate Program of Molecular and Systems Pharmacology (L.L., D.G.H.), Emory University School of Medicine, Atlanta; Department of Biology and Physics (J.C.S.), Kennesaw State University; Wallace H. Coulter Department of Biomedical Engineering (H.J.), Georgia Tech and Emory University, Atlanta; and Atlanta Veterans Administration Hospital (D.G.H.), Decatur.
* To whom correspondence should be addressed. E-mail: dharr02{at}emory.edu.
Rationale: GTP cyclohydrolase I (GTPCH-1) is the rate-limiting enzyme involved in de novo biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for NO 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: We sought 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 small interfering RNA or overexpressing plasmids, GFRP was shown to modulate phosphorylation of GTPCH-1, BH4 levels, and 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 downregulation prevents endothelial NO synthase uncoupling in response to oscillatory shear. Finally oscillatory shear was associated with impaired GTPCH-1 phosphorylation and reduced BH4 levels in vivo.
Conclusions: 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.
Key words: shear stress • eNOS uncoupling • electron spin resonance • high-performance liquid chromatography • coimmunoprecipitation • transfection • site-directed mutagenesis