Tetrahydrobiopterin Synthesis in Endothelial Cells is Regulated by Caveolin‐1

Abstract

GTP Cyclohydrolase-I (GTPCHI) is the rate-limiting enzyme for the synthesis of tetrahydrobiopterin (BH4), which is an essential cofactor for the enzymatic activity of endothelial nitric oxide synthase (eNOS). To better understand the role of GTPCHI in maintaining eNOS function, we analyzed GTPCHI subcellular localization and activity in human umbilical cord vein endothelial cells (HUVEC). Western blot analysis showed similar levels of GTPCHI protein expression in both Triton X-100 -soluble and -insoluble fractions, suggesting membrane targeting of GTPCHI. Further analysis of membrane-associated proteins using sucrose gradient ultracentrifugation revealed that GTPCHI protein expression was localized to caveolar microdomains along with caveolin-1(Cav-1) and eNOS. HPLC analysis for GTPCHI activity confirmed the localization of GTPCHI to caveolar microdomains as well as golgi-associated membrane fractions. Using an in vitro GST pull-down assay with recombinantGST-caveolin-1 fusion protein incubated with the lysate of HUVEC, we demonstrated specific binding of GTPCHI to caveolin-1. To determine if caveolin-1 may regulate GTPCHI activity in vivo, we analyzed aortas from Cav-1 −/ − mice and were able to detect a two-fold increase in BH4 production and GTPCHI activity in the aortas of Cav-1 −/ − mice when compared to eNOS −/ − mice or wild type mice. These studies reveal that BH4 synthesis can occur in specialized membrane compartments in close proximity to eNOS and may be subjected to posttranslational regulation by caveolin-1 which has an inhibitory effect on GTPCHI enzyme activity. Funded by NIH grant HL-53524.