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G-protein-coupled receptor kinase interactor-1 (GIT1) is a new endothelial nitric-oxide synthase (eNOS) interactor with functional effects on vascular homeostasis.

ABSTRACT: Endothelial cell nitric-oxide (NO) synthase (eNOS), the enzyme responsible for synthesis of NO in the vasculature, undergoes extensive post-translational modifications that modulate its activity. Here we have identified a novel eNOS interactor, G-protein-coupled receptor (GPCR) kinase interactor-1 (GIT1), which plays an unexpected role in GPCR stimulated NO signaling. GIT1 interacted with eNOS in the endothelial cell cytoplasm, and this robust association was associated with stimulatory eNOS phosphorylation (Ser(1177)), enzyme activation, and NO synthesis. GIT1 knockdown had the opposite effect. Additionally, GIT1 expression was reduced in sinusoidal endothelial cells after liver injury, consistent with previously described endothelial dysfunction in this disease. Re-expression of GIT1 after liver injury rescued the endothelial phenotype. These data emphasize the role of GPCR signaling partners in eNOS function and have fundamental implications for vascular disorders involving dysregulated eNOS.

SUBMITTER: Liu S

PROVIDER: S-EPMC3320980 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Previous studies have demonstrated that G protein-coupled receptor kinase interacting-1 protein (GIT1) associates with endothelial nitric oxide synthase (eNOS) to regulate nitric oxide production in sinusoidal endothelial cells (SECs). Here, we hypothesized that GIT1's tightly associated binding partner, β-PIX (p21-activated kinase-interacting exchange factor β, ARHGEF7) is specifically important in the regulation of eNOS activity. We examined β-PIX expression in normal rat liver by immunohistochemistry and explored β-PIX protein-protein interactions using immunoprecipitation and immunoblotting. The role of β-PIX in regulating eNOS enzymatic activity was studied in GIT1-deficient SECs. Finally, structural analysis of interaction sites in GIT1 and β-PIX required to regulate eNOS activity were mapped. β-PIX was expressed primarily in SECs in normal liver and was either absent or expressed at extremely low levels in other liver cells (stellate cells, Kupffer cells, and hepatocytes). β-PIX interacted with GIT1 and eNOS to form a trimolecular signaling module in normal SECs and was important in stimulating eNOS activity. Of note, GIT1-β-PIX interaction led to synergistic enhancement of eNOS activity, and β-PIX-driven increase in eNOS activity was GIT1 dependent. Disruption of β-PIX or GIT1 in normal SECs using β-PIX siRNA or GIT1-deficient SECs led to reduced eNOS activity. Finally, specific GIT1 domains [Spa2 homology domain (SHD) and synaptic localization domain (SLD), aa 331-596] and the β-PIX COOH terminal (aa 496-555) appeared to be critical in the regulation eNOS activity. The data indicate that β-PIX regulates eNOS phosphorylation and function in normal SECs and highlight the importance of the GIT1/β-PIX/eNOS trimolecular complex in normal liver SEC function.NEW & NOTEWORTHY β-PIX is a multidomain protein known to be a GIT1 binding partner. We report here that in the normal liver, the distribution and cellular localization of β-PIX are restricted largely to sinusoidal endothelial cells. Furthermore, β-PIX interacts with eNOS and GIT1 promotes eNOS activity and NO production and therefore exerts a novel posttranslational regulatory function on eNOS activity in sinusoidal endothelial cells. We also have identified specific molecular domains important in GIT1 and β-PIX's interaction with eNOS, which may represent novel therapeutic targets in the control of sinusoidal blood flow and intrahepatic resistance.

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G-protein-coupled receptor kinase interactor-1 (GIT1) is a new endothelial nitric-oxide synthase (eNOS) interactor with functional effects on vascular homeostasis.

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