Caveolin 1 and G-Protein-Coupled Receptor Kinase-2 Coregulate Endothelial Nitric Oxide Synthase Activity in Sinusoidal Endothelial Cells.
Liver injury leads to a vasculopathy in which post-translational modifications of endothelial nitric oxide synthase (eNOS) lead to impaired nitric oxide synthesis. We hypothesized that caveolin 1 (CAV1), a well-known eNOS interactor, regulates eNOS activity in sinusoidal endothelial cells (SECs) via its interaction with G-protein-coupled receptor kinase-2 (GRK2) that also post-translationally modifies eNOS. Liver injury with portal hypertension was established using bile duct ligation in rats. CAV1 function was modified using a CAV1 scaffolding domain construct and cDNAs encoding wild-type CAV1, and CAV1 phosphorylation was increased in injured SECs, resulting in increased GRK2-CAV1 interaction and decreased eNOS activity. In injured SECs, endothelin-1 blocked CAV1 phosphorylation induced by CAV1 scaffolding domain, indicating that CAV1 interaction with GRK2 is inversely regulated by endothelin-1 and CAV1 scaffolding domain after liver injury. In addition, after transduction with DNA encoding wild-type CAV1 into SECs isolated from Cav1-deficient mice, GRK2 association with CAV1 was evident, whereas transduction with a dominant negative CAV1 mutated at tyrosine 14 reduced the interaction. Finally, isoproterenol-induced GRK2 phosphorylation enhanced CAV1-GRK2 interaction and reduced eNOS activity. Our data suggest a novel mechanism and model in which CAV1 phosphorylation facilitates CAV1 scaffolding and GRK2-CAV1 interaction, thus clustering eNOS within a complex that inhibits eNOS activity. This process takes place in injured, but not in normal, SECs.
Department of Medicine, Medical University of South Carolina, Charleston, South Carolina.
Songling
S
Liu
Department of Medicine, Duke University Medical Center, Durham, North Carolina.
Richard T
RT
Premont
Department of Medicine, Medical University of South Carolina, Charleston, South Carolina.
Shweta
S
Singh
Department of Medicine, Medical University of South Carolina, Charleston, South Carolina. Electronic address: rockey@musc.edu.
Don C
DC
Rockey
D000818:Animals; D051242:Caveolin 1 / Q000737:chemistry / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D019332:Endothelin-1 / Q000378:metabolism; D054768:G-Protein-Coupled Receptor Kinases / Q000378:metabolism*; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D008099:Liver / Q000293:injuries / Q000473:pathology*; D008297:Male; D018345:Mice, Knockout; D008954:Models, Biological; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D000072417:Protein Domains; D017207:Rats, Sprague-Dawley; D044023:Receptor, Endothelin B / Q000378:metabolism
D051242:Caveolin 1; D019332:Endothelin-1; D044023:Receptor, Endothelin B; D019000:Phosphotyrosine; D052250:Nitric Oxide Synthase Type III; D054768:G-Protein-Coupled Receptor Kinases; D007545:Isoproterenol