Beta-arrestin 2 is required for B1 receptor-dependent post-translational activation of inducible nitric oxide synthase.
A major source of "high-output" NO in inflammation is inducible nitric oxide synthase (iNOS). iNOS is primarily transcriptionally regulated and is thought to function as an uncontrolled generator of high NO. We found that iNOS in cytokine-stimulated human lung microvascular endothelial cells (HLMVECs) is highly regulated post-translationally via activation of the B1 kinin G protein-coupled receptor (B1R). We report here that B1R-mediated iNOS activation was significantly inhibited by knockdown of beta-arrestin 2 with siRNA in cytokine-treated HLMVECs or HEK293 cells transfected with iNOS and B1R. In contrast, beta-arrestin 1 siRNA had no effect. The prolonged phase of B1R-dependent ERK activation was also inhibited by beta-arrestin 2 knockdown. Furthermore, robust ERK activation by the epidermal growth factor receptor (a beta-arrestin 2 independent pathway) had no effect on iNOS-derived NO production. beta-arrestin 2 and iNOS coimmunoprecipitated, and there was significant fluorescence resonance energy transfer between CFP-iNOS and beta-arrestin 2-YFP (but not beta-arrestin 1-YFP) that increased 3-fold after B1R stimulation. These data show that beta-arrestin 2 mediates B1R-dependent high-output NO by scaffolding iNOS and ERK to allow post-translational activation of iNOS. This could play a critical role in mediating endothelial function in inflammation.
Kuhr
Frank K
FK
Department of Pharmacology, University of Illinois at Chicago College of Medicine, Chicago, IL 60612, USA.
Zhang
Yongkang
Y
Brovkovych
Viktor
V
Skidgel
Randal A
RA
D019390:Arrestins / Q000502:physiology*; D002460:Cell Line; D004730:Endothelium, Vascular / Q000166:cytology; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D007249:Inflammation; D008168:Lung / Q000098:blood supply; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D043783:Receptor, Bradykinin B1 / Q000378:metabolism*; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C000605253:ARRB1 protein, human; C000605254:ARRB2 protein, human; D019390:Arrestins; D043783:Receptor, Bradykinin B1; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; D048049:Extracellular Signal-Regulated MAP Kinases