ß‐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 ß‐arrestin 2 with siRNA in cytokine‐treated HLMVECs or HEK293 cells transfected with iNOS and B1R In contrast, ß‐arrestin 1 siRNA had no effect. The prolonged phase of B1R‐dependent ERK activation was also inhibited by ß‐arrestin 2 knockdown. Furthermore, robust ERK activation by the epidermal growth factor receptor (a ß‐arrestin 2 independent pathway) had no effect on iNOS‐derived NO production. ß‐arrestin 2 and iNOS coimmunoprecipitated, and there was significant fluorescence resonance energy transfer between CFP‐iNOS and ß‐arrestin 2‐YFP (but not ß‐arrestin 1‐YFP) that increased 3‐fold after B1R stimulation. These data show that ß‐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, F. K, Zhang, Y., Brovkovych, V., Skidgel, R A. ß‐Arrestin 2 is required for B1 receptor‐dependent post‐translational activation of inducible nitric oxide synthase. FASEBJ. 24, 2475–2483 (2010). www.fasebj.org