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Dynamic regulation of endothelial NOS mediated by competitive interaction with -actinin-4 and calmodulin

Yukio Hiroi^*,1, Zhongmin Guo^*,1, Yuxin Li^*, Alan H. Beggs and James K. Liao^*,2

^* Vascular Medicine Research, Brigham & Women’s Hospital, and

Program in Genomics and Division of Genetics, Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts, USA

²Correspondence: Vascular Medicine Research, Brigham & Women’s Hospital, 65 Landsdowne Street, Room 275, Boston, MA 02139, USA. E-mail: jliao{at}rics.bwh.harvard.edu

Alpha-actinins are critical components of the actin cytoskeleton. Here we show that -actinins serve another important biological function by binding to and competitively inhibiting calcium-dependent activation of endothelial NOS (eNOS). -actinin-2 was found to associate with eNOS in a yeast two-hybrid screen. In vascular endothelial cells, which only express -actinin-1 and -4, -actinin-4 interacted and colocalized with eNOS. Addition of -actinin-4 directly inhibited eNOS recombinant protein, and overexpression of -actinin-4 inhibited eNOS activity in eNOS-transfected COS-7 cells and bovine aortic endothelial cells (BAECs). In contrast, knockdown of -actinin-4 by siRNA increased eNOS activity in BAECs. The -actinin-4-binding site on eNOS was mapped to a central region comprising the calmodulin-binding domain, and the eNOS-binding site on -actinin-4 was mapped to the fourth spectrin-like rod domain, R4. Treatment of endothelial cells with a calcium ionophore, A23187, decreased -actinin-4-eNOS interaction, leading to translocation of -actinin-4 from plasma membrane to cytoplasm. Indeed, addition of calmodulin displaced -actinin-4 binding to eNOS and increased eNOS activity. These findings indicate that eNOS activity in vascular endothelial cells is tonically and dynamically regulated by competitive interaction with -actinin-4 and calmodulin.—Hiroi, Y., Guo, Z., Li, Y., Beggs, A. H., Liao, J. K. Dynamic regulation of endothelial NOS mediated by competitive interaction with -actinin-4 and calmodulin.

Key Words: endothelium • nitric oxide • ionophore