NOSTRIN: A protein modulating nitric oxide release and subcellular distribution of endothelial nitric oxide synthase

Abstract

Activity and localization of endothelial nitric oxide synthase (eNOS) is regulated in a remarkably complex fashion, yet the complex molecular machinery mastering stimulus-induced eNOS translocation and trafficking is poorly understood. In a search by the yeast two-hybrid system using the eNOS oxygenase domain as bait, we have identified a previously uncharacterized eNOS-interacting protein, dubbed NOSTRIN (for eNOS traffic inducer). NOSTRIN contains a single polypeptide chain of 506-aa residues of 58 kDa with an N-terminal cdc15 domain and a Cterminal SH3 domain. NOSTRIN mRNA is abundant in highly vascularized tissues such as placenta, kidney, lung, and heart, and NOSTRIN protein is expressed in vascular endothelial cells. Coimmunoprecipitation experiments demonstrated the eNOS-NOSTRIN interaction in vitro and in vivo, and NOSTRIN's SH3 domain was essential and sufficient for eNOS binding. NOSTRIN colocalized extensively with eNOS at the plasma membrane of confluent human umbilical venous endothelial cells and in punctate cytosolic structures of CHO-eNOS cells. NOSTRIN overexpression induced a profound redistribution of eNOS from the plasma membrane to vesicle-like structures matching the NOSTRIN pattern and at the same time led to a significant inhibition of NO release. We conclude that NOSTRIN contributes to the intricate protein network controlling activity, trafficking, and targeting of eNOS. N itric oxide (NO) is a potent mediator in biological processes such as neurotransmission, inflammatory response, and vascular homeostasis (1). The prime source of NO in the cardiovascular system is endothelial NO synthase (eNOS), which is tightly regulated with respect to activity and localization. For example, coordinated phosphorylation contributes to activity control of eNOS because of activating and inhibiting phosphorylation at S1179 and T495, respectively (2-6). Myristoylation and dual palmitoylation at its extreme N terminus target eNOS to the cytoplasmic face of the Golgi complex and to the plasma membrane (7), where eNOS is thought to be fully capable of activation (8, 9). Misrouting of acylation-deficient eNOS impairs NO production (10, 11), indicating that correct subcellular targeting is critical for stimulus-dependent activation of the enzyme (8). Posttranslational modifications are efficiently complemented by multiple proteinprotein interactions that help regulate eNOS activity with respect to time and space. For instance, chaperone hsp90 bound to eNOS may mediate vascular endothelial growth factor-induced eNOS phosphorylation by promoting the interaction between eNOS and Akt (12, 13). At the plasma membrane, eNOS is complexed to and inhibited by the master components of caveolae, i.e., caveolin-1 in endothelial cells (9, 14) and caveolin-3 in cardiac myocytes (15). After stimulus-induced [Ca 2ϩ ] i increase, the Ca 2ϩ -calmodulin complex displaces eNOS from caveolin (16), stimulates eNOS to produce NO, and subsequently leads to the redistribution of eNOS from plasma membrane caveolae (17). The complexity...

Luminal flow induces eNOS activation and translocation in the rat thick ascending limb. II. Role of PI3-kinase and Hsp90

“…First, Hsp90 may act as a scaffolding protein, increasing the interaction of eNOS with Akt (13), and, second, Hsp90 may directly modify eNOS enzymatic activity by changing its structure via direct binding (48). While the exact mechanism by which Hsp90 influences eNOS activity is still unclear, our data suggest that besides these two mechanisms, Hsp90 may regulate eNOS activity by modulating its subcellular localization as shown for other proteins that interact with eNOS (59). Because Hsp90 is also involved in regulated trafficking of other proteins, such as the glucocorticoid receptor (6,42,43), it is possible that regulated trafficking of eNOS is dependent on the same protein machinery involved in Hsp90-mediated trafficking of the glucocorticoid receptor; however, this remains to be determined.…”

Targeting of Endothelial Nitric-oxide Synthase to the Cytoplasmic Face of the Golgi Complex or Plasma Membrane Regulates Akt- Versus Calcium-dependent Mechanisms for Nitric Oxide Release

“…Confluent endothelial cells have more plasma membrane and less Golgi eNOS than do subconfluent cells and release more NO in response to agonist challenge (42). The eNOS-binding proteins NOSIP and NOSTRIN displace plasma membrane eNOS to internal membranes (unlikely Golgi) and reduce calcium-dependent NO release (47,48). And finally, depletion of plasma membrane cholesterol with ␤-methyl cyclodextrin and treatment with oxidized low density lipoprotein reduce plasma membrane eNOS to an intracellular compartment (non-Golgi) and attenuate NO release (49,50).…”

“…vascular endothelial growth factor, insulin, and estrogen, would preferentially active Golgi eNOS, and conversely, calcium-dependent agonists such as thapsigargin and bradykinin would preferentially activate plasma membrane eNOS. This concept is strengthened by the recent discoveries of two novel eNOS-binding partners, NOSIP and NOSTRIN (47,48), and an indirect regulator, CHIP (57). Overexpression of both NOSIP and NOSTRIN influences eNOS activity only in vivo by redistributing eNOS from the plasma membrane to intracellular sites.…”

Recent Advances in the Regulation of Nitric Oxide in the Kidney

“…53 It is likely that these regulatory proteins alter NOS 3 activity in all endothelial cells, including those in the kidney. However, their significance in the regulation of NOS 3 activity and/or expression in renal epithelial and interstitial cells has not been thoroughly investigated except for the interaction of NOS 3 and heat shock protein 90.…”