Intracellular trafficking of endothelial nitric oxide synthase – function of NOSTRIN

In the cardiovascular system, eNOS is the major enzyme producing nitric oxide (NO), which plays an important role in vascular homeostasis. eNOS activity is tightly regulated and closely correlates with its subcellular localisation: At the plasma membrane, eNOS is kept in an inactive state by interaction with caveolin-1, the master component of caveolae. Ca++ stimulation releases eNOS from caveolin and thereby induces NO production and redistribution of eNOS from plasma membrane caveolae. In addition, eNOS has been shown to localise to different subcellular compartments, e.g. the Golgi apparatus, cell-cell contacts and intracellular vesicles [reviewed in Oess et al., 2006]. But so far, the transport mechanisms are poorly understood. Recently, we identified a novel human protein, NOSTRIN (eNOS traffic Inducer), as a binding partner of eNOS. Overexpression of NOSTRIN in CHO cells expressing eNOS (CHO-eNOS) leads to redistribution of eNOS from the plasma membrane to vesicular structures containing NOSTRIN. This effect is accompanied by an attenuation of NO release [Zimmermann et al. 2002]. The interaction of NOSTRIN with eNOS is mediated by the single C-terminal SH3 domain of NOSTRIN. In addition to this domain, NOSTRIN contains an N-terminal F-BAR domain - formerly often referred to as Pombe cdc15 homology (PCH) domain [Lippincott & Li 2000] (fig. 1). Proteins of this family were shown to function in vesicular protein trafficking and actin organization.

Figure 1: Modular structure of NOSTRIN

 

In line with this, we found that NOSTRIN has a role in trafficking of eNOS. In this process, NOSTRIN functions as an adaptor protein for the large GTPase dynamin and the Arp2/3 activator N-WASP thereby presumably coordinating vesicle fission [Icking et al., 2005]. Further studies revealed that NOSTRIN-mediated endocytosis of eNOS occurs via caveolae which is facilitated by a direct interaction of NOSTRIN and caveolin [Schilling et al., 2006]. Currently, we study the role of NOSTRIN and eNOS in various mouse and human tissues under normal or pathological conditions.

 

Figure 2: Colocalization of NOSTRIN, caveolin and dynamin in CHO-eNOS cells expressing

 

 

 

 

 

 

One protein – many functions?

Function and physiological role of NOSIP, a nitric oxide synthase interacting protein

The main focus of this project is the functional characterization of the nitric oxide synthase (NOS) interacting protein NOSIP. NOSIP was identified as an interacting protein of endothelial nitric oxide synthase (eNOS), by a yeast two hybrid screen, using the eNOS oxygenase domain as bait. NOSIP binds NOS in vitro and in vivo and the interaction is inhibited by a caveolin scaffolding domain peptide, suggesting that NOSIP might interfere with caveolar association of eNOS. Indeed overexpression of NOSIP causes a redistribution of eNOS from the plasma membrane to intracellular compartments, concomitant with a drastic decrease of nitric oxide (NO) production (Fig. 1). Our research is currently focused on (I) the characterization of the biochemical and physiological properties of NOSIP (II) the elucidation of the mechanism of NOS translocation and inhibition, (III) the regulation of the NOSIP/NOS interaction and its physiologic consequence, and (IV) the importance of the interaction of NOSIP with proteins other than NOS and its role as integral part of functional protein complexes.

Implications of NO reactivity

Nitric oxide (NO) is a gaseous radical with unique biological functions essential for e.g. the cardiovascular system, neurotransmission and host defense. For two decades it was believed that NO was able to diffuse freely across relatively long distances and to traverse major parts of the cell, if not multiple cell layers. However, NO has been proven to be extremely reactive. It reacts with other oxygen species, heavy metals, as well as with cystein and tyrosine residues in proteins. In accordance it is now widely accepted that once NO is generated, it is very short-lived (with a half-life of a few milliseconds in vivo) and diffuses only over short distances. This urges for the local production of NO and the localization of NO synthases (NOS) in the proximity of their downstream targets.

 

Fig. 1. Model for the localisation and regulation of eNOS in endothelial cells. eNOS is targeted to the plasma membrane by myristoylation and double palmitoylation. In caveolae it is bound and inhibited by caveolin-1. Increase of intracellular calcium – e.g. in response to stimulation of the bradykinin receptor – leads to formation of Ca2+-calmodulin, which competes with caveolin for eNOS binding and releases eNOS from the inhibitory caveolin interaction. Furthermore eNOS can also be activated by phosphorylation e.g. by the protein kinase Akt and the eNOS/Akt interaction is thought to be fostered by hsp90. Proteins coloured in green represent structural proteins of cell-cell contacts:α , α-catenin; β, β-catenin; γ, γ-catenin (plakoglobin); CAM, Ca-Calmodulin; VE, VE-cadherin; P, PECAM-1; vi, vinculin; VA, VASP; d, desmoplakin; PM, plasma membrane (Govers et al., 2004)

Localization of endothelial nitric oxide synthase Endothelial NOS (eNOS) is known to reside in different intracellular localizations: at the plasma membrane and in caveolae, intracellular vesicles, at the Golgi complex, in the cytosol and at cell-cell contacts. Neither the state of activity and the means of activation in these different compartments, nor the mechanisms responsible for eNOS targeting and transport are fully understood. Currently we characterize the involvement of NOSIP in these processes.

Dynamic regulation of NOSIP Not only eNOS but also NOSIP seems to be regulated on different levels including induction of expression, dynamic regulation of subcellular localization, oligomerization and reversible phosphorylation. The characterization of the subcellular targeting of NOSIP, its dynamic regulation and the physiological consequence in respect to NO bioavailability are our key interests.

NOSIP – NOS independent functions?

Recent results indicate that NOSIP interacts specifically with other proteins than NOS and we think it well possible that NOSIP has other functions in addition to the modulating effect on NOS localization and activity. To characterize the complex protein networks in which NOSIP participates and to define its biological function and physiological role(s) are our major aims at this time.