Consecutive to disruption of the caveolin-eNOS complex induced by agonist stimulation or shear stress, eNOS has been proposed to traffic intracellularly (Fig. 11.3). Prabhakar et al. showed that eNOS is de-palmitoylated after prolonged agonist stimulation, and is no longer selectively sequestered in the caveolae . The acyl-protein thioesterase 1 (APT1) has, indeed, been shown specifically to promote eNOS depalmitoylation through a Ca2+-calmodulin-dependent pathway . The translocated enzyme then partitions into noncaveolar plasma/intra-cellular membranes, and also in the cytosol. The interaction of eNOS with Hsp90 and consecutive (de)phosphorylation (see below) is probably also involved in trafficking of the enzyme, but the molecular mechanisms underlying these processes are still not clearly delineated. By contrast, several lines of evidence have indicated that, subsequent to the translocation of the enzyme and after the decline in [Ca2+]i to basal levels, eNOS may once again interact with caveolin and is then re-targeted to the caveolae, the process being accelerated (or stabilized) by enzyme palmitoyla-tion . The long-chain fatty acyl CoA synthetase was recently identified as a key modulator of eNOS re-palmitoylation . Re-association of eNOS with caveolin could occur either at the plasma (caveolar) or perinuclear membrane levels, or
even in the cytosol through which caveolin complexes may shuttle between the caveolae and an internalized caveolar vesicle/trans-Golgi network (see Fig. 11.3).
The lag time between the agonist-induced disruption of the caveolin-eNOS interaction and the heterocomplex re-formation is thought to be nonexclusively associated with eNOS activation. Indeed, the arginine transporter CAT1 being located in the caveolae , the dissociation of the enzyme from its proximity as well as from several receptors/effectors localized in the caveolae [22,23], is likely to serve as a feedback mechanism for eNOS activation. Also, because NO activates molecular targets outside the endothelial cell, it seems likely that the intracellular locale of eNOS could affect the signaling roles of its product (e. g., the paracrine effects of NO), and thereby modulate the response to extracellular signals. NO finds, indeed, most of its targets in the proximal myocyte layers or circulating blood cells such as platelets and red cells. Similarly, it appears logical that the luminal surface of the endothelium, which is directly exposed to the blood flow and therefore expected to be sensitive to hemodynamic forces, would be a primary site for the documented flow-responsive eNOS activity.
The group of Müller-Esterl has identified, by using yeast two-hybrid screening, two new proteins named NOSIP ("eNOS interacting protein") and NOSTRIN ("eNOS traffic inducer") that specifically bind to the human eNOS oxygenase do main [24,25] (see Fig. 11.3). Although structurally unrelated, overexpression of both proteins in eNOS-expressing cells has similar effects: net dissociation of eNOS from the plasma membrane and inhibition of agonist-induced NO production. Some observations indicate that both effects are very likely to be related (e.g., NOSIP and NOSTRIN modulate eNOS enzyme activity by uncoupling eNOS from plasma membrane caveolae). For instance, NOSIP overexpression does not impact on eNOS activity measured in vitro using a citrulline assay. Also, eNOS binding sites for caveolin and NOSIP do overlap. Nonetheless, whether NOSTRIN and NOSIP promote eNOS translocation from the plasma membrane or inhibit the reverse transport (usually observed after prolonged stimulation) remains unknown.
Of note, although NOSIP and NOSTRIN share some of their functional features and are both particularly abundant in vascularized organs, they differ in other respects [24,25]. Accordingly, while NOSIP overexpression induces eNOS translocation to intracellular sites that co-localize with Golgi and cytoskeletal marker proteins (b-COP and tubulin, respectively), NOSTRIN overexpression largely targets the enzyme to vesicle-like structures spread all over the cytosol. Also, in endothelial cells, NOSIP is mostly found in the cytosol and the nucleus, whereas NOSTRIN is found exclusively in extranuclear locations and at the plasma membrane; in this context, a positive effect of NOSTRIN to address eNOS to the plasma membrane (caveolae) cannot be excluded.
Importantly, although this chapter is focused on the caveolin-related mode of regulation of eNOS activity, it should be emphasized that besides the proteinprotein interactions detailed above, eNOS is also regulated by phosphorylations . Among the putative phosphorylation sites within the eNOS sequence, two (Ser1177 and Thr495) are currently considered to be critical for eNOS activation. Ser1177 phosphorylation is proposed to improve electron flux through the enzyme and to increase its affinity for calmodulin, whereas dephosphorylation of Thr495 is thought to suppress the steric inhibition for calmodulin association to its binding site  (Fig. 11.3). Accordingly, the phosphorylation of eNOS on Ser1177 has been extensively documented as a major kinase-dependent mode of eNOS activation, whereas Thr495 has been reported to participate, when phosphorylated, in the tonic inhibition of eNOS activity. In addition, by directly examining changes in native eNOS post-translational regulation, we recently found that the scaffolding function of Hsp90 previously identified for Akt [27,28] also applied to the phosphatase calcineurin [29,30]. These Hsp90-driven protein-protein associations provide an explanation for the reciprocal regulation of eNOS on distant phosphorylation sites (e.g., Akt-dependent Ser1177 phosphorylation and calcineurin-mediated Thr495 dephosphorylation).
The regulation of eNOS activity/trafficking is very likely to result from the combination of its dynamic interaction with the different partners identified to date (including caveolin, NOSIP, NOSTRIN and Hsp90) and the phosphorylation pattern of the enzyme. In the following sections, the caveolin-eNOS interaction will be retained as the main thread, and reference will be made to other modes of regulation when necessary.
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