Caveolin A Scaffold for eNOS

Since NO is a very labile and highly reactive messenger molecule with autocrine and paracrine functions, the site of NO production should logically have a major influence on its biological activity. The discovery in 1996 of the location of eNOS in caveolae [6,7] was therefore viewed as the proof of concept that compartmentation of the enzyme is critical to fine-tune NO synthesis. This specific locale of eNOS had been suspected based on the double acylation process that characterizes eNOS: myristoylation on glycine (at position 2) and palmitoylation on two cysteines (at positions 15 and 26). In fact, by using cultured endothelial cells, eNOS was shown to be preferentially located in caveolae (versus the rest of the plasma membrane), with each acylation process enhancing the caveolar enrichment some 10-fold [6]. This discovery was rapidly followed by the identification of a tight regulation between eNOS and caveolin, the structural protein of caveolae. It has been reported that, in endothelial cells and cardiac myocytes, eNOS was quantitatively associated with caveolin-1 and caveolin-3, respectively [8]. The determinants of this interaction subsequently became the focus of studies conducted by several independent groups. It was shown, by exploring the differential effects of detergents, that although eNOS thiopalmitoylation is not absolutely required to induce formation of the caveolin-eNOS complex, acylation largely facilitates the interaction between both proteins [9]. The sequences involved in this mutual interaction were also identified, based on the studies of Lisanti et al., who found a region within the caveolin sequence that could act as a scaffold for many caveolar proteins [10].

This so-called "caveolin scaffolding domain" (CSD) is a region spanning 20 residues in the caveolin sequence (mapping to residues 81-101 in the human caveolin-1 sequence) [10]. Using a glutathione S-transferase (GST)-CSD fusion protein as a bait to select peptide ligands from a bacteriophage display library, Lisanti and colleagues identified a "caveolin binding motif" (CBM) that appeared to be present in whole or in part in many proteins located in caveolae (FXFXXXX FXXF, where F represents an aromatic amino acid) [11]. That the interaction with caveolin involves these consensus sequences is, however, only documented for a few caveolar residents but among them, stands eNOS [12,13]. Several laboratories have indeed investigated the molecular determinants of the caveolin-eNOS interaction using in-vitro binding assay systems with GST fusion proteins (including deletion mutants) and an in-vivo yeast two-hybrid system [9,13-15]. The major conclusions from these studies are that eNOS and caveolin-1 interact directly rather than indirectly, and that this interaction involves multiple sites: the oxygenase and reductase domains of eNOS and the two cytoplasmic domains of caveolin-1. The CBM (sequence 350-358 FPAAPFSGW) that recognizes the CSD is located in the oxygenase domain of eNOS, between the heme and the calmodulin binding domains. This location is adjacent to a glutamate residue (Glu361) necessary for the binding of l-arginine, which suggests that caveolin may interfere with heme iron reduction, similarly to l-arginine-based NOS inhibitors (see Fig. 11.1).

This latter observation led us and others to investigate whether the caveolin-eNOS interaction was inhibitory. Like all known NO synthases, eNOS enzyme activity is dependent on calmodulin binding, the activation of which requires an increase in intracellular calcium. With the discovery of the caveolin-eNOS interaction, it appeared that calmodulin acts, in fact, as a direct allosteric competitor promoting the disruption of the heteromeric complex formed between eNOS and caveolin in a Ca2+-dependent fashion [13,15,16] (Fig. 11.1). Both the CSD and the CBM domains were shown to be involved. Accordingly, peptides corresponding to the CSD domain were shown to interact directly with the enzyme and markedly inhibit NOS activity in endothelial cells [13]. Likewise, site-directed mutagenesis of the CBM was found to block the ability of caveolin-1 to suppress NO release in transfection experiments [12].

Using full-length eNOS or truncated enzyme which only expresses the oxygenase domain, studies conducted by Ghosh et al. [17] led to the establishment of a model, according to which caveolin interaction with the oxygenase domain helps to target the eNOS-caveolin complex to caveolae. In contrast, caveolin interaction with the reductase domain is primarily responsible for antagonizing calmodulin binding and for slowing electron transfer from the reductase, thus inhibiting heme iron reduction and NO synthesis (see Fig. 11.1).

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