NOSTRIN functions as a homotrimeric adaptor protein facilitating internalization of eNOS

Abstract

Intracellular trafficking of endothelial nitric oxide synthase (eNOS) between different compartments is incompletely understood. Recently, we described a novel eNOS-interacting protein, NOSTRIN, which upon overexpression drives eNOS away from the plasma membrane towards intracellular compartments. Sequence similarity of NOSTRIN and pacsins/syndapins suggested a role for NOSTRIN in endocytosis. Accordingly, we show here that NOSTRIN interacts with the large GTPase dynamin and the actin nucleation promoting factor N-WASP by means of its SH3 domain, which also represents the docking site for eNOS. Via a coiled-coil region in the C-terminal portion of the protein, NOSTRIN oligomerizes, mainly forming trimers, which would allow simultaneous interaction with multiple binding partners of the SH3 domain. Consistent with this notion, expression of dynamin-2-GFP in CHO cells stably expressing eNOS (CHO-eNOS) results in recruitment of eNOS to dynamin-positive structures, only when NOSTRIN is present as well. Similarly, when N-WASP-GFP and NOSTRIN are co-expressed in CHO-eNOS cells, both proteins strongly co-localize with eNOS and are recruited to structures running along actin filaments. If, however, the actin cytoskeleton is depolymerized by cytochalasin D, NOSTRIN and eNOS are associated with extended structures in the cell periphery, possibly being unable to leave the plasma membrane. Together, these results indicate that NOSTRIN may facilitate endocytosis of eNOS by coordinating the function of dynamin and N-WASP.

Translocation of Endothelial Nitric-Oxide Synthase Involves a Ternary Complex with Caveolin-1 and NOSTRIN

“…In the absence of NOSTRIN, there was hardly any overlap of caveolin-1 or eNOS with dynamin-2-GFP ( Figure 7, E-H). However, in the presence of NOSTRIN, eNOS localization was clearly changed in favor of vesicular and tubular structures (Figure 7, I-L) previously demonstrated to be positive for NOSTRIN (Icking et al, 2005). In these NOSTRIN-expressing cells, eNOS colocalized extensively with dynamin-2-GFP and caveolin-1 ( Figure 7L, inset).…”

“…Importantly, whereas eNOS interacts with the SH3 domain of NOSTRIN (positions 434 -506), binding of caveolin to NOSTRIN depends on residues 323-434 of NOSTRIN, allowing for formation of a ternary complex of NOSTRIN, caveolin, and eNOS. At present, we do not know the precise stoichiometry of the protein complex but given that caveolin-1 oligomerizes to large complexes of up to 600 kDa (Monier et al, 1995) and that eNOS and NOSTRIN form homodimers and -trimers, respectively (Icking et al, 2005), the resultant macromolecular complex may provide a platform for dynamic recruitment of proteins involved in caveolar function. Previous reports suggested that interaction of eNOS and caveolin may be regulated (Andries et al, 1998).…”

“…Another binding partner of the SH3 domain of NOSTRIN is dynamin (Icking et al, 2005), which led us to ask whether NOSTRIN could serve to recruit the GTPase to the ternary complex. Using CHO-eNOS cells recombinantly expressing dynamin-2-GFP and NOSTRIN, we found that the two proteins colocalized extensively with caveolin-1 (Figure 7, A-D, inset).…”

“…For NOSIP, targeting of eNOS to the cytoskeleton seems to be one of the protein's major functions (Schleicher et al, 2005). Overexpression of NOSTRIN, in contrast, leads to translocation of eNOS from the PM to intracellular vesicular structures (Zimmermann et al, 2002), possibly involving an endocytic process (Icking et al, 2005).…”

“…For NOSIP, targeting of eNOS to the cytoskeleton seems to be one of the protein's major functions (Schleicher et al, 2005). Overexpression of NOSTRIN, in contrast, leads to translocation of eNOS from the PM to intracellular vesicular structures (Zimmermann et al, 2002), possibly involving an endocytic process (Icking et al, 2005).Within membranes, eNOS associates with raft-like membrane microdomains, specifically with caveolae at the PM (Shaul et al, 1996). The major constituent of caveolae is caveolin-1 (Fra et al, 1995), which is expressed in many cell types, including endothelial and epithelial cells.…”