ABSTRACT
Nitric oxide (NO) is an essential vasodilator. In vascular diseases, oxidative stress attenuates NO signaling by both chemical scavenging of free NO and oxidation and downregulation of its major intracellular receptor, the alphabeta heterodimeric heme-containing soluble guanylate cyclase (sGC). Oxidation can also induce loss of the heme of sGC, as well as the responsiveness of sGC to NO. sGC activators such as BAY 58-2667 bind to oxidized/heme-free sGC and reactivate the enzyme to exert disease-specific vasodilation. Here, we show that oxidation-induced downregulation of sGC protein extends to isolated blood vessels. Mechanistically, degradation was triggered through sGC ubiquitination and proteasomal degradation. The heme-binding site ligand BAY 58-2667 prevented sGC ubiquitination and stabilized both alpha and beta subunits. Collectively, our data establish oxidation-ubiquitination of sGC as a modulator of NO/cGMP signaling and point to a new mechanism of action for sGC activating vasodilators by stabilizing their receptor, oxidized/heme-free sGC.
Subject(s)
Guanylate Cyclase/metabolism , Heme/metabolism , Nitric Oxide/pharmacology , Proteasome Endopeptidase Complex/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Vasodilator Agents/pharmacology , Blood Vessels , Cell Line , Cyclic GMP/metabolism , Humans , Oxidation-Reduction , Soluble Guanylyl Cyclase , UbiquitinationABSTRACT
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.
