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1.
EMBO J ; 31(15): 3309-22, 2012 Aug 01.
Article in English | MEDLINE | ID: mdl-22751148

ABSTRACT

F-BAR proteins are multivalent adaptors that link plasma membrane and cytoskeleton and coordinate cellular processes such as membrane protrusion and migration. Yet, little is known about the function of F-BAR proteins in vivo. Here we report, that the F-BAR protein NOSTRIN is necessary for proper vascular development in zebrafish and postnatal retinal angiogenesis in mice. The loss of NOSTRIN impacts on the migration of endothelial tip cells and leads to a reduction of tip cell filopodia number and length. NOSTRIN forms a complex with the GTPase Rac1 and its exchange factor Sos1 and overexpression of NOSTRIN in cells induces Rac1 activation. Furthermore, NOSTRIN is required for fibroblast growth factor 2 dependent activation of Rac1 in primary endothelial cells and the angiogenic response to fibroblast growth factor 2 in the in vivo matrigel plug assay. We propose a novel regulatory circuit, in which NOSTRIN assembles a signalling complex containing FGFR1, Rac1 and Sos1 thereby facilitating the activation of Rac1 in endothelial cells during developmental angiogenesis.


Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Blood Vessels/embryology , DNA-Binding Proteins/physiology , Fibroblast Growth Factors/metabolism , Neovascularization, Physiologic/genetics , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Animals , Animals, Genetically Modified , Animals, Newborn , Blood Vessels/growth & development , Blood Vessels/physiology , CHO Cells , Cells, Cultured , Cricetinae , Cricetulus , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Embryo, Mammalian , Embryo, Nonmammalian , Fibroblast Growth Factors/physiology , Mice , Mice, Knockout , Models, Biological , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Receptor, Fibroblast Growth Factor, Type 1/physiology , Signal Transduction/genetics , Signal Transduction/physiology , Zebrafish/embryology , Zebrafish/genetics
2.
Circ Res ; 105(1): 33-41, 2009 Jul 02.
Article in English | MEDLINE | ID: mdl-19478201

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 , Ubiquitination
3.
FEBS Lett ; 582(2): 327-31, 2008 Jan 23.
Article in English | MEDLINE | ID: mdl-18155168

ABSTRACT

Endothelium-derived nitric oxide (NO) activates the heterodimeric heme protein soluble guanylate cyclase (sGC) to form cGMP. In different disease states, sGC levels and activity are diminished possibly involving the sGC binding chaperone, heat shock protein 90 (hsp90). Here we show that prolonged hsp90 inhibition in different cell types reduces protein levels of both sGC subunits by about half, an effect that was prevented by the proteasome inhibitor MG132. Conversely, acute hsp90 inhibition affected neither basal nor NO-stimulated sGC activity. Thus, hsp90 is a molecular stabilizer for sGC tonically preventing proteasomal degradation rather than having a role in short-term activity regulation.


Subject(s)
Guanylate Cyclase/metabolism , HSP90 Heat-Shock Proteins/metabolism , Animals , Cyclic GMP/metabolism , Dimerization , Enzyme Activation , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Humans , PC12 Cells , Rats , Spodoptera
5.
Mol Biol Cell ; 17(9): 3870-80, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16807357

ABSTRACT

Recently, we characterized a novel endothelial nitric-oxide synthase (eNOS)-interacting protein, NOSTRIN (for eNOS-trafficking inducer), which decreases eNOS activity upon overexpression and induces translocation of eNOS away from the plasma membrane. Here, we show that NOSTRIN directly binds to caveolin-1, a well-established inhibitor of eNOS. Because this interaction occurs between the N terminus of caveolin (positions 1-61) and the central domain of NOSTRIN (positions 323-434), it allows for independent binding of each of the two proteins to eNOS. Consistently, we were able to demonstrate the existence of a ternary complex of NOSTRIN, eNOS, and caveolin-1 in Chinese hamster ovary (CHO)-eNOS cells. In human umbilical vein endothelial cells (HUVECs), the ternary complex assembles at the plasma membrane upon confluence or thrombin stimulation. In CHO-eNOS cells, NOSTRIN-mediated translocation of eNOS involves caveolin in a process most likely representing caveolar trafficking. Accordingly, trafficking of NOSTRIN/eNOS/caveolin is affected by altering the state of actin filaments or cholesterol levels in the plasma membrane. During caveolar trafficking, NOSTRIN functions as an adaptor to recruit mediators such as dynamin-2 essential for membrane fission. We propose that a ternary complex between NOSTRIN, caveolin-1, and eNOS mediates translocation of eNOS, with important implications for the activity and availability of eNOS in the cell.


Subject(s)
Caveolin 1/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Multiprotein Complexes/metabolism , Nitric Oxide Synthase Type III/metabolism , Actins/metabolism , Adaptor Proteins, Signal Transducing , Animals , CHO Cells , Cell Membrane/metabolism , Cells, Cultured , Cholesterol/metabolism , Cricetinae , Cytoskeleton/metabolism , DNA-Binding Proteins , Dynamins/metabolism , Endothelial Cells/cytology , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Mice , NIH 3T3 Cells , Protein Binding , Protein Transport
6.
FEBS Lett ; 580(1): 223-8, 2006 Jan 09.
Article in English | MEDLINE | ID: mdl-16376344

ABSTRACT

NOSTRIN, an NO synthase binding protein, belongs to the PCH family of proteins, exposing a typical domain structure. While its SH3 domain and the C-terminal coiled-coil region cc2 have been studied earlier, the function of the N-terminal half comprising a Cdc15 domain with an FCH (Fes/CIP homology) region followed by a coiled-coil stretch cc1 is unknown. Here, we show that the FCH region is necessary and sufficient for membrane association of NOSTRIN, whereas the Cdc15 domain further specifies subcellular distribution of the protein. Thus, the FCH region and the Cdc15 domain fulfill complementary functions in subcellular targeting of NOSTRIN.


Subject(s)
Cell Cycle Proteins/metabolism , GTP-Binding Proteins/metabolism , Nitric Oxide Synthase/metabolism , Adaptor Proteins, Signal Transducing , Cell Cycle Proteins/genetics , DNA-Binding Proteins , GTP-Binding Proteins/genetics , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins , Nitric Oxide Synthase/genetics , Protein Transport/physiology , src Homology Domains/genetics
7.
J Cell Sci ; 118(Pt 21): 5059-69, 2005 Nov 01.
Article in English | MEDLINE | ID: mdl-16234328

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.


Subject(s)
Adaptor Proteins, Vesicular Transport/chemistry , Adaptor Proteins, Vesicular Transport/physiology , Endocytosis/physiology , Nitric Oxide Synthase Type III/metabolism , Nitric Oxide Synthase/chemistry , Nitric Oxide Synthase/physiology , Actins/metabolism , Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport/metabolism , Animals , CHO Cells , COS Cells , Cell Cycle Proteins/chemistry , Chlorocebus aethiops , Cricetinae , DNA-Binding Proteins , Dynamins/metabolism , GTP-Binding Proteins/chemistry , Humans , Intracellular Signaling Peptides and Proteins , Multigene Family , Nitric Oxide Synthase/metabolism , Protein Binding/physiology , Protein Processing, Post-Translational , Protein Transport/physiology , Schizosaccharomyces pombe Proteins/chemistry , Sequence Homology, Amino Acid , Wiskott-Aldrich Syndrome Protein, Neuronal/metabolism , src Homology Domains/physiology
8.
Proc Natl Acad Sci U S A ; 99(26): 17167-72, 2002 Dec 24.
Article in English | MEDLINE | ID: mdl-12446846

ABSTRACT

Activity and localization of endothelial nitric oxide synthase (eNOS) is regulated in a remarkably complex fashion, yet the complex molecular machinery mastering stimulus-induced eNOS translocation and trafficking is poorly understood. In a search by the yeast two-hybrid system using the eNOS oxygenase domain as bait, we have identified a previously uncharacterized eNOS-interacting protein, dubbed NOSTRIN (for eNOS traffic inducer). NOSTRIN contains a single polypeptide chain of 506-aa residues of 58 kDa with an N-terminal cdc15 domain and a C-terminal SH3 domain. NOSTRIN mRNA is abundant in highly vascularized tissues such as placenta, kidney, lung, and heart, and NOSTRIN protein is expressed in vascular endothelial cells. Coimmunoprecipitation experiments demonstrated the eNOS-NOSTRIN interaction in vitro and in vivo, and NOSTRIN's SH3 domain was essential and sufficient for eNOS binding. NOSTRIN colocalized extensively with eNOS at the plasma membrane of confluent human umbilical venous endothelial cells and in punctate cytosolic structures of CHO-eNOS cells. NOSTRIN overexpression induced a profound redistribution of eNOS from the plasma membrane to vesicle-like structures matching the NOSTRIN pattern and at the same time led to a significant inhibition of NO release. We conclude that NOSTRIN contributes to the intricate protein network controlling activity, trafficking, and targeting of eNOS.


Subject(s)
Nitric Oxide Synthase/analysis , Nitric Oxide Synthase/physiology , Nitric Oxide/metabolism , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , CHO Cells , Carrier Proteins/physiology , Cricetinae , DNA-Binding Proteins , Endothelial Growth Factors/pharmacology , Endothelium, Vascular/metabolism , Intercellular Signaling Peptides and Proteins/pharmacology , Intracellular Signaling Peptides and Proteins , Lymphokines/pharmacology , Molecular Sequence Data , Nitric Oxide Synthase/chemistry , Nitric Oxide Synthase Type III , RNA, Messenger/analysis , Ubiquitin-Protein Ligases , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , src Homology Domains
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