NOSTRIN functions as a homotrimeric adaptor protein facilitating internalization of eNOS.
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.
Icking
Ann
A
Institute for Biochemistry II, University of Frankfurt Medical School, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.
Matt
Simone
S
Opitz
Nils
N
Wiesenthal
Anja
A
Müller-Esterl
Werner
W
Schilling
Kirstin
K
D000199:Actins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D018797:Cell Cycle Proteins / Q000737:chemistry; D002522:Chlorocebus aethiops; D006224:Cricetinae; D004268:DNA-Binding Proteins; D034281:Dynamins / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000737:chemistry; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D005810:Multigene Family; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011499:Protein Processing, Post-Translational; D021381:Protein Transport / Q000502:physiology; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000378:metabolism; D018909:src Homology Domains / Q000502:physiology
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; C082279:CDC15 protein; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C470345:NOSTRIN protein, human; D029702:Schizosaccharomyces pombe Proteins; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019204:GTP-Binding Proteins; D034281:Dynamins