Translocation of endothelial nitric-oxide synthase involves a ternary complex with caveolin-1 and NOSTRIN.
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.
Schilling
Kirstin
K
Institute of Biochemistry II, University of Frankfurt Medical School, D-60590 Frankfurt, Germany.
Opitz
Nils
N
Wiesenthal
Anja
A
Oess
Stefanie
S
Tikkanen
Ritva
R
Müller-Esterl
Werner
W
Icking
Ann
A
D000199:Actins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D016466:CHO Cells; D051242:Caveolin 1 / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D006224:Cricetinae; D003599:Cytoskeleton / Q000378:metabolism; D004268:DNA-Binding Proteins; D034281:Dynamins / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D046912:Multiprotein Complexes / Q000378:metabolism*; D041681:NIH 3T3 Cells; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D051242:Caveolin 1; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D046912:Multiprotein Complexes; C470345:NOSTRIN protein, human; D002784:Cholesterol; D052250:Nitric Oxide Synthase Type III; D034281:Dynamins