Interaction of the endothelial nitric oxide synthase with the CAT-1 arginine transporter enhances NO release by a mechanism not involving arginine transport.
eNOS (endothelial nitric oxide synthase) catalyses the conversion of L-arginine into L-citrulline and NO. Evidence has been presented previously that eNOS is associated with the CAT (cationic amino acid transporter)-1 arginine transporter in endothelial caveolae, and it has been proposed that eNOS-CAT-1 association facilitates the delivery of extracellular L-arginine to eNOS. Definitive proof of a protein-protein interaction between eNOS and CAT-1 is lacking, however, and it is also unknown whether the two proteins interact directly or via an adaptor protein. In the present study, we raised a polyclonal antibody against CAT-1, and show using reciprocal co-immunoprecipitation protocols that eNOS and CAT-1 do indeed form a complex in BAECs (bovine aortic endothelial cells). In vitro binding assays with GST (glutathione S-transferase)-CAT-1 fusion proteins and eNOS show that the two proteins interact directly and that no single CAT-1 intracellular domain is sufficient to mediate the interaction. Overexpression of CAT-1 in BAECs by adenoviral-mediated gene transfer results in significant increases in both L-arginine uptake and NO production by the cells. However, whereas increased L-arginine transport is reversed completely by the CAT-1 inhibitor, L-lysine, increased NO release is unaltered, suggesting that NO production in this in vitro model is independent of CAT-1-mediated transport. Furthermore, eNOS enzymic activity is increased in lysates of CAT-1-overexpressing cells accompanied by increased phosphorylation of eNOS at Ser-1179 and Ser-635, and decreased association of eNOS with caveolin-1. Taken together, these data suggest that direct interaction of eNOS with CAT-1 enhances NO release by a mechanism not involving arginine transport.
Li
Chunying
C
Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912, USA.
Huang
Wei
W
Harris
M Brennan
MB
Goolsby
Jonathan M
JM
Venema
Richard C
RC
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001011:Aorta / Q000166:cytology; D001120:Arginine / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D001920:Bradykinin / Q000494:pharmacology; D027201:Cationic Amino Acid Transporter 1 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002417:Cattle; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D006031:Glycosylation; D007106:Immune Sera / Q000276:immunology; D047468:Immunoprecipitation; D008239:Lysine / Q000494:pharmacology; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D014161:Transduction, Genetic
D027201:Cationic Amino Acid Transporter 1; C495541:Cav1 protein, mouse; D051242:Caveolin 1; D022461:Caveolins; D007106:Immune Sera; D009569:Nitric Oxide; D001120:Arginine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D008239:Lysine; D001920:Bradykinin
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