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.
Interaction of the endothelial nitric oxide synthase with the CAT-1 arginine transporter enhances NO release by a mechanism not involving arginine transport
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Chunying LI, Wei HUANG, M. Brennan HARRIS, Jonathan M. GOOLSBY, Richard C. VENEMA; Interaction of the endothelial nitric oxide synthase with the CAT-1 arginine transporter enhances NO release by a mechanism not involving arginine transport. Biochem J 15 March 2005; 386 (3): 567–574. doi: https://doi.org/10.1042/BJ20041005
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