Interaction of the endothelial nitric oxide synthase with the CAT-1 arginine transporter enhances NO release by a mechanism not involving arginine transport

Abstract

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.

Figure 1. Immunoblotting of BAEC lysates with anti-CAT-1-ID6 antibody, and glycoprotein detection and deglycosylation of CAT-1 proteins

Cultured BAECs were lysed, and lysates were immunoblotted with anti-CAT-1-ID6 antibody (A). Lysates were also subjected to immunoprecipitation with anti-CAT-1-ID6 antibody, and glycosylation of immunoprecipitated proteins was detected by the periodic acid–Schiff method (B). To investigate deglycosylation, lysates were also incubated without or with glycosidase and then immunoblotted with anti-CAT-1-ID6 (C). Similar results were obtained in three different experiments. Molecular masses are indicated in kDa.

Figure 2. Effects of pre-adsorption of anti-CAT-1-ID6 antibody with either GST only or GST-CAT-1-ID6 on reactivity of the antibody in immunoblots of BAEC lysates

Anti-CAT-1-ID6 antibody was pre-incubated with 0, 0.5, 5 and 50 μg of purified GST only (A) or GST-CAT-1-ID6 (B), and then used for immunoblotting of BAEC lysates. The experiment was carried out three times with similar results. Molecular masses are indicated in kDa.

Figure 3. Co-immunoprecipitation of CAT-1 and eNOS from endothelial cells

BAEC lysates were immunoprecipitated with either anti-CAT-1-ID6 or anti-eNOS antibody. Anti-CAT-1-ID6 immunoprecipitates were immunoblotted with anti-eNOS antibody (A), and anti-eNOS immunoprecipitates were immunoblotted with anti-CAT-1-ID6 antibody (B). Results shown are representative of six different experiments. Molecular masses are indicated in kDa.

Figure 4. In vitro binding of eNOS to a full-length-CAT-1 GST fusion protein

GST or a GST-CAT-1 fusion protein was expressed in E. coli and purified by affinity binding to glutathione–agarose. Proteins, pre-bound to beads, were incubated overnight at 4 °C with recombinant bovine eNOS, expressed and purified from a baculovirus system. Beads were washed six times in buffer containing 1 M NaCl. Bound proteins were eluted with reduced glutathione and immunoblotted with anti-eNOS antibody. Results shown are representative of six separate experiments. Molecular masses are indicated in kDa.

Figure 5. Effects of bradykinin (BK) on eNOS–CAT-1 complex formation in endothelial cells

BAECs were treated with bradykinin (1 μM) for 0, 1, 5, 10 and 30 min. BAEC lysates were prepared and were immunoprecipitated with either anti-CAT-1-ID6 or anti-eNOS antibody. Anti-CAT-1-ID6 immunoprecipitates were immunoblotted with anti-eNOS antibody (A), and anti-eNOS immunoprecipitates were immunoblotted with anti-CAT-1-ID6 antibody (B). Similar results were obtained in three separate experiments. Molecular masses are indicated in kDa.

Figure 6. Increased protein expression of CAT-1 and eNOS–CAT-1 association in endothelial cells by adenoviral-mediated gene transfer

(A) BAECs were infected for 24 h with Ad-β-Gal (negative control) or Ad-CAT-1. Lysates were prepared, and equal quantities of lysate proteins from each condition were immunoblotted with anti-CAT-1-ID6 antibody. Three separate experiments gave similar results. Molecular masses are indicated in kDa. (B) Lysates were prepared, and equal quantities of lysate protein from each condition were immunoprecipitated with anti-CAT-1-ID6 antibody. Immunoprecipitates were then immunoblotted with anti-eNOS antibody. Results shown are representative of three experiments.

Figure 7. Effects of CAT-1 overexpression and lysine on arginine uptake in endothelial cells

BAECs were infected for 24 h with Ad-β-Gal (negative control) or Ad-CAT-1. Cells from both conditions were incubated in the absence or presence of various concentrations of lysine, and the amount of [³H]arginine taken up by the cells in a 5 min incubation was quantified by liquid-scintillation counting (means±S.E.M., n=4; *P<0.05 compared with Ad-β-Gal control, **P<0.05 compared with Ad-CAT-1 control, one-way ANOVA).

Figure 8. Effects of CAT-1 overexpression on phosphorylation of eNOS at Ser-1179 and Ser-635, and eNOS association with caveolin-1

BAECs were infected for 24 h with Ad-β-Gal (negative control) or Ad-CAT-1. Lysates were prepared, and equal quantities of lysate proteins were immunoblotted with anti-phospho-Ser-1179 eNOS (A) or anti-phospho-Ser-635 eNOS (B). Lysates were also immunoprecipitated with anti-eNOS antibody and immunoblotted with anti-caveolin-1 antibody (C). Similar results were obtained in at least three experiments.