Interaction of neuronal nitric oxide synthase with alpha(1)-adrenergic receptor subtypes in transfected HEK-293 cells

BACKGROUND: The C-terminal four amino acids (GEEV) of human α(1A)-adrenergic receptors (ARs) have been reported to interact with the PDZ domain of neuronal nitric oxide synthase (nNOS) in a yeast two-hybrid system. The other two α(1)-AR subtypes have no sequence homology in this region, raising the possibility of subtype-specific protein-protein interactions. RESULTS: We used co-immunoprecipitation and functional approaches with epitope-tagged α(1)-ARs to examine this interaction and the importance of the C-terminal tail. Following co-transfection of HEK-293 cells with hexahistidine/Flag (HF)-tagged α(1A)-ARs and nNOS, membranes were solubilized and immunoprecipitated with anti-FLAG affinity resin or anti-nNOS antibodies. Immunoprecipitation of HFα(1A)-ARs resulted in co-immunoprecipitation of nNOS and vice versa, confirming that these proteins interact. However, nNOS also co-immunoprecipitated with HFα(1B)- and HFα(1D)-ARs, suggesting that the interaction is not specific to the α(1A) subtype. In addition, nNOS co-immunoprecipitated with each of the three HFα(1)-AR subtypes which had been C-terminally truncated, suggesting that this interaction does not require the C-tails; and with Flag-tagged β(1)- and β(2)-ARs. Treatment of PC12 cells expressing HFα(1A)-ARs with an inhibitor of nitric oxide formation did not alter norepinephrine-mediated activation of mitogen activated protein kinases, suggesting nNOS is not involved in this response. CONCLUSIONS: These results show that nNOS does interact with full-length α(1A)-ARs, but that this interaction is not subtype-specific and does not require the C-terminal tail, raising questions about its functional significance.