Interaction of neuronal nitric oxide synthase with alpha1-adrenergic receptor subtypes in transfected HEK-293 cells.
BACKGROUND
The C-terminal four amino acids (GEEV) of human alpha1A-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 alpha1-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 alpha1-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 alpha1A-ARs and nNOS, membranes were solubilized and immunoprecipitated with anti-FLAG affinity resin or anti-nNOS antibodies. Immunoprecipitation of HFalpha1A-ARs resulted in co-immunoprecipitation of nNOS and vice versa, confirming that these proteins interact. However, nNOS also co-immunoprecipitated with HFalpha1B- and HFalpha1D-ARs, suggesting that the interaction is not specific to the alpha1A subtype. In addition, nNOS co-immunoprecipitated with each of the three HFalpha1-AR subtypes which had been C-terminally truncated, suggesting that this interaction does not require the C-tails; and with Flag-tagged beta1- and beta2-ARs. Treatment of PC12 cells expressing HFalpha1A-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 alpha1A-ARs, but that this interaction is not subtype-specific and does not require the C-terminal tail, raising questions about its functional significance.
Department of Pharmacology, Emory University, Atlanta, GA 30322, USA. aspupo@ibb.unesp.br
Andre S
AS
Pupo
Kenneth P
KP
Minneman
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002478:Cells, Cultured; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D016716:PC12 Cells; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000502:physiology*; D051381:Rats; D018340:Receptors, Adrenergic, alpha-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
C547713:ADRA1A protein, human; C547716:ADRA1B protein, human; C547715:Adra1a protein, rat; C547718:Adra1b protein, rat; D000906:Antibodies; D004791:Enzyme Inhibitors; D018340:Receptors, Adrenergic, alpha-1; C496320:NOS1 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D020928:Mitogen-Activated Protein Kinases; D019331:NG-Nitroarginine Methyl Ester