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.