CLONING AND CHARACTERIZATION OF HUMAN INDUCIBLE NITRIC-OXIDE SYNTHASESPLICE VARIANTS - A DOMAIN, ENCODED BY EXON-8 AND EXON-9, IS CRITICALFOR DIMERIZATION

The inducible nitric oxide synthase (iNOS) contains an amino-terminal oxygenase domain, a carboxyterminal reductase domain, and an interveni ng calmodulin-binding region. For the synthesis of nitric oxide (NO), iNOS is active as a homodimer. The human iNOS mRNA is subject to alter native splicing, including deletion of exons 8 and 9 that encode amino acids 242-335 of the oxygenase domain. In this study, iNOS(8-9-) and full-length iNOS (iNOS(FL)) were cloned from bronchial epithelial cell s. Expression of iNOS(8-9-) in 293 cell line resulted in generation of iNOS(8-9-) mRNA and protein but did not lead to NO production. In con trast to iNOS(FL), iNOS(8-9-) did not form dimers, Similar to iNOS(FL) , iNOS(8-9-) exhibited NADPH-diaphorase activity and contained tightly bound calmodulin, indicating that the reductase and calmodulin-bindin g domains were functional. To identify sequences in exons 8 and 9 that are critical for dimerization, iNOS(FL) was used to construct 12 muta nts, each with deletion of eight residues in the region encoded by exo ns 8 and 9, In addition, two ''control'' iNOS deletion mutants were sy nthesized, lacking either residues 45-52 of the oxygenase domain or re sidues 1131-1138 of the reductase domain. Whereas both control deletio n mutants generated NO and formed dimers, none of the 12 other mutants formed dimers or generated NO. The region encoded by exons 8 and 9 is critical for iNOS dimer formation and NO production but not for reduc tase activity, This region could be a potential target for therapeutic interventions aimed at inhibiting iNOS dimerization and hence NO synt hesis.