TY - JOUR
T1 - Cloning and characterization of human inducible nitric oxide synthase splice variants
T2 - A domain, encoded by exons 8 and 9, is critical for dimerization
AU - Eissa, N. Tony
AU - Yuan, Jean W.
AU - Haggerty, Cynthia M.
AU - Choo, Esther K.
AU - Palmer, Celeste D.
AU - Moss, Joel
PY - 1998/6/23
Y1 - 1998/6/23
N2 - The inducible nitric oxide synthase (iNOS) contains an amino-terminal oxygenase domain, a carboxy-terminal reductase domain, and an intervening calmodulin-binding region. For the synthesis of nitric oxide (NO), iNOS is active as a homodimer. The human iNOS mRNA is subject to alternative splicing, including deletion of exons 8 and 9 that encode amino acids 242- 335 of the oxygenase domain. In this study, iNOS8-9- and full-length iNOS (iNOS(FL)) were cloned from bronchial epithelial cells. Expression of iNOS8-9- in 293 cell line resulted in generation of iNOS8-9- mRNA and protein but did not lead to NO production. In contrast to iNOS(FL), iNOS8- 9- did not form dimers. Similar to iNOS(FL), iNOS8-9-exhibited NADPH- diaphorase activity and contained tightly bound calmodulin, indicating that the reductase and calmodulin-binding domains were functional. To identify sequences in exons 8 and 9 that are critical for dimerization, iNOS(FL) was used to construct 12 mutants, each with deletion of eight residues in the region encoded by exons 8 and 9. In addition, two 'control' iNOS deletion mutants were synthesized, lacking either residues 45-52 of the oxygenase domain or residues 1131-1138 of the reductase domain. Whereas both control deletion 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 reductase activity. This region could be a potential target for therapeutic interventions aimed at inhibiting iNOS dimerization and hence NO synthesis.
AB - The inducible nitric oxide synthase (iNOS) contains an amino-terminal oxygenase domain, a carboxy-terminal reductase domain, and an intervening calmodulin-binding region. For the synthesis of nitric oxide (NO), iNOS is active as a homodimer. The human iNOS mRNA is subject to alternative splicing, including deletion of exons 8 and 9 that encode amino acids 242- 335 of the oxygenase domain. In this study, iNOS8-9- and full-length iNOS (iNOS(FL)) were cloned from bronchial epithelial cells. Expression of iNOS8-9- in 293 cell line resulted in generation of iNOS8-9- mRNA and protein but did not lead to NO production. In contrast to iNOS(FL), iNOS8- 9- did not form dimers. Similar to iNOS(FL), iNOS8-9-exhibited NADPH- diaphorase activity and contained tightly bound calmodulin, indicating that the reductase and calmodulin-binding domains were functional. To identify sequences in exons 8 and 9 that are critical for dimerization, iNOS(FL) was used to construct 12 mutants, each with deletion of eight residues in the region encoded by exons 8 and 9. In addition, two 'control' iNOS deletion mutants were synthesized, lacking either residues 45-52 of the oxygenase domain or residues 1131-1138 of the reductase domain. Whereas both control deletion 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 reductase activity. This region could be a potential target for therapeutic interventions aimed at inhibiting iNOS dimerization and hence NO synthesis.
UR - http://www.scopus.com/inward/record.url?scp=0032560499&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0032560499&partnerID=8YFLogxK
U2 - 10.1073/pnas.95.13.7625
DO - 10.1073/pnas.95.13.7625
M3 - Article
C2 - 9636200
AN - SCOPUS:0032560499
SN - 0027-8424
VL - 95
SP - 7625
EP - 7630
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 13
ER -