Oligomerization of endothelial nitric oxide synthase. Evidence for a dominant negative effect of truncation mutants.
Nitric oxide produced by the endothelial isoform of nitric oxide synthase (ecNOS) is a key determinant of vascular tone. In contrast to other nitric oxide synthase (NOS) isoforms, which have been characterized as soluble homodimeric enzymes, ecNOS is predominantly membrane-associated, a feature that has hindered direct biochemical analyses of its oligomeric structure. We investigated ecNOS oligomerization using co-immunoprecipitation experiments in transiently transfected COS-7 cells. When COS-7 cells co-transfected with constructs encoding wild-type ecNOS and an epitope-tagged myristoylation-deficient mutant were biosynthetically labeled with [3H]myristate, the antibody to the epitope tag specifically immunoprecipitated 3H-labeled ecNOS, reflecting enzyme oligomerization. In COS-7 cells transfected with cDNAs encoding epitope-tagged truncation mutants and untagged full-length ec-NOS, the wild-type enzyme could be immunoprecipitated by the antibody to the epitope tag. Co-immunoprecipitation of ecNOS with truncation mutants documented that both N- and C-terminal domains are involved in ecNOS oligomerization. When these truncation mutants are co-expressed with wild-type ecNOS, they exert a marked dominant negative effect on enzyme activity. Since NOS oligomerization itself may be subject to dynamic modulation, the regulation of ecNOS assembly may have implications for NO signaling in the vascular wall.
D000818:Animals; D001120:Arginine / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D002956:Citrulline / Q000378:metabolism; D004730:Endothelium, Vascular / Q000201:enzymology*; D000939:Epitopes; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D016296:Mutagenesis; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D016324:Sequence Tagged Sites; D014162:Transfection
D000939:Epitopes; D007527:Isoenzymes; D046911:Macromolecular Substances; D009227:Myristic Acids; D011994:Recombinant Proteins; D019814:Myristic Acid; D002956:Citrulline; D001120:Arginine; D019001:Nitric Oxide Synthase