OLIGOMERIZATION OF ENDOTHELIAL NITRIC-OXIDE SYNTHASE - EVIDENCE FOR ADOMINANT-NEGATIVE EFFECT OF TRUNCATION MUTANTS

Nitric oxide produced by the endothelial isoform of nitric oxide synth ase (ecNOS) is a key determinant of vascular tone, In contrast to othe r nitric oxide synthase (NOS) isoforms, which have been characterized as soluble homodimeric enzymes, ecNOS is predominantly membrane-associ ated, a feature that has hindered direct biochemical analyses of its o ligomeric structure, We investigated ecNOS oligomerization using co im munoprecipitation experiments in transiently transfected COS-7 cells. When COS-7 cells co transfected with constructs encoding wild-type ecN OS and an epitope-tagged myristoylation-deficient mutant were biosynth etically labeled with [H-3]myristate, the antibody to the epitope tag specifically immunoprecipitated H-3-labeled ecNOS, reflecting enzyme o ligomerization, In COS-7 cells transfected with cDNAs encoding epitope -tagged truncation mutants and untagged full-length ecNOS, the wild-ty pe enzyme could be immunoprecipitated by the antibody to the epitope t ag, Co-immunoprecipitation of ecNOS with truncation mutants documented that both N- and C-terminal domains are involved in ecNOS oligomeriza tion, When these truncation mutants are coexpressed with wild-type ecN OS, they exert a marked dominant negative effect on enzyme activity. S ince NOS oligomerization itself may be subject to dynamic modulation, the regulation of ecNOS assembly may have implications for NO signalin g in the vascular wall.