Calmodulin-dependent regulation of inducible and neuronal nitric-oxide synthase.
Neuronal and endothelial nitric-oxide synthases depend upon Ca2+/calmodulin for activation, whereas the activity of the inducible nitric-oxide synthase is Ca2+-independent, presumably due to tightly bound calmodulin. To study these different mechanisms, a series of chimeras derived from neuronal and inducible nitric- oxide synthases were analyzed. Chimeras containing only the oxygenase domain, calmodulin-binding region, or reductase domain of inducible nitric-oxide synthase did not confer significant Ca2+-independent activity. However, each chimera was more sensitive to Ca2+ than the neuronal isoform. The calmodulin-binding region of inducible nitric-oxide synthase with either its oxygenase or reductase domains resulted in significant, but not total, Ca2+-independent activity. Co-immunoprecipitation experiments showed no calmodulin associated with the former chimera in the absence of Ca2+. Trifluoperazine also inhibited this chimera in the absence of Ca2+. The combined interactions of calmodulin bound to inducible nitric-oxide synthase calmodulin-binding region with the oxygenase domain may be weaker than with the reductase domain. Thus, Ca2+-independent activity of inducible nitric-oxide synthase appears to result from the concerted interactions of calmodulin with both the oxygenase and reductase domains in addition to the canonical calmodulin-binding region. The neuronal isoform is not regulated by a unique autoinhibitory element in its reductase domain.
Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235, USA.
S J
SJ
Lee
J T
JT
Stull
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D020134:Catalytic Domain; D004790:Enzyme Induction; D005786:Gene Expression Regulation; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D002147:Calmodulin; D011993:Recombinant Fusion Proteins; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D002118:Calcium