In the past, the regulation of NOS activity has been reported to involve Ca-calmodulin binding for endothelial (eNOS) and neuronal (nNOS). Inducible forms (iNOS) of this enzyme are also known.
The authors used the Gal4-based two hybrid system to screen for proteins which bind nNOS. Using a vector expressing nNOS and Gal4 DNA binding domain-cDNA and rat hippocampus cDNA library fused to Gal 4-activated domain, they found a protein which they named PIN (protein inhibitor of nNOS). Using the first step of PIN clones as probes they screened a rat brain cDNA library to obtain a 615 bp with an open reading frame of 270 bp. Thus PIN is an 89 aa protein, i.e., it has a molecular weight of 10 kDa. PIN mRNA levels are kidney > brain > other peripheral tissues. PIN has very good identity with a hypothetical protein based on genome sequence of C. elegans and with proteins expressed in Drosophila, Chlamydomonas, Schistosoma and Arabidopsis.
They used truncated nNOS-Gal 4 DNA binding domain fusions to determine that amino acids 163-245 of nNOS were sufficient for binding to PIN.
PIN-glutathione transferase (GST) fusion protein (but not GST) binds nNOS to form a nNOS dimers.
Rat cerebellum contained both monomers and dimers of nNOS. PIN-GST inhibited the nNOS activity with a Ki of 1 while Ki for PIN was 5 uM.
PIN binds nNOS but not eNOS or iNOS.
It provides evidence for additional regulation of nNOS-synthase.
This work immediately raises several questions:
There is one recent paper which suggests that eNOS may bind a 90 kDa protein. The tyrosine phosphorylation of the 90 kDa protein upon stimulation by bradykinin results in translocation of eNOS (Venema et al. 1996. Bradykinin-stimulated protein tyrosine phosphorylation promotes endothelial nitric oxide synthase translocation to the cytoskeleton. Biochim Biophy Res Comm 226:703-710.)
The Ki of 1-5 uM suggests very rapid dissociation of PIN from nNOS. Formation of a ternary complex involving 2 nNOS molecules and one PIN molecule would thus make this a very slow nNOS inhibition step which can be readily reversed.
The neurotransmitter functions of nitric oxide are dependent on dynamic regulation of its biosynthetic enzyme, neuronal nitric oxide synthase (nNOS). By means of a yeast two-hybrid screen, a 10-kD protein was identified that physically interacts with and inhibits the activity of nNOS. This inhibitor, designated PIN, appears to be one of the most conserved proteins in nature, showing 92 percent amino acid identity with the nematode and rat homolog. Binding of PIN destabilizes the nNOS dimer, a conformation necessary for activity. These results suggest that PIN may regulate numerous biological processes through its effects on nitric oxide synthase activity.