NIDD, a Novel DHHC-containing Protein, Targets Neuronal Nitric-oxide Synthase (nNOS) to the Synaptic Membrane through a PDZ-dependent Interaction and Regulates nNOS Activity*

Targeting of neuronal nitric-oxide synthase (nNOS) to appropriate sites in a cell is mediated by interactions with its PDZ domain and plays an important role in specifying the sites of reaction of nitric oxide (NO) in the central nervous system. Here we report the identifica-tion and characterization of a novel nNOS-interacting DHHC domain-containing protein with dendritic mRNA (NIDD) (GenBank TM accession number AB098078), which increases nNOS enzyme activity by targeting the nNOStothesynapticplasmamembraneinaPDZdomain-dependent manner. The deduced NIDD protein con-sisted of 392 amino acid residues and possessed five transmembrane segments, a zinc finger DHHC domain, and a PDZ-binding motif ( (cid:1) EDIV) at its C-terminal tail. In vitro pull-down assays suggested that the C-terminal tail region of NIDD specifically interacted with the PDZ domain of nNOS. The PDZ dependence was confirmed by an experiment using a deletion mutant, and the interaction was further confirmed by co-sedimentation assays using COS-7 cells transfected with NIDD and nNOS. Both NIDD and nNOS were enriched in synaptosome de- transiently transfected We assayed , lysed ability L

Nitric oxide (NO) 1 works as a volatile messenger and plays important physiological roles in the central nervous system (1,2). N-Methyl-D-aspartate-type glutamate receptor (NMDAR) plays a critical role in synaptic plasticity, the basis for learning and memory. NMDAR-mediated increases in intracellular Ca 2ϩ trigger various cellular responses, such as activation of the Ras-mitogen-activated protein kinase pathway, which transmits the glutamate signal to the nucleus and ultimately leads to long lasting neuronal responses (3). Recent studies (4 -6) have suggested that NO is a key molecule linking the NMDAR-mediated increase in cytoplasmic Ca 2ϩ and long term potentiation (LTP), a physiological model for learning and memory. NO is thought to mediate synaptic plasticity in the hippocampus and influence LTP and thus enhance memory formation. The role of NO in memory formation is also suggested by the observation that the formation of olfactory memory is dependent on NO production (7). In the brain, the production and release of NO is induced by glutamate acting on both NMDAR and ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor. NO can modulate synaptic plasticity through a cGMP-dependent increase of glutamate release. NO also enhances glutamate release and a subsequent increase in the response of neuronal cell synapses to glutamate. This positive feedback loop appears to be particularly relevant to the hippocampal LTP (8 -11).
In the brain, NO is generated from L-arginine mainly by Ca 2ϩ /calmodulin-dependent neuronal NO synthase (nNOS) (12). nNOS is unique compared with other NO synthases (NOSs), such as inducible NOS and epithelial NOS (eNOS), in that it contains a PDZ (postsynaptic density protein 95/discslarge/zona occlusens-1) domain at its N terminus. This domain may play a role in the precise localization of nNOS within the cell.
We have surveyed the postsynaptic density (PSD) fractionassociated mRNAs by random amplification and sequencing (13). Among them, the DemA20-5 cDNA fragment of 148 bp was not identified by our initial data base search. In this study, we cloned a full-length DemA20-5 cDNA and re-named it a novel nNOS-interacting DHHC domain-containing protein with dendritic mRNA (NIDD) (GenBank TM accession number AB098078). We also report the regulation by NIDD of nNOS activity via targeting of nNOS to the postsynaptic membrane through a PDZ domain-dependent interaction. Our results suggest that NIDD plays an important role in the regulation of synaptic plasticity by specifying the NO signaling pathway at postsynaptic sites.

EXPERIMENTAL PROCEDURES
Materials-pGEM-T easy vector was purchased from Promega (Madison, WI). Polyclonal rabbit anti-nNOS, monoclonal mouse anti-nNOS, monoclonal mouse anti-eNOS, mouse anti-PSD-95, and anti-flotillin1 antibodies were from Transduction Laboratories (Lexington, KY). Anti-SAP97 antibody was from Stressgen (Victoria, British Columbia, Canada); mouse anti-His.G antibody was from Invitrogen; antiactin antibody was from Sigma; secondary antibodies raised against rabbit and mouse IgG and conjugated with horseradish peroxidase were from Calbiochem and Cappel (West Chester, PA), and those conjugated with rhodamine and fluorescein isothiocyanate were from Molecular Probes (Eugene, OR). The Maxi-DNA purification kit was from Qiagen (Tokyo, Japan), and polyethyleneimine-coated slide glasses were from Iwaki Glass (Funabashi, Japan). Neurobasal medium supplemented with B27, pcDNA4/HisMax, pcDNA3.1, and LipofectAMINE 2000 were from Invitrogen. Calcium ionophore A23187 and the nNOS detection kit were from Sigma. pGEX4T-1, glutathione-Sepharose 4B, Hybond-N nylon membranes, and L-[ 3 H]arginine were from Amersham Biosciences. All other chemicals were of reagent grade.
cDNA Cloning and Sequence Analysis-Full-length cDNA of NIDD was cloned from a rat hippocampal cDNA library by several rounds of rapid amplification of cDNA 5Ј end based on the sequence of the initially isolated DemA20-5 cDNA fragment (13). The cDNA was cloned into pGEM-T easy vector, and the sequences of both strands were determined by the chain termination method with a BigDye terminator cycle sequencing kit. Data base analyses were performed with the BLAST program (National Center for Biotechnology Information, National Institutes of Health, Bethesda) and SMART server (smart.embl-heidelberg.de/).
RNA Analyses-In situ hybridization in cultured neurons was performed as described previously (13). Our in situ hybridization protocol was judged to be appropriate because mRNA for the ␣-subunit of Ca 2ϩ / calmodulin-dependent protein kinase was localized in dendrites and that for ␤-tubulin was restricted to soma under the conditions used previously (13,14). A cRNA probe (nt 217-402 of NIDD) was generated from the linearized plasmid using T7 and Sp6 polymerase according to the protocol of the digoxigenin-labeled cRNA synthesis kit.
In situ hybridization in brain sections was performed as described previously (15). Antisense oligonucleotide probes (nt 958 -1003 and 1363-1407 for NIDD and nt 4468 -4512 and 4704 -4748 for nNOS; GenBank TM accession number X59949) were labeled with ␣-35 S-dATP by using terminal deoxynucleotidyltransferase. The sagittal and coronal sections were made from Wistar rat brains (7 weeks old) using a cryostat. Hybridization was performed overnight at 42°C in a solution consisting of 50% deionized formamide, 4ϫ SSC, 1ϫ Denhardt's solution, 1% sodium N-lauroyl sarcosinate, 0.1 M phosphate buffer, pH 7.2, and 200 g/ml yeast tRNA, 10% dextran sulfate, 100 mM dithiothreitol, and a mixture of two 35 S-dATP-labeled oligonucleotide probes. After washing, the sections were autoradiographed using NTB2 nuclear track emulsion for 1 month at 4°C.
For Northern blotting, poly(A) ϩ RNA (2 g) isolated from rat forebrain was size-fractionated on a formamide-containing 1% agarose gel and capillary-blotted to a Hybond-N nylon membrane. The filter was hybridized with the random-primed 32 P-labeled probe (nt 4276 -5688 of NIDD). The blot was then exposed on an imaging plate overnight, and the hybridization signals were visualized with a BAS-1500 Phosphor-Imager (Fuji Film, Tokyo, Japan).
For the nNOS pull-down assay, one adult rat cerebellum (250 mg) was homogenized in 2.5 ml of Tris-HCl buffer (25 mM, pH 7.4) containing 1 mM EDTA, 1 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride (TEE). Cerebellar proteins were solubilized by incubation with 1% Triton X-100 on a rocking platform at 4°C for 1 h. For the NIDD pull-down assay, proteins (200 g) of the synaptic plasma membrane (SPM) fraction were solubilized with 1% Triton X-100 at 4°C for 1 h. After clarification by centrifugation at 15,000 ϫ g for 15 min and 1:10 dilution in TEE buffer, the supernatant was incubated at 4°C for 2 h with 30 g of GST fusion proteins or GST alone coupled to glutathione-Sepharose 4B. After incubation, the pulled down materials were washed four times, and the bound proteins were analyzed by Western blotting. The pull-down assays for PSD-95, SAP97, and GRIP were performed using PSD proteins (20 g) as described previously (16).
Generation of Affinity-purified Anti-NIDD Antibody and Western Blotting-Rabbit anti-NIDD antiserum was raised against a fusion protein of GST with NIDD-NT (GST-NIDD-NT). Antibody was affinitypurified with GST-NIDD-NT chemically immobilized to glutathione-Sepharose 4B after removal of anti-GST antibody with GST-glutathione-Sepharose 4B. Western blotting was performed as described previously (18,19).
Subcellular Fractionation and Sucrose Density Gradient Flotation Assay-Subcellular fractions were prepared from the forebrains of Wistar rats (6 weeks old, male) as described previously (18,19). For the sucrose density gradient flotation assay, proteins (500 g) of the SPM fraction were suspended by repeated passage through a 26-gauge needle in 1.85 ml of TNE buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 200 M phenylmethylsulfonyl fluoride, 10 g/ml pepstatin A, 10 g/ml leupeptin, and 10 g/ml aprotinin) containing 0.15% Triton X-100 or 0.5% Rubrol and incubated on a rocking platform at 4°C for 30 min. The sample was adjusted to 40% sucrose with 80% sucrose in TNE buffer, placed at the bottom of a centrifuge tube, and overlaid successively with 3.75 ml each of TNE buffer containing 30% sucrose and 5% sucrose, respectively. The sample was then centrifuged at 256,000 ϫ g max for 30 h at 4°C. After centrifugation, 11 fractions of 1 ml each were collected sequentially from the top, and the pellet was resuspended in 1 ml of TNE buffer. Proteins in each fraction were precipitated in 10% trichloroacetic acid solution and resuspended in 200 l of TNE buffer. Equal volumes of the fractions were separated and analyzed by Western blotting.
Cell Culture and Transfection of cDNAs-The cerebral cortex or hippocampus of rats (embryonic day 18, Wistar) was dissociated and plated onto a polyethyleneimine-coated glass slide. Neurons were cultured essentially as described previously (20) in Neurobasal medium supplemented with B27. COS-7 cells were cultured as described previously (14). cDNAs encoding full-length NIDD, nNOS, and eNOS were amplified by PCR and inserted into appropriate restriction sites of mammalian expression vectors, pcDNA4/HisMax and pcDNA3.1, respectively. The primer sets used were as follows: CGCGGATCCAT-GAAGCCAGTAAAAAAAAAG and CGCTCGAGTTAGACAATGTC-CTCGGCAGG for NIDD; CGGAATTCATGGAAGAGAACACGTTTG and CGCTCGAGTTAGGAGCTGAAAACCTC for nNOS; and GCAT-GGGCAACTTGAAGAGTG and GAAGAGGGCAGCAGGATG for eNOS (GenBank TM accession number AB176831). The restriction enzyme sites introduced are underlined. Plasmid DNAs were isolated using a Maxi-DNA purification kit. Each plasmid was transfected into COS-7 cells at 90% confluence with the aid of LipofectAMINE 2000. The efficiency of transient transfection in these studies was consistently ϳ60% as assessed by fluorescence derived from a control plasmid expressing green fluorescent protein.
Immunocytochemistry-Cultures were fixed with 4% paraformaldehyde for 20 min at room temperature, and the cells were permeabilized with 0.25% Triton X-100. The cells were incubated at 4°C overnight with primary antibodies diluted in PBS containing 5% normal goat serum. Slides were incubated with rhodamine-or fluorescein isothiocyanate-conjugated secondary antibodies for 1 h at room temperature. Cells were observed with a Leica confocal laser scanning microscope.
NOS Enzyme Assay-nNOS activity in COS-7 cells was assayed by All experiments were done in triplicate and repeated at least three times. Statistical evaluation of the data was performed by Student's two-tailed t test. All data are reported as means Ϯ S.E. A p value of less than 0.05 was considered significant.

RESULTS
Cloning and Characterization of NIDD cDNA-We have cloned a full-length cDNA of DemA20-5, whose mRNA is associated with the PSD fraction (13), and we named it NIDD. Two clones were obtained for NIDD, a long form and a short form (NIDD-L and NIDD-S, respectively). The full-length cDNAs of NIDD-L and NIDD-S were 5867 and 5756 bp long, respectively, and the encoded proteins comprised 429 and 392 amino acids, respectively. NIDD-L contained an additional 111 nucleotides at nt 1009 of NIDD-S, as shown in Fig. 1a. Domain search analysis using the SMART program revealed that NIDD protein contained five transmembrane domains and a zinc finger DHHC domain, which is highly conserved from yeast to humans and possibly involved in protein-protein or protein-DNA interactions (25). In addition, NIDD possessed an ϪEDIV motif at its C-terminal tail, which was expected to bind to the PDZ domain of nNOS (26,27). Homology search using the BLAST program revealed that NIDD had orthologs in the mouse and human genomes (GenBank TM accession numbers XM_285173 and NM_173570, respectively), but there was no homology to any known proteins whose function has been specified. The nucleotide sequence surrounding the first AUG codon in NIDD mRNA agreed well with the Kozak consensus sequence (28) and NIDD mRNA possessed an in-frame stop codon upstream of the first AUG. The human homolog was located on chromosome 3p13. 31.
Analyses of NIDD Transcript-Because NIDD was originally isolated from mRNAs associated with the PSD fraction, we considered that NIDD mRNA was likely to be localized to neuronal dendrites. To test this possibility, we performed in situ hybridization using cultured hippocampal neurons (Fig.  2a). As expected, the antisense probe revealed that the expression pattern of NIDD mRNA showed a typical dendritic distribution. Dendritic mRNA signals were observed in nearly all the cells with neuron-like morphology.
Northern blotting showed a 7-kb major band and an 8-kb minor band (Fig. 2b), which suggested the presence of isoforms. The developmental changes of NIDD expression were examined by RT-PCR (Fig. 2c). The level of NIDD mRNA was high 1 day and 1 week after birth and reduced to about half that level 2 weeks after birth and thereafter. This pattern was confirmed by repeated experiments using different lots of tissue samples. We next determined the expression levels of NIDD mRNAs in various tissues by RT-PCR (Fig. 2d). NIDD was highly expressed in the brain, whereas very low levels of expression were detected in muscle, spleen, lung, and testis.
Analyses of NIDD Protein Expression-To examine the expression of NIDD, we carried out immunoblotting analysis with affinity-purified anti-NIDD antibody. The specificity of the antibody was confirmed by its specific interaction with His-tagged NIDD protein expressed in COS-7 cells (data not shown). The antibody detected doublet bands of 40 kDa in the brain. These doublet bands may be NIDD-S and NIDD-L, judging from their sizes. The 40-kDa bands were not detected in other tissues examined (Fig. 3). The brain-specific expression of NIDD protein was in good agreement with the high level of NIDD mRNA expression in the brain (Fig. 2c). The antibody also detected a weaker band of 42 kDa in muscle and a band of 90 kDa in brain, spleen, lung, and testis. The identities of these 42-and 90-kDa bands are not known at present.
Specific Interaction between NIDD and PDZ Domain of nNOS-We examined the interaction between nNOS and NIDD by pull-down assays, because NIDD has at its C-terminal tail an ϪEDIV motif, which was expected to bind to the PDZ domain of nNOS (26, 27, 29 -31) (Fig. 4). As expected, nNOS was pulled down by NIDD-CT but not by GST alone or by GST-NIDD-del3, a deletion mutant in which the last three amino acid residues of GST-NIDD-CT were deleted (Fig. 4a). Thus, the interaction occurs via the PDZ-binding motif of NIDD. The binding of NIDD-CT was specific for the PDZ domain of nNOS, because it did not pull down other PDZ domaincontaining proteins, such as PSD-95, SAP97, or GRIP1 (Fig.  4b). Conversely, NIDD was pulled down by GST fusion protein with the PDZ domain of nNOS (GST-nNOS-PDZ) but not by GST alone (Fig. 4c).
Attempts to co-immunoprecipitate NIDD with nNOS, and vice versa, from detergent extracts of rat brain or co-transfected COS-7 cells, have been unsuccessful because of the insolubility of NIDD in nondenaturing detergents (data not shown). In particular, His-NIDD expressed in COS-7 cells aggregated even in the presence of 1% Triton X-100 (data not shown) as observed in Abl-phinlin 2 (Aph2), another zinc finger-DHHC-containing protein involved in endoplasmic reticulum stress-induced apoptosis (32). Therefore, we adopted a co-sedimentation assay, instead of co-immunoprecipitation, to examine further the interaction between NIDD and nNOS. COS-7 cells were co-transfected with nNOS (fixed amounts) and His-tagged NIDD (graded amounts). After 48 h of transfection, the cells were lysed in PBS, and the supernatant and the pellet fractions were subjected to immunoblotting with anti-His.G and anti-nNOS antibodies. As shown in Fig. 5, coexpression of His-NIDD caused a dose-dependent shift of nNOS from the supernatant to the pellet, suggesting an interaction between nNOS and NIDD in COS-7 cells.
Localization of NIDD at Synaptic Sites-The subcellular localization of NIDD was examined by immunoblotting (Fig. 6). NIDD showed a distribution pattern similar to that of nNOS, except for the soluble fraction (Fig. 6a). NIDD and nNOS proteins were enriched in synaptic fractions such as the synaptosome and SPM fractions, indicating that the two proteins could be co-localized at synaptic sites. NIDD and nNOS were also present in the PSD fraction, although the amounts detected were reduced or minimal, respectively. Most interesting, a 90-kDa NIDD-immunoreactive protein was highly concentrated in the PSD fraction but scarce in the synaptosome and SPM fractions. The NIDD distribution in the SPM was further examined by sucrose density gradient centrifugation (Fig. 6b). The NIDD was distributed in both lipid raft fractions (Fig. 6b,  lanes 4 -6), although most of it was recovered in the top and 2nd fractions under the conditions used. Treatment of SPM protein with 1% of Triton X-100, or even 0.25%, caused a shift of the distribution of NIDD to the soluble fraction (Fig. 6b,  lanes 8 -11) and the pellet with no NIDD in the top, the 2nd, and raft fractions (data not shown). Thus, raft distribution became evident under conditions using a relatively low amount of the detergent. Raft localization of NIDD was also shown in the experiment using 0.5% Rubrol. nNOS was distributed in both the raft and non-raft fractions under the conditions we used.
The subcellular distribution of NIDD was further examined in primary cultures of rat cortical neurons (Fig. 7). The immunoreactivities for NIDD and nNOS were distributed in and co-localized in the dendrites as well as soma.
Comparison of mRNA Distribution between NIDD and nNOS-To clarify further the relationship between NIDD and nNOS, we compared their distributions in the brain by in situ hybridization analysis. Prominent expression of NIDD mRNA was detected in the hippocampal pyramidal cell layer of the CA1 region, tenia tecta, piriform cortex, and olfactory mitral cells and tufted cells (Fig. 8, a, c, e, and g). Moderate expression was observed in the anterior olfactory nuclei, caudate putamen, nucleus accumbens, cerebral cortex, amygdaloid nuclei, hippocampal pyramidal cell layer of the CA3 region, and dentate granule cell layer (Fig. 8, a, e and g). Weak expression was detected in the septal nuclei, medial habenular nucleus, paraventricular thalamic nucleus, anterior medial preoptic nucleus, medial preoptic area, ventromedial hypothalamic nucleus, locus coeruleus, and cerebellar Purkinje cell layer (Fig. 8, a and  g). The expression of NIDD mRNAs was summarized in Table  I. Dendritic localization of the NIDD mRNA was not clearly evident with this in situ hybridization protocol, possibly due to lower expression level in the dendrites compared with that in somatic areas. Autoradiographic detection of mRNA in tissue sections appeared to be unsuitable for the detection of dendritically distributed mRNAs with respect to resolution and sensitivity.
As compared with the expression pattern of nNOS mRNA, NIDD mRNA was expressed in some restricted populations of NOS mRNA-expressing neuronal cells such as olfactory mitral cells, hippocampal pyramidal cells, dentate granule cells, presubiculum, piriform cortex, amygdaloid nuclei, medial habenular nucleus, paraventricular thalamic nucleus, and ventromedial hypothalamic nucleus (Fig. 8). The expression profile of nNOS mRNA was similar to that reported previously (33).
Effect of NIDD on the nNOS Enzyme Activity-Because previous reports showed that nNOS-interacting proteins, such as protein inhibitor of nNOS, regulated nNOS activity by altering the subcellular distribution of nNOS (34), we examined the effects of NIDD on nNOS activity. To investigate whether the interaction of nNOS with NIDD modulates nNOS activity, COS-7 cells were transfected with cDNAs encoding nNOS and NIDD. After 48 h of transfection, nNOS activity was assessed by measuring the conversion of radiolabeled L-arginine to Lcitrulline. Co-transfection of NIDD into nNOS-expressing cells markedly enhanced nNOS enzyme activity (Fig. 9a). To determine whether NIDD could directly activate nNOS enzyme, we measured nNOS activity in the cell lysates. Co-expression of NIDD caused a significant increase in nNOS activity (Fig. 9b). To further confirm the specificity of NIDD to nNOS, we performed additional experiments to study the effect of NIDD on eNOS activity. As shown in Fig. 9, the eNOS activity was not affected by co-transfection with NIDD. The equal expression and cell density were monitored by immunoblotting with the indicated antibodies (Fig. 9c). These results indicate a functional association between nNOS and NIDD. DISCUSSION We identified a novel nNOS-interacting synaptic membrane protein, NIDD. The major finding of this study was that nNOS distribution and activity are regulated through specific interaction with NIDD. We also found a unique molecular structure (Fig. 1), relatively specific expression in the brain (Fig. 4), early expression in the postnatal period (Fig. 2c), and dendritic distribution of the mRNA of NIDD (Fig. 2a).
The interaction of NIDD with nNOS is mediated by the PDZ-binding motif at the end of the C terminus of NIDD and the PDZ domain of nNOS, which was proven by the pull-down experiment (Fig. 4). Notably, the C-terminal sequence of NIDD is specific to the PDZ domain of nNOS (Fig. 4, a and b). The specificity of the interaction between the NIDD C-terminal sequence and nNOS PDZ domain is supported by findings reported previously (27). This type of interaction with nNOS is also seen in the interaction with C-terminal PDZ ligand of nNOS (CAPON) (29), C-terminal binding protein (31), and phosphofructokinase-M (30).  NIDD is a transmembrane protein ( Fig. 1) that is co-localized with nNOS in the synaptosome, SPM, and synaptic lipid raft fractions (Fig. 6). Co-localization of these proteins at synaptic sites was also confirmed in cultured cortical neurons (Fig. 7). Moreover, co-expression of NIDD in the nNOS-expressing COS-7 cells caused a shift of nNOS from the supernatant to the pellet fraction (Fig. 5). All these findings suggest that NIDD and nNOS interact in vivo and that NIDD plays a role in targeting nNOS to the synaptic membrane. The functional result of the interaction could be the regulation of NO production through nNOS at the synaptic membrane.
Membrane association of nNOS is important for its enzyme activity, because biochemical studies indicate that up to 60% of total NOS activity in the brain is found in the membrane fraction (35), and targeting of eNOS to the plasma membrane is accompanied by a large increase in NO production (36). Conversely, an nNOS-interacting protein, protein inhibitor of nNOS, inhibits nNOS enzyme activity by recruiting nNOS from the membrane to the cytosol (34). The relationship between membrane association and enzyme activity of nNOS may also apply to the NIDD-nNOS interaction. As expected, NO production by nNOS in COS-7 cells is significantly increased when the cells are co-transfected with the membrane protein NIDD (Fig. 9). Thus, NIDD regulates the nNOS enzyme activity.
In general, one of the roles of the nNOS-interacting proteins is to target the nNOS to appropriate sites and restrict the effect of NO to the region proximal to the targeted sites. NO produced by nNOS can diffuse into neighboring cells and activate soluble guanylyl cyclase or covalently modify cysteine residues of target proteins (S-nitrosylation). For example, ␣1-syntrophin targets nNOS to the sarcolemma (37). PSD-95 and PSD-93 target nNOS to postsynaptic sites and couple the nNOS activation to Ca 2ϩ entry through NMDAR, which is also targeted by PSD-95 and PSD-93 (38). A brain-specific adaptor protein, CAPON, competes with PSD-95 for the PDZ domain in nNOS and recruits nNOS to a small monomeric G-protein, Dexras 1 (29,39). This may facilitate the nitrosylation and subsequent activation of Dexras 1 (29,39,40). This generalization may also be true in the case of NIDD. The effects of NIDD on nNOS are limited to some restricted brain areas because the localization of NIDD, as far as the mRNA distribution is considered, overlapped that of nNOS only in restricted areas in the brain. We are waiting for further studies regarding this point.
In summary, we have cloned and characterized a novel nNOS-interacting synaptic membrane protein, NIDD, which increases the nNOS enzyme activity by targeting it to the synaptic membrane in a PDZ domain-dependent manner. The results suggest that NIDD plays an important role in regulating synaptic plasticity by regulating the NO signaling pathway through specification of the sites of nNOS action. FIG. 9. Effects of NIDD on nNOS enzyme activity. COS-7 cells transfected with indicated cDNAs were cultured in 6-well tissue culture plates. The pcDNA3.1 or pcDNA4 parent vector was also transfected to keep the applied DNA quantity constant. The enzyme activity was assayed in two ways 48 h after the transfection by counting L-[ 3 H]citrulline. a, cells were incubated with L-[ 3 H]arginine, and after 10 min, they were harvested and lysed for the assay. b, cells were harvested and lysed, and incubated with L-[ 3 H]arginine at 25°C for 15 min. c, expression of the transfected DNA was confirmed by Western blotting of the cell lysates, processed in parallel with the cells used for the activity assay. The immunoblot for actin was used as a monitor to show that cultures with equal cell density were used in these assays. Assays of nNOS and eNOS in (a or b) were carried out simultaneously. All experiments were done in triplicate and repeated at least three times with essentially the same results. Student's t test. *, p Ͻ 0.05.