Semaphorin 4B interacts with the post-synaptic density protein PSD-95/SAP90 and is recruited to synapses through a C-terminal PDZ-binding motif

The semaphorins are a large family of proteins that act as guidance signals for axons and dendrites. The class 4 semaphorins are integral membrane proteins that are widely expressed throughout the nervous system. Here, we show that a subclass of these semaphorins is characterized by a PDZ-binding motif at their carboxy-terminus. This sequence mediates the interaction with the post-synaptic density protein PSD-95/SAP90. Co-expression of Sema4B with PSD-95 in COS 7 cells results in the clustering of Sema4B. Sema4B co-localizes with PSD-95 at synaptic contacts between cultured hippocampal neurons. This synaptic localization depends on the presence of the PDZ-binding motif.     

Sema4c, a transmembrane semaphorin, interacts with a post-synaptic density protein, PSD-95

Semaphorins are known to act as chemorepulsive molecules that guide axons during neural development. Sema4C, a group 4 semaphorin, is a transmembrane semaphorin of unknown function. The cytoplasmic domain of Sema4C contains a proline-rich region that may interact with some signaling proteins. In this study, we demonstrate that Sema4C is enriched in the adult mouse brain and associated with PSD-95 isoforms containing PDZ (PSD-95/DLG/ZO-1) domains, such as PSD-95/SAP90, PSD-93/chapsin110, and SAP97/DLG-1, which are concentrated in the post-synaptic density of the brain. In the neocortex, S4C is enriched in the synaptic vesicle fraction and Triton X-100 insoluble post-synaptic density fraction. Immunostaining for Sema4C overlaps that for PSD-95 in superficial layers I-IV of the neocortex. In neocortical culture, S4C is colocalized with PSD-95 in neurons, with a dot-like pattern along the neurites. Sema4C thus may function in the cortical neurons as a bi-directional transmembrane ligand through interacting with PSD-95.     

FAK and PYK2 interact with SAP90/PSD-95-Associated Protein-3

Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2) are two related non-receptor tyrosine kinases highly expressed in brain. Although they are both involved in synaptic plasticity, little is known about their specific neuronal partners. Using a yeast two-hybrid screen and GST pull-down assays we show that SAPAP3 (SAP90/PSD-95-Associated Protein-3) interacts with FAK (residues 676-840) and PYK2. The three proteins partly co-distribute in the same sucrose gradient fractions as the post-synaptic density protein PSD-95 and Src. Our results suggest that SAPAP3 is an anchoring protein for FAK and PYK2 in post-synaptic densities and may contribute to the synaptic function of these tyrosine kinases.     

MAGUIN, a novel neuronal membrane-associated guanylate kinase-interacting protein

Postsynaptic density (PSD)-95/Synapse-associated protein (SAP) 90 and synaptic scaffolding molecule (S-SCAM) are neuronal membrane-associated guanylate kinases. Because PSD-95/SAP90 and S-SCAM function as synaptic scaffolding proteins, identification of ligands for these proteins is important to elucidate the structure of synaptic junctions. Here, we report a novel protein interacting with the PDZ domains of PSD-95/SAP90 and S-SCAM and named it MAGUIN-1 (membrane-associated guanylate kinase-interacting protein-1). MAGUIN-1 has one sterile alpha motif, one PDZ, and one plekstrin homology domain. MAGUIN-1 is localized at the plasma membrane via the plekstrin homology domain and the C-terminal region and interacts with PSD-95/SAP90 and S-SCAM via a C-terminal PDZ domain-binding motif. MAGUIN-1 has a short isoform, MAGUIN-2, which lacks a PDZ domain-binding motif. MAGUINs are expressed in neurons and localized in the cell body and neurites and are coimmunoprecipitated with PSD-95/SAP90 and S-SCAM from rat crude synaptosome. MAGUIN-1 may play an important role with PSD-95/SAP90 and S-SCAM to assemble the components of synaptic junctions.     

Three isoforms of synaptic scaffolding molecule and their characterization. Multimerization between the isoforms and their interaction with N-methyl-D-aspartate receptors and SAP90/PSD-95-associated protein

The synaptic scaffolding molecule (S-SCAM) has been identified as a protein interacting with SAP90/PSD-95-associated protein (SAPAP) (also called guanylate kinase-associated protein/hDLG-associated protein). S-SCAM has six PDZ (we have numbered them PDZ-0 to -5), two WW, and one guanylate kinase (GK) domains and interacts with N-methyl-D-aspartate (NMDA) receptor via PDZ-5 and SAPAP via the GK domain. We have identified here shorter isoforms of S-SCAM that start at the 164th or 224th methionine, and we renamed the original one, S-SCAMalpha, the middle one, S-SCAMbeta, and the shortest one, S-SCAM-gamma. S-SCAMbeta and -gamma have five PDZ (PDZ-1 to -5), two WW, and one GK domains. S-SCAMalpha interacted with S-SCAMbeta and -gamma through the region containing PDZ-4 and -5. The region containing both of PDZ-4 and -5 is sufficient for the clustering of NMDA receptors and forms a dimer in gel filtration, suggesting that S-SCAM forms multimers via the interaction between the C-terminal PDZ domains and assembles NMDA receptors into clusters. S-SCAMbeta and -gamma also interacted with SAPAP, suggesting that the N-terminal region of the GK domain is not necessary for the interaction. Finally, we have identified the interaction of the PDZ domains of S-SCAM with the GK domain of PSD-95/SAP90. S-SCAM, PSD-95/SAP90, and SAPAP are colocalized at least in some part in brain. Therefore, S-SCAM, PSD-95/SAP90, and SAPAP may form a complex in vivo.     

Differential interaction of NMDA receptor subtypes with the post-synaptic density-95 family of membrane associated guanylate kinase proteins

NMDA receptors are a subclass of ionotropic glutamate receptors. They are trafficked and/or clustered at synapses by the post-synaptic density (PSD)-95 membrane associated guanylate kinase (MAGUK) family of scaffolding proteins that associate with NMDA receptor NR2 subunits via their C-terminal glutamate serine (aspartate/glutamate) valine motifs. We have carried out a systematic study investigating in a heterologous expression system, the association of the four major NMDA receptor subtypes with the PSD-95 family of MAGUK proteins, chapsyn-110, PSD-95, synapse associated protein (SAP) 97 and SAP102. We report that although each PSD-95 MAGUK was shown to co-immunoprecipitate with NR1/NR2A, NR1/NR2B, NR1/NR2C and NR1/NR2D receptor subtypes, they elicited differential effects with regard to the enhancement of total NR2 subunit expression which then results in an increased cell surface expression of NMDA receptor subtypes. PSD-95 and chapsyn-110 enhanced NR2A and NR2B total expression which resulted in increased NR1/NR2A and NR1/NR2B receptor cell surface expression whereas SAP97 and SAP102 had no effect on total or cell surface expression of these subtypes. PSD-95, chapsyn-110, SAP97 and SAP102 had no effect on either total NR2C and NR2D subunit expression or cell surface NR1/NR2C and NR1/NR2D expression. A comparison of PSD-95α, PSD-95β and PSD-95α^C3S,C5S showed that PSD-95-enhanced cell surface expression of NR1/NR2A receptors was dependent upon the PSD-95 N-terminal C3,C5 cysteines. These observations support differential interaction of NMDA receptor subtypes with different PSD-95 MAGUK scaffolding proteins. This has implications for the stabilisation, turnover and compartmentalisation of NMDA receptor subtypes in neurones during development and in the mature brain.     

Interaction between SAP97 and PSD-95, two Maguk proteins involved in synaptic trafficking of AMPA receptors

Synapse-associated protein 97 (SAP97) and postsynaptic density 95 (PSD-95) are closely related membrane-associated guanylate kinase homologs (Maguks) implicated in the synaptic targeting and anchoring of alpha-amino-5-methyl-3-hydroxy-4-isoxazolepropionic acid (AMPA)-selective glutamate receptors. Prompted by accumulating evidence for an oligomeric nature of Maguks, we examined the potential of SAP97 and PSD-95 to form heteromeric complexes. SAP97 and PSD-95 coimmunoprecipitated from rat brain detergent extracts and subsequent glutathione S-transferase pull-down and immunoprecipitation experiments showed that the interaction is mediated by binding of the N-terminal segment of SAP97 (SAP97(NTD)) to the Src homology 3 domain of PSD-95 (PSD-95(SH3)). In cultured hippocampal neurons, expression of green fluorescent protein-tagged PSD-95 triggered accumulation of SAP97 in synaptic spines, which was totally inhibited by coexpression of PSD-95(SH3). Furthermore, overexpression of green fluorescent protein-PSD-95 induced dendritic clustering of GluR-A subunit-containing AMPA receptors, which was strongly inhibited by cotransfection with SAP97(NTD) and PSD-95(SH3) constructs. Our results demonstrated a direct interaction between SAP97 and PSD-95 and suggested that this association may play a functional role in the trafficking and clustering of AMPA receptors.     

Synamon, a novel neuronal protein interacting with synapse-associated protein 90/postsynaptic density-95-associated protein.

Guanylate kinase-associated protein (GKAP)/SAP90/PSD-95-associated protein (SAPAP)/DLG-associated protein (DAP) is a protein of the postsynaptic density (PSD), and binds to the guanylate kinase domain of PSD-95/synapse-associated protein (SAP) 90 and synaptic scaffolding molecule. GKAP/SAPAP/DAP recruits PSD-95/SAP90 and its interacting protein, brain-enriched guanylate kinase-interacting protein, into the Triton X-100-insoluble fraction in transfected cells, suggesting that GKAP/SAPAP/DAP may link several PSD components to the Triton X-100-insoluble structures in the PSD. We have identified here a novel neuronal GKAP/SAPAP/DAP-binding protein and named it synamon. Synamon has seven ankyrin repeats at the NH(2) terminus followed by one src homology 3 domain and one PSD-95/Dlg-A/ZO-1 domain, and several proline-rich regions at the carboxyl terminus. Synamon interacts with the COOH-terminal region of GKAP/SAPAP/DAP via the middle region containing a PSD-95/Dlg-A/ZO-1 domain. Synamon was coimmunoprecipitated with SAPAP from rat crude synaptosomes and colocalized with SAPAP in primary cultured rat hippocampal neurons. Because synamon is composed of various protein-interacting modules, it may also interact with proteins other than GKAP/SAPAP/DAP to organize the architecture of the PSD.     

Characterisation of de novo MAPK10/JNK3 truncation mutations associated with cognitive disorders in two unrelated patients

The c-Jun N-terminal kinases (JNKs) are stress-activated serine-threonine kinases that have recently been linked to various neurological disorders. We previously described a patient with intellectual disability (ID) and seizures (Patient 1), carrying a de novo chromosome translocation affecting the CNS-expressed MAPK10/JNK3 gene. Here, we describe a second ID patient (Patient 2) with a similar translocation that likewise truncates MAPK10/JNK3, highlighting a role for JNK3 in human brain development. We have pinpointed the breakpoint in Patient 2, which is just distal to that in Patient 1. In both patients, the rearrangement resulted in a predicted protein interrupted towards the C-terminal end of the kinase domain. We demonstrate that these truncated proteins, although capable of weak interaction with various known JNK scaffolds, are not capable of phosphorylating the classical JNK target c-Jun in vitro, which suggests that the patient phenotype potentially arises from partial loss of JNK3 function. We next investigated JNK3-binding partners to further explore potential disease mechanisms. We identified PSD-95, SAP102 and SHANK3 as novel interaction partners for JNK3, and we demonstrate that JNK3 and PSD-95 exhibit partially overlapping expression at synaptic sites in cultured hippocampal neurons. Moreover, JNK3, like JNK1, is capable of phosphorylating PSD-95 in vitro, whereas disease-associated mutant JNK3 proteins do not. We conclude that reduced JNK3 activity has potentially deleterious effects on neuronal function via altered regulation of a set of post-synaptic proteins.     

PSD-95 and SAP97 exhibit distinct mechanisms for regulating K(+) channel surface expression and clustering

Mechanisms of ion channel clustering by cytoplasmic membrane-associated guanylate kinases such as postsynaptic density 95 (PSD-95) and synapse-associated protein 97 (SAP97) are poorly understood. Here, we investigated the interaction of PSD-95 and SAP97 with voltage-gated or Kv K(+) channels. Using Kv channels with different surface expression properties, we found that clustering by PSD-95 depended on channel cell surface expression. Moreover, PSD-95-induced clusters of Kv1 K(+) channels were present on the cell surface. This was most dramatically demonstrated for Kv1.2 K(+) channels, where surface expression and clustering by PSD-95 were coincidentally promoted by coexpression with cytoplasmic Kvbeta subunits. Consistent with a mechanism of plasma membrane channel-PSD-95 binding, coexpression with PSD-95 did not affect the intrinsic surface expression characteristics of the different Kv channels. In contrast, the interaction of Kv1 channels with SAP97 was independent of Kv1 surface expression, occurred intracellularly, and prevented further biosynthetic trafficking of Kv1 channels. As such, SAP97 binding caused an intracellular accumulation of each Kv1 channel tested, through the accretion of SAP97 channel clusters in large (3-5 microm) ER-derived intracellular membrane vesicles. Together, these data show that ion channel clustering by PSD-95 and SAP97 occurs by distinct mechanisms, and suggests that these channel-clustering proteins may play diverse roles in regulating the abundance and distribution of channels at synapses and other neuronal membrane specializations.     

Association of membrane-associated guanylate kinase-interacting protein-1 with Raf-1

Membrane-associated guanylate kinase-interacting protein (MAGUIN)-1 was identified as a protein interacting with synaptic scaffolding molecule (S-SCAM) and postsynaptic density (PSD)-95/synapse-associated protein (SAP)90. MAGUIN-1 has a chimerical molecular structure composed of one sterile alpha motif, one PSD-95/Dlg-A/ZO-1 (PDZ), and one pleckstrin homology (PH) domain, and interacts with the PDZ domains of S-SCAM and PSD-95/SAP90 via its carboxyl-terminal PDZ-binding motif. MAGUIN-1 is considered as a mammalian homologue of Drosophila CNK, which is a Raf-interacting protein implicated in the regulation of eye development. Here we have tested whether MAGUIN-1 interacts directly with Raf-1. MAGUIN-1 and Raf-1 were coimmunoprecipitated from rat brain. MAGUIN-1 binds to the kinase domain of Raf-1, and Raf-1 binds to the middle region of MAGUIN-1 containing the PH domain. However, in contrast to the dominant active mutant of Ki-Ras, which interacts with Raf-1, recruits it to the plasma membrane from the cytosol, and activates it, MAGUIN-1 neither activates Raf-1 nor recruits it to the plasma membrane. MAGUIN-1 may link Raf-1 to components of synapses assembled by PSD-95/SAP90 and S-SCAM.     

Interaction of NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase protein, with calmodulin and PSD-95/SAP90. A possible regulatory role in molecular clustering at synaptic sites

NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase family protein, is known to bind to C-terminal ends of N-methyl-D-aspartate receptor 2B (NR2B) through its PDZ (PSD-95/Dlg/ZO-1) domains. NE-dlg/SAP102 and NR2B colocalize at synaptic sites in cultured rat hippocampal neurons, and their expressions increase in parallel with the onset of synaptogenesis. We have identified that NE-dlg/SAP102 interacts with calmodulin in a Ca2+-dependent manner. The binding site for calmodulin has been determined to lie at the putative basic alpha-helix region located around the src homology 3 (SH3) domain of NE-dlg/SAP102. Using a surface plasmon resonance measurement system, we detected specific binding of recombinant NE-dlg/SAP102 to the immobilized calmodulin with a Kd value of 44 nM. However, the binding of Ca2+/calmodulin to NE-dlg/SAP102 did not modulate the interaction between PDZ domains of NE-dlg/SAP102 and the C-terminal end of rat NR2B. We have also identified that the region near the calmodulin binding site of NE-dlg/SAP102 interacts with the GUK-like domain of PSD-95/SAP90 by two-hybrid screening. Pull down assay revealed that NE-dlg/SAP102 can interact with PSD-95/SAP90 in the presence of both Ca2+ and calmodulin. These findings suggest that the Ca2+/calmodulin modulates interaction of neuronal membrane-associated guanylate kinase proteins and regulates clustering of neurotransmitter receptors at central synapses.     

The glutamate transporter GLT1b interacts with the scaffold protein PSD-95

The glutamate transporter (GLT1) regulates glutamate concentrations in glutamatergic synapses and it is expressed in at least two isoforms, GLT1a and GLT1b. In this work, we show that the C-terminus of GLT1b is able to interact with the PDZ domains of a number of proteins. Notably, one of them might be the scaffold protein post-synaptic density (PSD-95). GLT1b formed co-immunoprecipitable complexes with PSD-95 in solubilizated rat brain extracts, complexes that also contained NMDA receptors. Co-transfection of GLT1b, PSD-95, and NMDA receptor subunits in heterologous expression systems recapitulated in vitro the interactions among these proteins that had been observed in the rat brain extracts and revealed the importance of the GLT1b C-terminal PDZ binding motif in tethering this transporter to PSD-95. Significantly, co-expression of GLT1b and PSD-95 increased the V _max of the transporter by decreasing the rate of GLT1b endocytosis. Moreover, GLT1b transfected into primary cultured neurons or glia formed protein clusters that co-localized with co-transfected PSD-95, clusters that in these neurons accumulated preferentially in dendritic spines. We hypothesize that the GLT1b/PSD-95 interaction, characterized here in vitro , might anchor this transporter close to the post-synaptic glutamate receptors, thereby permitting the fine regulation of glutamate concentrations in this microenvironment. This tight association might also facilitate the regulation of GLT1b through the signaling pathways initiated by the activation of glutamate receptors.     

Acute Physiological Stress Promotes Clustering of Synaptic Markers and Alters Spine Morphology in the Hippocampus

GluA2-containing AMPA receptors and their association with protein kinase M zeta (PKMζ) and post-synaptic density-95 (PSD-95) are important for learning, memory and synaptic plasticity processes. Here we investigated these synaptic markers in the context of an acute 1h platform stress, which can disrupt spatial memory retrieval for a short-term memory on the object placement task and long-term memory retrieval on a well-learned radial arm maze task. Acute stress increased serum corticosterone and elevated the expression of synaptic PKMζ while decreasing synaptic GluA2. Using co-immunoprecipitation, we found that this stressor promotes the clustering of GluA2, PKMζ and PSD-95, which is consistent with effects reported from overexpression of PKMζ in cell culture. Because PKMζ overexpression has also been shown to induce spine maturation in culture, we examined how stress impacts synaptic markers within changing spines across various hippocampal subfields. To achieve this, we employed a new technique combining Golgi staining and immmunohistochemistry to perform 3D reconstruction of tertiary dendrites, which can be analyzed for differences in spine types and the colocalization of synaptic markers within these spines. In CA1, stress increased the densities of long-thin and mushroom spines and the colocalization of GluA2/PSD-95 within these spines. Conversely, in CA3, stress decreased the densities of filopodia and stubby spines, with a concomitant reduction in the colocalization of GluA2/PSD-95 within these spines. In the outer molecular layer (OML) of the dentate gyrus (DG), stress increased both stubby and long-thin spines, together with greater GluA2/PSD-95 colocalization. These data reflect the rapid effects of stress on inducing morphological changes within specific hippocampal subfields, highlighting a potential mechanism by which stress can modulate memory consolidation and retrieval.     

nArgBP2, a novel neural member of ponsin/ArgBP2/vinexin family that interacts with synapse-associated protein 90/postsynaptic density-95-associated protein (SAPAP).

Postsynaptic density (PSD)-95/synapse-associated protein (SAP) 90 and synaptic scaffolding molecule (S-SCAM) are synaptic membrane-associated guanylate kinases. Both the proteins interact with SAP90/PSD-95-associated protein (SAPAP) (also called guanylate kinase-associated protein/Dlg-associated protein). SAPAP is a protein highly enriched in the PSD fraction and may link PSD-95/SAP90 and S-SCAM to Triton X-100-insoluble structures. We found here a novel SAPAP-interacting protein, which was specifically expressed in neural tissue and was present in the postsynaptic density fraction in brain. This protein had a sorbin homology domain in the N terminus, a zinc finger motif in the middle region, and three src homology (SH) 3 domains in the C terminus and was homologous to the ponsin/ArgBP2/vinexin family proteins. We named this protein nArgBP2 because it was the most homologous to ArgBP2. nArgBP2 is a neural member of a growing family of SH3-containing proteins. nArgBP2 bound to the proline-rich region of SAPAP via its third SH3 domain and was coimmunoprecipitated with SAPAP from the extract of rat brain. Furthermore, nArgBP2 was colocalized with SAPAP at synapses in cerebellum. nArgBP2 bound to not only SAPAP but also vinculin and l-afadin, known to bind to ponsin and vinexin. nArgBP2 may be implicated in the protein network around SAPAP in the PSD.     

Structural differences between the SH3-HOOK-GuK domains of SAP90/PSD-95 and SAP97

The SH3-HOOK-GUK domains of the postsynaptic scaffolding proteins SAP90/PSD-95 and SAP97 are established targets of synaptic plasticity processes in the brain. A crucial molecular mechanism involved is the transition of this domain to different conformational states. We purified the SH3-HOOK-GUK domain of both proteins to examine variations in protein conformation and stability. As monitored by circular dichroism and differential scanning calorimetry, SAP97 (T_m = 64 °C) is significantly more thermal stable than SAP90/PSD-95 (T_m = 52 °C) and follows a bimodal phase transition. GdmCl-induced equilibrium unfolding of both proteins follows the two-state transitions and thus does not involve the accumulation of stable intermediate state(s). Equilibrium unfolding of SAP97 is highly cooperative from a native state to an unfolded state. In contrast, SAP90/PSD-95 follows a non-cooperative transition from native to unfolded states. A highly cooperative unfolding reaction in case of SAP97 indicates that the protein existed initially as a compact, well-folded structure, while the gradual, non-cooperative melting reaction in case of SAP90/PSD-95 indicates that the protein is in comparison more flexible.     

Interaction of the tyrosine kinase Pyk2 with the N-methyl-D-aspartate receptor complex via the Src homology 3 domains of PSD-95 and SAP102

The protein-tyrosine kinase Pyk2/CAKbeta/CADTK is a key activator of Src in many cells. At hippocampal synapses, induction of long term potentiation requires the Pyk2/Src signaling pathway, which up-regulates the activity of N-methyl-d-aspartate-type glutamate receptors. Because localization of protein kinases close to their substrates is crucial for effective phosphorylation, we investigated how Pyk2 might be recruited to the N-methyl-d-aspartate receptor complex. This interaction is mediated by PSD-95 and its homolog SAP102. Both proteins colocalize with Pyk2 at postsynaptic dendritic spines in the cerebral cortex. The proline-rich regions in the C-terminal half of Pyk2 bind to the SH3 domain of PSD-95 and SAP102. The SH3 and guanylate kinase homology (GK) domain of PSD-95 and SAP102 interact intramolecularly, but the physiological significance of this interaction has been unclear. We show that Pyk2 effectively binds to the Src homology 3 (SH3) domain of SAP102 only when the GK domain is removed from the SH3 domain. Characterization of PSD-95 and SAP102 as adaptor proteins for Pyk2 fills a critical gap in the understanding of the spatial organization of the Pyk2-Src signaling pathway at the postsynaptic site and reveals a physiological function of the intramolecular SH3-GK domain interaction in SAP102.     

Tamalin is a scaffold protein that interacts with multiple neuronal proteins in distinct modes of protein-protein association

Tamalin is a scaffold protein that comprises multiple protein-interacting domains, including a 95-kDa postsynaptic density protein (PSD-95)/discs-large/ZO-1 (PDZ) domain, a leucine-zipper region, and a carboxyl-terminal PDZ binding motif. Tamalin forms a complex with metabotropic glutamate receptors and guanine nucleotide exchange factor cytohesins and promotes intracellular trafficking and cell surface expression of group 1 metabotropic glutamate receptors. In the present study, using several different approaches we have shown that tamalin interacts with multiple neuronal proteins through its distinct protein-binding domains. The PDZ domain of tamalin binds to the PDZ binding motifs of SAP90/PSD-95-associated protein and tamalin itself, whereas the PDZ binding motif of tamalin is capable of interacting with the PDZ domain of S-SCAM. In addition, tamalin forms a complex with PSD-95 and Mint2/X11beta/X11L by mechanisms different from the PDZ-mediated interaction. Tamalin has the ability to assemble with these proteins in vivo; their protein complex with tamalin was verified by coimmunoprecipitation of rat brain lysates. Interestingly, the distinct protein-interacting domains of tamalin are evolutionarily conserved, and mRNA expression is developmentally up-regulated at the postnatal period. The results indicate that tamalin exists as a key element that forms a protein complex with multiple postsynaptic and protein-trafficking scaffold proteins.     

Characterization of signaling pathway for the translocation of neuronal nitric oxide synthase to the plasma membrane by PACAP

In the central nervous system, the activation of neuronal nitric oxide synthase (nNOS) is closely associated with activation of NMDA receptor, and trafficking of nNOS may be a prerequisite for efficient NO production at synapses. We recently demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) and NMDA synergistically caused the translocation of nNOS to the membrane and stimulated NO production in PC12 (pheochromocytoma) cells. However, the mechanisms responsible for trafficking and activation of nNOS are largely unknown. To address these issues, here we constructed a yellow fluorescent protein (YFP)-tagged nNOS N-terminal (1–299 a.a.) mutant, nNOSNT-YFP, and visualized its translocation in PC12 cells stably expressing it. PACAP enhanced the translocation synergistically with NMDA in a time- and concentration-dependent manner. The translocation was blocked by inhibitors of protein kinase A (PKA), protein kinase C (PKC), and Src kinase; and the effect of PACAP could be replaced with PKA and PKC activators. The β-finger region in the PSD-95/disc large/zonula occludens-1 domain of nNOS was required for the translocation of nNOS and its interaction with post-synaptic density-95 (PSD-95), and NO formation was attenuated by dominant negative nNOSNT-YFP. These results demonstrate that PACAP stimulated nNOS translocation mediated by PKA and PKC via PAC₁-receptor (a PACAP receptor) and suggest cross-talk between PACAP and NMDA for nNOS activation by Src-dependent phosphorylation of NMDA receptors.