BACKGROUND:Synapse-associated protein (SAP) 90/Postsynaptic density (PSD)-95-associated protein (SAPAP) (also called Guanylate kinase-associated protein/hDLG-associated protein) interacts with the guanylate kinase domains of PSD-95 and synaptic scaffolding molecule (S-SCAM) via the middle region containing 5 repeats of 14 amino acids. SAPAP also binds the recently identified proteins, nArgBP2 and synamon (also called Shank 1a), via the proline-rich region and the C-terminus, respectively. SAPAP is highly enriched in the Triton X-100-insoluble PSD fraction, and recruits PSD-95 into the Triton X-100-insoluble fraction in transfected cells. We have further characterized here the Triton X-100-insolubility of SAPAP and tried to identify the Triton X-100-insoluble structures which SAPAP interacts with. RESULTS:N-Methyl-D-aspartate receptors were recruited into the Triton X-100-insoluble fraction with PSD-95 by SAPAP. The N-terminal region of SAPAP was Triton X-100-insoluble, whereas the middle and C-terminal regions were Triton X-100-soluble. We identified proteins interacting with 35S-methionine-labelled SAPAP in the overlay assay, determined their amino acid sequences, and found them to be neurofilaments. SAPAP interacted with neurofilaments via the N-terminal region, was co-immunoprecipitated with neurofilaments from the rat brain, and co-localized with neurofilaments in transfected cells. CONCLUSION:SAPAP associates with neurofilaments via the N-terminal region and may link various components of the PSD to neurofilaments.

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 withN-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-SCAMα, the middle one, S-SCAMβ, and the shortest one, S-SCAM-γ. S-SCAMβ and -γ have five PDZ (PDZ-1 to -5), two WW, and one GK domains. S-SCAMα interacted with S-SCAMβ and -γ 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-SCAMβ and -γ 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.

A neural plakophilin-related armadillo repeat protein (NPRAP)/delta-catenin interacts with one of Alzheimer disease-related gene products, presenilin 1. We have previously reported the interaction of NPRAP/delta-catenin with synaptic scaffolding molecule, which is involved in the assembly of synaptic components. NPRAP/delta-catenin also interacts with E-cadherin and beta-catenin and is implicated in the organization of cell-cell junctions. p0071, a ubiquitous isoform of NPRAP/delta-catenin, is localized at desmosomes in HeLa and A431 cells and at adherens junctions in Madin-Darby bovine kidney cells. We have identified here a novel protein interacting with NPRAP/delta-catenin and p0071 and named this protein plakophilin-related armadillo repeat protein-interacting PSD-95/Dlg-A/ZO-1 (PDZ) protein (PAPIN). PAPIN has six PDZ domains and binds to NPRAP/delta-catenin and p0071 via the second PDZ domain. PAPIN and p0071 are ubiquitously expressed in various tissues and are localized at cell-cell junctions in normal rat kidney cells and bronchial epithelial cells. PAPIN may be a scaffolding protein connecting components of epithelial junctions with p0071.

Synaptic scaffolding molecule (S-SCAM) is a multiple PDZ domain-containing protein, which interacts with neuroligin, a cell adhesion molecule, and the NMDA receptor. In this study, we searched for S-SCAM-interacting proteins and obtained a neuralplakophilin-related armadillo-repeat protein (NPRAP)/delta-catenin. NPRAP/delta-catenin bound to the last PDZ domain of S-SCAM via its carboxyl-terminus in three different cell-free assay systems, was coimmunoprecipitated with S-SCAM from rat crude synaptosomes, and was localized at the excitatory synapses in rat hippocampal neurons. NPRAP/delta-catenin may be implicated in the molecular organization of synaptic junctions through the interaction with S-SCAM.

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.

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 α motif, one PDZ, and one plekstrin homology domain. MAGUIN-1 is localized at the plasma membranevia 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.

PSD-95/SAP90 is a synaptic membrane-associated guanylate kinase with three PDZ, one SH3, and one guanylate kinase (GK) domain. PSD-95/SAP90 binds various proteins through the PDZ domains and organizes synaptic junctions. PSD-95/SAP90 also interacts with the postsynaptic density (PSD) fraction-enriched protein, named SAPAP (also called GKAP and DAP), through the GK domain. SAPAP is Triton X-100-insoluble and recruits PSD-95/SAP90 into the Triton X-100-insoluble fraction in the transfected cells, suggesting that SAPAP may fix PSD-95/SAP90 to the PSD. Here we report a novel protein interacting with the GK domain of PSD-95/SAP90, BEGAIN. BEGAIN is specifically expressed in brain and enriched in the PSD fraction. BEGAIN is Triton X-100-soluble in the transfected cells but is recruited to the Triton X-100-insoluble fraction by SAPAP when coexpressed with PSD-95/SAP90. BEGAIN may be a novel PSD component associated with the core complex of PSD-95/SAP90 and SAPAP.

InCaenorhabditis elegans,the vulval induction is mediated by tyrosine kinase receptor/Ras signal transduction pathway composed of thelin-3, let-23,andlet-60products. In addition to these gene products, thelin-2, lin-7,andlin-10products are also implicated in this pathway.Lin-2encodes a MAGUK andlin-7encodes a small protein with one PDZ domain. Thelin-10product has no homology to known proteins. Here, we have cloned a rat homologue of thelin-10product and characterized it. Rat lin-10 is ubiquitously expressed in various rat tissues and distributed in both the cytosol and membrane fractions. In brain, however, rat lin-10 is distributed only in the membrane fraction and enriched in the synaptic plasma membrane and postsynaptic density fractions. These results suggest that rat lin-10 is involved at least in synaptic functions in brain.

We have recently isolated a rat homologue of the Caenorrhabditis elegans lin-10 product. Although rat lin-10 is expressed in the cytosol and membrane fractions of various tissues, it is distributed only in the membrane fraction in brain where it is enriched in the synaptic plasma membrane and postsynaptic density fractions. We have isolated here a rat lin-10-interacting protein from rat brain and identified it to be neurabin-II/spinophilin, which has recently been isolated as a protein interacting with protein phosphatase I and F-actin. Neurabin-II/spinophilin is ubiquitously expressed but enriched in brain, especially in the synaptic plasma membrane and postsynaptic density fractions. We discuss the physiological significance of the interaction of rat lin-10 with neurabin-II/spinophilin.

At synaptic junctions, pre- and postsynaptic membranes are connected by cell adhesion and have distinct structures for specialized functions. The presynaptic membranes have a machinery for fast neurotransmitter release, and the postsynaptic membranes have clusters of neurotransmitter receptors. The molecular mechanism of the assembly of synaptic junctions is not yet clear. Pioneering studies identified postsynaptic density (PSD)-95/SAP90 as a prototypic synaptic scaffolding protein to maintain the structure of synaptic junctions. PSD-95/SAP90 belongs to a family of membrane-associated guanylate kinases and binds N-methyl-D-aspartate receptors, potassium channels, and neuroligins through the PDZ domains and GKAP/SAPAP/DAP through the guanylate kinase (GK) domain. We performed here a yeast two-hybrid screening for SAPAP-interacting molecules and identified a novel protein that has an inverse structure of membrane-associated guanylate kinases with an NH2-terminal GK-like domain followed by two WW and five PDZ domains. It binds SAPAP through the GK-like domain and NMDA receptors and neuroligins through the PDZ domains. We named this protein S-SCAM (synaptic scaffolding molecule) because S-SCAM may assemble receptors and cell adhesion proteins at synaptic junctions.