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 NH 2 -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.Effective neurotransmission requires the precise localization of the receptors for neurotransmitters opposite to the presynaptic active zone. The postsynaptic membrane has a dense thickening of submembranous cytoskeleton, called postsynaptic density (PSD) 1 (for review, see Refs. 1 and 2). PSDs play essential roles in the localization of the receptors. Recent studies have identified several molecules that may function in the accumulation and clustering of various receptors at PSDs. PSD-95/SAP90 and its isoforms interact with NMDA receptors and Shaker type potassium channels (3-10), whereas glutamate receptor-interacting protein and Homer interact with ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid and metabotropic glutamate receptors, respectively (11, 12). The common feature for these molecules is that they have a modular PDZ domain for protein-protein interactions (for review, see Refs. 13-16).PDZ domains were originally recognized as the repeats of about 90 amino acids in PSD-95/SAP90 and named after three proteins containing such repeats, PSD-95/SAP90, Drosophila discs-large tumor suppressor protein (Dlg-A), and a tight junction protein, 4,[17][18][19]. The PDZ domains bind to COOH-terminal sequences of various proteins. The first and second PDZ domains of PSD-95/SAP90 interact with NMDA receptors and Shaker type potassium channels (5, 6). PSD-95/ SAP90 is multimerized through the NH 2 -terminal disulfide linkage and clusters these receptors and channels (20). The third PDZ domain of PSD-95/SAP90 binds to the cytoplasmic domain of neuronal cell adhesion molecules, called neuroligins (21).PSD-95/SAP90 has one SH3 domain and one guanylate kinase (GK) domain in addition to the PDZ domains. The SH3 domain was defined as a signaling module in Src ty...

Membrane-associated guanylate kinase (MAGI)-1/BAIassociated protein (BAP) 1 and Synapse-associated protein (SAP) 97/human Discs-large tumor suppressor gene (hDLG) are ubiquitous isoforms of synaptic scaolding molecule (S-SCAM) and Postsynaptic density (PSD)-95/SAP90, both of which are implicated in the structures of synapses, respectively. SAP97/hDLG is localized at epithelial junctions and may function as a scaolding protein, but the subcellular localization or the function of MAGI-1/BAP1 has not been clari®ed. In intestinal epithelial cells, MAGI-1/BAP1 was localized at tight junctions, whereas SAP97/hDLG was localized diusely at cell ± cell junctions. In Madine Darby canine kidney (MDCK) cells, MAGI-1/BAP1 was colocalized with ZO-1, whereas SAP97/hDLG was colocalized with E-cadherin. In MDCK cells, dominant active and negative mutants of Rac1 small G protein changed the amounts of SAP97/hDLG at cell ± cell junctions, but not that of MAGI-1/BAP1. When MDCK cells were switched to a low Ca 2+ medium, E-cadherin disappeared from the plasma membrane, and cells were dissociated. The phorbol 12-myristate 13-acetate-treatment after the low Ca 2+ switch induced a tight junction-like structure. MAGI-1/BAP1 was recruited with ZO-1 to this structure, but SAP97/hDLG or E-cadherin was not. These ®ndings suggest that MAGI-1/BAP1 is a component of tight junctions of epithelial cells, and that its role is dierent from that of SAP97/hDLG.

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 ␣ 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 domainbinding 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.Synaptic junctions are interneuronal cell-cell junctions differentiated for neurotransmission. Neurotransmitters are released from the synaptic vesicles into the synaptic cleft and bind to the receptors accumulated at the postsynapse, opening the ion channels and generating the second messengers involved in synaptic plasticity (reviewed in Ref. 1). The components required for this process are organized at the synaptic junctions to play specific roles in felicitous orders. Several scaffolding proteins are reported to be involved in the assembly of components of synaptic junctions (reviewed in Refs. 2-6). Postsynaptic density (PSD) 1 -95/synapse-associated protein (SAP) 90 has three PSD-95/Dlg-A/ZO-1 (PDZ) domain, one Src homology 3 domain, and one guanylate kinase (GK) domain (7,8). The PDZ domain is a protein-interacting module (reviewed in Refs. 9 -12), and PSD-95/SAP90 binds the C termini of N-methyl-D-aspartate (NMDA) receptors, K ϩ channels, neuroligins, synGAP, and CRIPT through distinct PDZ domains to assemble these components at the synaptic junctions (13-18). PSD-95/SAP90 interacts with SAP90/PSD-95-associated protein (SAPAP) (also called GK-associated protein and hDLGassociated protein) (19 -21), and a recently identified protein, BEGAIN (brain-enriched guanylate kinase-interacting protein) via the GK domain (22). Glutamate receptor-interacting protein has seven PDZ domains and binds ␣ amino-3-hydroxy-5-methyl-4-isoxazaole propionic acid receptors via the fourth and fifth PDZ domains (23). The ligands for other PDZ domains of glutamate receptor-interacting protein have not been so far reported. Synaptic scaffolding molecule (S-SCAM) was originally identified as a SAPAP-interacting protein (24). We have first reported that S-SCAM has one GK, two WW, and five PDZ domains (24). The GK domain of S-SCAM is shorter than that of PSD-95/SAP90. The WW domain is a protein-interacting modul...

The postsynaptic density (PSD) 1 is a submembranous structure at the postsynaptic membrane mainly at the excitatory synapses (for review, see Refs. 1-3). The neurotransmitter receptors are assembled and fixed at the PSD, and several molecules implicated in the synaptic plasticity are also enriched. PSD-95/synapse-associated protein (SAP) 90 is involved in the molecular organization of these components of the PSD (Refs. 4 and 5; for review, see Refs. 6 -11) and essential for learning and memory (12). PSD-95/SAP90 is a member of membrane-associated guanylate kinases (for review, see Ref. 13), and binds to guanylate kinase-associated protein (GKAP)/ SAP90/PSD-95-associated protein (SAPAP)/DLG-associated protein (DAP) and brain-enriched guanylate kinase-interacting protein (BEGAIN) via the guanylate kinase domain (14 -17). In this paper, we have described GKAP/SAPAP/DAP as SAPAP just for simplicity. SAPAP is Triton X-100-insoluble in both the brain homogenate and the transfected cells. It links PSD-95/ SAP90 to the Triton X-100-insoluble structures of transfected Chinese hamster ovary cells (17). BEGAIN is also recruited by SAPAP into the Triton X-100-insoluble fraction with PSD-95/ SAP90 in Chinese hamster ovary cells (17). SAPAP interacts with another neuronal membrane-associated guanylate kinase, synaptic scaffolding molecule (S-SCAM), which also provides scaffolds for the components of synaptic junctions (18). Thereby, SAPAP is a key protein in the architecture of the PSD. The detergent-insolubility of SAPAP is conferred by its NH 2 -terminal and middle regions. The middle region contains 5 repeats of 14 amino acids (aa) and is involved in the interaction with PSD-95/SAP90 (14 -16). A proline-rich region that fits to the consensus motif for the binding of the src homology 3 (SH3) domain exists immediately after the middle region (19). The carboxyl-terminal region is Triton X-100-soluble and well conserved among the four isoforms of SAPAP, suggesting that it may have some function. We have searched for a SAPAPbinding protein, identified a novel neuronal protein, which binds to the carboxyl-terminal region of SAPAP, and named it synamon. MATERIALS AND METHODSYeast Two-hybrid Screening and cDNA Cloning-Rat brain yeast two-hybrid library was constructed using pVP16 vector and screened (20). Rat brain cDNA libraries (Stratagene and CLONTECH) were screened with the [␣-32 P]dCTP-labeled random-primed probes (20). Construction of Expression Vectors-Various expression vectors were constructed by conventional molecular biology techniques and polymerase chain reaction method using pBTM116, pCMV Myc, pClneo Myc, and pGex4T-1 (Amersham Pharmacia Biotech). pBTM116 SAPAP2-1 contains full-length SAPAP2. The following constructs contain the following aa residues of SAPAP1: pCMV Myc SAPAP1-1, 1-992; pCMV Myc SAPAP1-2, 1-477; pCMV Myc SAPAP1-6, 478 -992; pCMV Myc SAPAP1-8, 568 -992; pClneo Myc SAPAP1-8, 800 -992; and pClneo Myc SAPAP1-22, 800 -985. The following constructs contain the following aa residues of synamon: pGex4T-1 s...

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.Synaptic junctions accumulate molecules involved in neurotransmissions. The organization of components of synaptic junctions is orchestrated by several scaffolding proteins (reviewed in Refs. 1-5). PSD-95/SAP90 is a prototypic synaptic scaffolding protein and has three isoforms, SAP97, PSD-93/ chapsyn, and SAP102 (6 -12). They have three PDZ, one SH3, and one guanylate kinase (GK) 1 domain. The PDZ domain is a protein-interacting module (reviewed in Refs. 13-16), and those of PSD-95/SAP90 bind to the C termini of NMDA receptors, K ϩ channels, neuroligins, synGAP, and CRIPT (8 -12, 17-21). The SH3 domain is also a protein-interacting module (reviewed in Ref. 22), but the molecules interacting with the SH3 domain of PSD-95/SAP90 have not been identified. The GK domain shows a homology to the yeast guanylate kinase. Three groups including ours have recently identified a protein interacting with the GK domain of PSD-95/SAP90 and named it GKAP, SAPAP, and DAP, respectively (23-25). SAPAP is neuronal-specific and tightly attached to the synaptic plasma membranes, and in the transfected cells, SAPAP recruits PSD-95/SAP90 to the plasma membranes. We have subsequently identified a protein interacting with SAPAP and named it S-SCAM (26). S-SCAM has five PDZ domains and binds to NMDA receptors and neuroligins. PSD-95/SAP90, SAPAP, and S-SCAM may cooperate to assemble receptors and cell adhesion molecules at the synaptic junctions.In the yeast two-hybrid screening using the GK domain of PSD-95/SAP90 as bait, we have identified two novel proteins in addition to SAPAP. One of them is ubiquitously expressed, and the other is specifically expressed in brain. In this study, we have characterized this brain-specific protein and named it BEGAIN (brain-enriched guanylate-kinase domain-associated protein). BEGAIN is expressed in neurons and rather enriched at synaptic junctions. BEGAIN may be involved also in the organization of the components of synaptic junctions. MATERIALS AND METHODSYeast Two-hybrid Screening and cDNA Cloning-Rat brain yeast two-hybrid library was constructed ...

No abstract

No abstract

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 identi®ed 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.