Formation of complexes between Ca2+.calmodulin and the synapse-associated protein SAP97 requires the SH3 domain-guanylate kinase domain-connecting HOOK region

Mammalian synapse-associated protein SAP97, a structural and functional homolog of Drosophila Dlg, is a membrane-associated guanylate kinase (MAGUK) that is present at pre- and postsynaptic sites as well as in epithelial cell-cell contact sites. It is a multidomain scaffolding protein that shares with other members of the MAGUK protein family a characteristic modular organization composed of three sequential protein interaction motifs known as PDZ domains, followed by an Src homology 3 (SH3) domain, and an enzymatically inactive guanylate kinase (GK)-like domain. Specific binding partners are known for each domain, and different modes of intramolecular interactions have been proposed that particularly involve the SH3 and GK domains and the so-called HOOK region located between these two domains. We identified the HOOK region as a specific site for calmodulin binding and studied the dynamics of complex formation of recombinant calmodulin and SAP97 by surface plasmon resonance spectroscopy. Binding of various SAP97 deletion constructs to immobilized calmodulin was strictly calcium-dependent. From the rate constants of association and dissociation we determined an equilibrium dissociation constant K(d) of 122 nm for the association of calcium-saturated calmodulin and a SAP97 fragment, which encompassed the entire SH3-HOOK-GK module. Comparative structure-based sequence analysis of calmodulin binding regions from various target proteins predicts variable affinities for the interaction of calmodulin with members of the MAGUK protein family. Our findings suggest that calmodulin could regulate the intramolecular interaction between the SH3, HOOK, and GK domains of SAP97.     

Intramolecular interactions regulate SAP97 binding to GKAP

Membrane-associated guanylate kinase homologs (MAGUKs) are multidomain proteins found to be central organizers of cellular junctions. In this study, we examined the molecular mechanisms that regulate the interaction of the MAGUK SAP97 with its GUK domain binding partner GKAP (GUK-associated protein). The GKAP-GUK interaction is regulated by a series of intramolecular interactions. Specifically, the association of the Src homology 3 (SH3) domain and sequences situated between the SH3 and GUK domains with the GUK domain was found to interfere with GKAP binding. In contrast, N-terminal sequences that precede the first PDZ domain in SAP97, facilitated GKAP binding via its association with the SH3 domain. Utilizing crystal structure data available for PDZ, SH3 and GUK domains, molecular models of SAP97 were generated. These models revealed that SAP97 can exist in a compact U-shaped conformation in which the N-terminal domain folds back and interacts with the SH3 and GUK domains. These models support the biochemical data and provide new insights into how intramolecular interactions may regulate the association of SAP97 with its binding partners.     

Identification of multiple binding partners for the amino-terminal domain of synapse-associated protein 97

Multiprotein complexes mediate static and dynamic functions to establish and maintain cell polarity in both epithelial cells and neurons. Membrane-associated guanylate kinase (MAGUK) proteins are thought to be scaffolding molecules in these processes and bind multiple proteins via their obligate postsynaptic density (PSD)-95/Disc Large/Zona Occludens-1, Src homology 3, and guanylate kinase-like domains. Subsets of MAGUK proteins have additional protein-protein interaction domains. An additional domain we identified in SAP97 called the MAGUK recruitment (MRE) domain binds the LIN-2,7 amino-terminal (L27N) domain of mLIN-2/CASK, a MAGUK known to bind mLIN-7. Here we show that SAP97 binds two other mLIN-7 binding MAGUK proteins. One of these MAGUK proteins, DLG3, coimmunoprecipitates with SAP97 in lysates from rat brain and transfected Madin-Darby canine kidney cells. This interaction requires the MRE domain of SAP97 and surprisingly, both the L27N and L27 carboxyl-terminal (L27C) domains of DLG3. We also demonstrate that SAP97 can interact with the MAGUK protein, DLG2, but not the highly related protein, PALS2. The ability of SAP97 to interact with multiple MAGUK proteins is likely to be important for the targeting of specific protein complexes in polarized cells.     

MAGUKs: beyond ionic channel anchoring

A family of anchoring proteins named MAGUK (for membrane associated guanylate kinase) has emerged as a key element in the organization of protein complexes in specialized membrane regions. These proteins are characterized by the presence of multipe protein-protein interaction domains including PDZ and SH3 domains. The MAGUK family comprises the post-synaptic density 95 (PSD-95) protein and closely related molecules such as chapsyn-110, synapse-associated protein 102 (SAP-102), and SAP-97. These are located either on the pre- and/or post-synaptic sides of synapses or at cell-cell adhesion sites of epithelial cells. MAGUK proteins interact with glutamate receptors and various ionic channels. For instance, an interaction has been reported between the first two PDZ domains of MAGUK proteins and several channels via a consensus sequence Thr/Ser-X-Val/Leu usually located at their carboxy terminus. The role of these anchoring proteins in channel function is not fully understood. MAGUK proteins enhance the current density by increasing the number of functional channels to the sarcolemma. They can also facilitate signaling between channels and several enzymes or G protein-dependent signaling pathways. In the heart also, MAGUK proteins are abundantly expressed and they interact with various channels including Shaker Kv1.5 and connexins.     

Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules

Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tissue developments, cell-cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood. Here we show that the GK domain of DLG1/SAP97 binds to asymmetric cell division regulatory protein LGN in a phosphorylation-dependent manner. The structure of the DLG1 SH3-GK tandem in complex with a phospho-LGN peptide reveals that the GMP-binding site of GK has evolved into a specific pSer/pThr-binding pocket. Residues both N- and C-terminal to the pSer are also critical for the specific binding of the phospho-LGN peptide to GK. We further demonstrate that the previously reported GK domain-mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/SAPAP1 and SPAR, are also phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs also function as phospho-peptide-binding modules. The discovery of the phosphorylation-dependent MAGUK GK/target interactions indicates that MAGUK scaffold-mediated signalling complex organizations are dynamically regulated.     

Synapse associated protein 102 (SAP102) binds the C-terminal part of the scaffolding protein neurobeachin

Neurobeachin (Nbea) is a multidomain scaffold protein abundant in the brain, where it is highly expressed during development. Nbea-null mice have severe defects in neuromuscular synaptic transmission resulting in lethal paralysis of the newborns. Recently, it became clear that Nbea is important also for the functioning of central synapses, where it is suggested to play a role in trafficking membrane proteins to both, the pre- and post-synaptic sites. So far, only few binding partners of Nbea have been found and the precise mechanism of their trafficking remains unclear. Here, we used mass spectrometry to identify SAP102, a MAGUK protein implicated in trafficking of the ionotropic glutamate AMPA- and NMDA-type receptors during synaptogenesis, as a novel Nbea interacting protein in mouse brain. Experiments in heterologous cells confirmed this interaction and revealed that SAP102 binds to the C-terminal part of Nbea that contains the DUF, PH, BEACH and WD40 domains. Furthermore, we discovered that introducing a mutation in Nbea's PH domain, which disrupts its interaction with the BEACH domain, abolishes this binding, thereby creating an excellent starting point to further investigate Nbea-SAP102 function in the central nervous system.     

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 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.     

Structural requirements for calmodulin binding to membrane-associated guanylate kinase homologs

Effector molecules such as calmodulin modulate the interactions of membrane-associated guanylate kinase homologs (MAGUKs) and other scaffolding proteins of the membrane cytoskeleton by binding to the Src homology 3 (SH3) domain, the guanylate kinase (GK) domain, or the connecting HOOK region of MAGUKs. Using surface plasmon resonance, we studied the interaction of members of all four MAGUK subfamilies--synapse-associated protein 97 (SAP97), calcium/calmodulin-dependent serine protein kinase (CASK), membrane palmitoylated protein 2 (MPP2), and zona occludens (ZO) 1--and calmodulin to determine interaction affinities and localize the binding site. The SH3-GK domains of the proteins and derivatives thereof were expressed in E. coli and purified. In all four proteins, high-affinity calmodulin binding was identified. CASK was shown to contain a Ca2+-dependent calmodulin binding site within the HOOK region, overlapping with a protein 4.1 binding site. In ZO1, a Ca2+-dependent calmodulin binding site was detected within the GK domain. The equilibrium dissociation constants for MAGUK-calmodulin interaction were found to range from 50 nM to 180 nM. Sequence analyses suggest that binding sites for calmodulin have evolved independently in at least three subfamilies. For ZO1, pulldown of GST-calmodulin was shown to occur in a calcium-dependent manner; moreover, molecular modeling and sequence analyses predict conserved basic residues to be exposed on one side of a helix. Thus, calmodulin binding appears to be a common feature of MAGUKs, and Ca2+-activated calmodulin may serve as a general regulator to affect the interactions of MAGUKs and various components of the cytoskeleton.     

hCASK and hDlg associate in epithelia, and their src homology 3 and guanylate kinase domains participate in both intramolecular and intermolecular interactions

Membrane-associated guanylate kinase (MAGUK) proteins act as molecular scaffolds organizing multiprotein complexes at specialized regions of the plasma membrane. All MAGUKs contain a Src homology 3 (SH3) domain and a region homologous to yeast guanylate kinase (GUK). We showed previously that one MAGUK protein, human CASK (hCASK), is widely expressed and associated with epithelial basolateral plasma membranes. We now report that hCASK binds another MAGUK, human discs large (hDlg). Immunofluorescence microscopy demonstrates that hCASK and hDlg colocalize at basolateral membranes of epithelial cells in small and large intestine. These proteins co-precipitate from lysates of an intestinal cell line, Caco-2. The GUK domain of hCASK binds the SH3 domain of hDlg in both yeast two-hybrid and fusion protein binding assays, and it is required for interaction with hDlg in transfected HEK293 cells. In addition, the SH3 and GUK domains of each protein participate in intramolecular binding that in vitro predominates over intermolecular binding. The SH3 and GUK domains of human p55 display the same interactions in yeast two-hybrid assays as those of hCASK. Not all SH3-GUK interactions among these MAGUKs are permissible, however, implying specificity to SH3-GUK interactions in vivo. These results suggest MAGUK scaffold assembly may be regulated through effects on intramolecular SH3-GUK binding.     

Subunit-specific Regulation of N-Methyl-d-aspartate (NMDA) Receptor Trafficking by SAP102 Protein Splice Variants

Synapse-associated protein 102 (SAP102) is a scaffolding protein abundantly expressed early in development that mediates glutamate receptor trafficking during synaptogenesis. Mutations in human SAP102 have been reported to cause intellectual disability, which is consistent with its important role during early postnatal development. SAP102 contains PDZ, SH3, and guanylate kinase (GK)-like domains, which mediate specific protein-protein interactions. SAP102 binds directly to N-methyl-d-aspartate receptors (NMDARs), anchors receptors at synapses, and facilitates transduction of NMDAR signals. Proper localization of SAP102 at the postsynaptic density is essential to these functions. However, how SAP102 is targeted to synapses is unclear. In the current study we find that synaptic localization of SAP102 is regulated by alternative splicing. The SAP102 splice variant that possesses a C-terminal insert (I2) between the SH3 and GK domains is highly enriched at dendritic spines. We also show that there is an intramolecular interaction between the SH3 and GK domains in SAP102 but that the I2 splicing does not influence SH3-GK interaction. Previously, we have shown that SAP102 expression promotes spine lengthening. We now find that the spine lengthening effect is independent of the C-terminal alternative splicing of SAP102. In addition, expression of I2-containing SAP102 isoforms is regulated developmentally. Knockdown of endogenous I2-containing SAP102 isoforms differentially affect NMDAR surface expression in a subunit-specific manner. These data shed new light on the role of SAP102 in the regulation of NMDAR trafficking.     

Targeting of PKA to glutamate receptors through a MAGUK-AKAP complex

Compartmentalization of glutamate receptors with the signaling enzymes that regulate their activity supports synaptic transmission. Two classes of binding proteins organize these complexes: the MAGUK proteins that cluster glutamate receptors and AKAPs that anchor kinases and phosphatases. In this report, we demonstrate that glutamate receptors and PKA are recruited into a macromolecular signaling complex through direct interaction between the MAGUK proteins, PSD-95 and SAP97, and AKAP79/150. The SH3 and GK regions of the MAGUKs mediate binding to the AKAP. Cell-based studies indicate that phosphorylation of AMPA receptors is enhanced by a SAP97-AKAP79 complex that directs PKA to GluR1 via a PDZ domain interaction. As AMPA receptor phosphorylation is implicated in regulating synaptic plasticity, these data suggest that a MAGUK-AKAP complex may be centrally involved.     

Plasma membrane Ca2+-atpase isoforms 2b and 4b interact promiscuously and selectively with members of the membrane-associated guanylate kinase family of PDZ (PSD95/Dlg/ZO-1) domain-containing proteins

Spatial and temporal regulation of intracellular Ca(2+) signaling depends on localized Ca(2+) microdomains containing the requisite molecular components for Ca(2+) influx, efflux, and signal transmission. Plasma membrane Ca(2+)-ATPase (PMCA) isoforms of the "b" splice type contain predicted PDZ (PSD95/Dlg/ZO-1) interaction domains. The COOH-terminal tail of PMCA2b isolated the membrane-associated guanylate kinase (MAGUK) protein SAP97/hDlg as a binding partner in a yeast two-hybrid screen. The related MAGUKs SAP90/PSD95, PSD93/chapsyn-110, SAP97, and SAP102 all bound to the COOH-terminal tail of PMCA4b, whereas only the first three bound to the tail of PMCA2b. Coimmunoprecipitations confirmed the interaction selectivity between PMCA4b and SAP102 as opposed to the promiscuity of PMCA2b and 4b in interacting with other SAPs. Confocal immunofluorescence microscopy revealed the exclusive presence and colocalization of PMCA4b and SAP97 in the basolateral membrane of polarized Madin-Darby canine kidney epithelial cells. In hippocampal neurons, PMCA2b was abundant throughout the somatodendritic compartment and often extended into the neck and head of individual spines where it colocalized with SAP90/PSD95. These data show that PMCA "b" splice forms interact promiscuously but also with specificity with different members of the PSD95 family of SAPs. PMCA-SAP interactions may play a role in the recruitment and maintenance of the PMCA at specific membrane domains involved in local Ca(2+) regulation.     

A novel and conserved protein-protein interaction domain of mammalian Lin-2/CASK binds and recruits SAP97 to the lateral surface of epithelia

Mammalian Lin-2 (mLin-2)/CASK is a membrane-associated guanylate kinase (MAGUK) and contains multidomain modules that mediate protein-protein interactions important for the establishment and maintenance of neuronal and epithelial cell polarization. The importance of mLin-2/CASK in mammalian development is demonstrated by the fact that mutations in mLin-2/CASK or SAP97, another MAGUK protein, lead to cleft palate in mice. We recently identified a new protein-protein interaction domain, called the L27 domain, which is present twice in mLin-2/CASK. In this report, we further define the binding of the L27C domain of mLin-2/CASK to the L27 domain of mLin-7 and identify the binding partner for L27N of mLin-2/CASK. Biochemical analysis reveals that this L27N domain binds to the N terminus of SAP97, a region that was previously reported to be essential for the lateral membrane recruitment of SAP97 in epithelia. Our colocalization studies, using dominant-negative mLin-2/CASK, show that the association with mLin-2/CASK is crucial for lateral localization of SAP97 in MDCK cells. We also report the identification of a novel isoform of Discs Large, a Drosophila melanogaster orthologue of SAP97, which contains a region highly related to the SAP97 N terminus and which binds Camguk, a Drosophila orthologue of mLin-2/CASK. Our data identify evolutionarily conserved protein-protein interaction domains that link mLin-2/CASK to SAP97 and account for their common phenotype when mutated in mice.     

Dual role of CaMKII-dependent SAP97 phosphorylation in mediating trafficking and insertion of NMDA receptor subunit NR2A

Synapse Associated Protein 97 (SAP97), a member of membrane-associated guanylate kinase (MAGUK) protein family, has been involved in the correct targeting and clustering of ionotropic glutamate receptors (iGluRs) at postsynaptic sites. Calcium/calmodulin kinase II (CaMKII) phosphorylates SAP97 on two major sites in vivo; one located in the N-terminal domain (Ser39) and the other in the first postsynaptic density disc large ZO1 (PDZ) domain (Ser232). CaMKII-mediated phosphorylation of SAP97-Ser39 is necessary and sufficient to drive SAP97 to the postsynaptic compartment in cultured hippocampal neurons. CaMKII-dependent phosphorylation of Ser232 disrupts SAP97 interaction with NR2A subunit, thereby regulating synaptic targeting of this NMDA receptor subunit. Here we show by means of phospho-specific antibodies that SAP97-Ser39 phosphorylation represents the driving force to release SAP97/NR2A complex from the endoplasmic reticulum. Ser39 phosphorylation does not interfere with SAP97 capability to bind NR2A. On the contrary, SAP97-Ser232 phosphorylation occurs within the postsynaptic compartment and is responsible for both the disruption of NR2A/SAP97 complex and, consequently, for NR2A insertion in the postsynaptic membrane. Thus, CaMKII-dependent phosphorylation of SAP97 in different time frames and locations within the neurons controls both NR2A trafficking and insertion.     

Protracted and asynchronous accumulation of PSD95‐family MAGUKs during maturation of nascent dendritic spines

The formation and stabilization of new dendritic spines is a key component of the experience‐dependent neural circuit plasticity that supports learning, but the molecular maturation of nascent spines remains largely unexplored. The PSD95‐family of membrane‐associated guanylate kinases (PSD‐MAGUKs), most notably PSD95, has a demonstrated role in promoting spine stability. However, nascent spines contain low levels of PSD95, suggesting that other members of the PSD‐MAGUK family might act to stabilize nascent spines in the early stages of spiny synapse formation. Here, we used GFP‐fusion constructs to quantitatively define the molecular composition of new spines, focusing on the PSD‐MAGUK family. We found that PSD95 levels in new spines were as low as those previously associated with rapid subsequent spine elimination, and new spines did not achieve mature levels of PSD95 until between 12 and 20 h following new spine identification. Surprisingly, we found that the PSD‐MAGUKs PSD93, SAP97, and SAP102 were also substantially less enriched in new spines. However, they accumulated in new spines more quickly than PSD95: SAP102 enriched to mature levels within 3 h, SAP97 and PSD93 enriched gradually over the course of 6 h. Intriguingly, when we restricted our analysis to only those new spines that persisted, SAP97 was the only PSD‐MAGUK already present at mature levels in persistent new spines when first identified. Our findings uncover a key structural difference between nascent and mature spines, and suggest a mechanism for the stabilization of nascent spines through the sequential arrival of PSD‐MAGUKs. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1161–1174, 2017     

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.     

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.     

ZO-3, a Novel Member of the MAGUK Protein Family Found at the Tight Junction, Interacts with ZO-1 and Occludin

A 130-kD protein that coimmunoprecipitates with the tight junction protein ZO-1 was bulk purified from Madin-Darby canine kidney (MDCK) cells and subjected to partial endopeptidase digestion and amino acid sequencing. A resulting 19–amino acid sequence provided the basis for screening canine cDNA libraries. Five overlapping clones contained a single open reading frame of 2,694 bp coding for a protein of 898 amino acids with a predicted molecular mass of 98,414 daltons. Sequence analysis showed that this protein contains three PSD-95/SAP90, discs-large, ZO-1 (PDZ) domains, a src homology (SH3) domain, and a region similar to guanylate kinase, making it homologous to ZO-1, ZO-2, the discs large tumor suppressor gene product of Drosophila, and other members of the MAGUK family of proteins. Like ZO-1 and ZO-2, the novel protein contains a COOH-terminal acidic domain and a basic region between the first and second PDZ domains. Unlike ZO-1 and ZO-2, this protein displays a proline-rich region between PDZ2 and PDZ3 and apparently contains no alternatively spliced domain. MDCK cells stably transfected with an epitope-tagged construct expressed the exogenous polypeptide at an apparent molecular mass of ~130 kD. Moreover, this protein colocalized with ZO-1 at tight junctions by immunofluorescence and immunoelectron microscopy. In vitro affinity analyses demonstrated that recombinant 130-kD protein directly interacts with ZO-1 and the cytoplasmic domain of occludin, but not with ZO-2. We propose that this protein be named ZO-3.     

Identification of an intramolecular interaction between the SH3 and guanylate kinase domains of PSD-95.

Postsynaptic density-95 (PSD-95/SAP-90) is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins that assemble protein complexes at synapses and other cell junctions. MAGUKs comprise multiple protein-protein interaction motifs including PDZ, SH3 and guanylate kinase (GK) domains, and these binding sites mediate the scaffolding function of MAGUK proteins. Synaptic binding partners for the PDZ and GK domains of PSD-95 have been identified, but the role of the SH3 domain remains elusive. We now report that the SH3 domain of PSD-95 mediates a specific interaction with the GK domain. The GK domain lacks a poly-proline motif that typically binds to SH3 domains; instead, SH3/GK binding is a bi-domain interaction that requires both intact motifs. Although isolated SH3 and GK domains can bind in trans, experiments with intact PSD-95 molecules indicate that intramolecular SH3/GK binding dominates and prevents intermolecular associations. SH3/GK binding is conserved in the related Drosophila MAGUK protein DLG but is not detectable for Caenorhabditis elegans LIN-2. Many previously identified genetic mutations of MAGUKs in invertebrates occur in the SH3 or GK domains, and all of these mutations disrupt intramolecular SH3/GK binding.