Crystal structure of the Homer 1 family conserved region reveals the interaction between the EVH1 domain and own proline-rich motif

PSD-Zip45 (also named Homer 1c/Vesl-1L) is a synaptic scaffolding protein, which interacts with neurotransmitter receptors and other scaffolding proteins to target them into post-synaptic density (PSD), a specialized protein complex at the synaptic junction. Binding of the PSD-Zip45 to the receptors and scaffolding proteins results in colocalization and clustering of its binding partners in PSD. It has an Ena/VASP homology 1 (EVH1) domain in the N terminus for receptor binding, two leucine zipper motifs in the C terminus for clustering, and a linking region whose function is unclear despite the high level of conservation within the Homer 1 family. The X-ray crystallographic analysis of the largest fragment of residues 1-163, including an EVH1 domain reported here, demonstrates that the EVH1 domain contains an alpha-helix longer than that of the previous models, and that the linking part included in the conserved region of Homer 1 (CRH1) of the PSD-Zip45 interacts with the EVH1 domain of the neighbour CRH1 molecule in the crystal. The results suggest that the EVH1 domain recognizes the PPXXF motif found in the binding partners, and the SPLTP sequence (P-motif) in the linking region of the CRH1. The two types of binding are partly overlapped in the EVH1 domain, implying a mechanism to regulate multimerization of Homer 1 family proteins.     

Synaptic targeting of the postsynaptic density protein PSD-95 mediated by a tyrosine-based trafficking signal

Synaptic function requires proper localization of proteins at synaptic sites. Targeting of the postsynaptic density protein 95 (PSD-95) relies on multiple signals within the protein, including twelve C-terminal amino acids. We now show that this C-terminal targeting domain of PSD-95 mediates postsynaptic localization through a short tyrosine-based motif followed by a pair of hydrophobic amino acids. Consistent with a role in cellular trafficking, the tyrosine motif resembles the canonical motif for interactions with clathrin adaptor proteins. In fact, we find that the C-terminal targeting domain of PSD-95 is sufficient to mediate clathrin-dependent endocytosis when appended to a transmembrane protein. Furthermore, systematic mutagenesis reveals that endocytosis mediated by this domain depends on both the tyrosine motif and the dihydrophobic amino acid pair. Thus, postsynaptic targeting of PSD-95 requires a tyrosine-based signal that can mediate clathrin-coated vesicle formation.     

Synaptic targeting of the postsynaptic density protein PSD-95 mediated by lipid and protein motifs

During synaptic development, proteins aggregate at specialized pre- and postsynaptic structures. Mechanisms that mediate protein clustering at these sites remain unknown. To investigate this process, we analyzed synaptic targeting of a postsynaptic density protein, PSD-95, by expressing green fluorescent protein- (GFP-) tagged PSD-95 in cultured hippocampal neurons. We find that postsynaptic clustering relies on three elements of PSD-95: N-terminal palmitoylation, the first two PDZ domains, and a C-terminal targeting motif. In contrast, disruptions of PDZ3, SH3, or guanylate kinase (GK) domains do not affect synaptic targeting. Palmitoylation is sufficient to target the diffusely expressed SAP-97 to synapses, and palmitoylation cannot be replaced with alternative membrane association motifs, suggesting that a specialized synaptic lipid environment mediates postsynaptic clustering. The requirements for PDZ domains and a C-terminal domain of PSD-95 indicate that protein-protein interactions cooperate with lipid interactions in synaptic targeting.     

Insulin receptor substrate of 53 kDa links postsynaptic shank to PSD-95

The insulin receptor substrate of 53 kDa (IRSp53) is a target of the small GTPase cdc42 which is strongly enriched in the postsynaptic density of excitatory synapses. IRSp53 interacts with the postsynaptic shank1 scaffolding molecule in a cdc42 regulated manner. The functional significance of the cdc42/IRSp53 pathway in postsynaptic sites is however, unclear. Here we identify PSD-95 as a second synaptic interaction partner of IRSp53. Interaction is mediated by a C-terminal PDZ binding motif in IRSp53 and the second PDZ domain of PSD-95. In HEK cells, overexpressed IRSp53 induces filopodia and targets PSD-95 into these processes. Immunoprecipitation and immunocytochemistry experiments demonstrate that the interaction occurs at postsynaptic sites in the brain. By virtue of its PDZ-binding and SH3 domains, IRSp53 is capable of inducing the formation of a triple complex (shank1/IRSp53/PSD-95).     

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.     

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.     

Shank, a novel family of postsynaptic density proteins that binds to the NMDA receptor/PSD-95/GKAP complex and cortactin.

NMDA receptors are linked to intracellular cytoskeletal and signaling molecules via the PSD-95 protein complex. We report a novel family of postsynaptic density (PSD) proteins, termed Shank, that binds via its PDZ domain to the C terminus of PSD-95-associated protein GKAP. A ternary complex of Shank/GKAP/PSD-95 assembles in heterologous cells and can be coimmunoprecipitated from rat brain. Synaptic localization of Shank in neurons is inhibited by a GKAP splice variant that lacks the Shank-binding C terminus. In addition to its PDZ domain, Shank contains a proline-rich region that binds to cortactin and a SAM domain that mediates multimerization. Shank may function as a scaffold protein in the PSD, potentially cross-linking NMDA receptor/PSD-95 complexes and coupling them to regulators of the actin cytoskeleton.     

The Shank family of postsynaptic density proteins interacts with and promotes synaptic accumulation of the beta PIX guanine nucleotide exchange factor for Rac1 and Cdc42

The Shank/ProSAP family of multidomain proteins is known to play an important role in organizing synaptic multiprotein complexes. Here we report a novel interaction between Shank and beta PIX, a guanine nucleotide exchange factor for the Rac1 and Cdc42 small GTPases. This interaction is mediated by the PDZ domain of Shank and the C-terminal leucine zipper domain and the PDZ domain-binding motif at the extreme C terminus of beta PIX. Shank colocalizes with beta PIX at excitatory synaptic sites in cultured neurons. In brain, Shank forms a complex with beta PIX and beta PIX-associated signaling molecules including p21-associated kinase (PAK), an effector kinase of Rac1/Cdc42. Importantly, overexpression of Shank in cultured neurons promotes synaptic accumulation of beta PIX and PAK. Considering the involvement of Rac1 and PAK in spine dynamics, these results suggest that Shank recruits beta PIX and PAK to spines for the regulation of postsynaptic structure.     

GKAP, a Novel Synaptic Protein That Interacts with the Guanylate Kinase-like Domain of the PSD-95/SAP90 Family of Channel Clustering Molecules

The molecular mechanisms underlying the organization of ion channels and signaling molecules at the synaptic junction are largely unknown. Recently, members of the PSD-95/SAP90 family of synaptic MAGUK (membrane-associated guanylate kinase) proteins have been shown to interact, via their NH₂-terminal PDZ domains, with certain ion channels (NMDA receptors and K⁺ channels), thereby promoting the clustering of these proteins. Although the function of the NH₂-terminal PDZ domains is relatively well characterized, the function of the Src homology 3 (SH3) domain and the guanylate kinase-like (GK) domain in the COOH-terminal half of PSD-95 has remained obscure. We now report the isolation of a novel synaptic protein, termed GKAP for guanylate kinase-associated protein, that binds directly to the GK domain of the four known members of the mammalian PSD-95 family. GKAP shows a unique domain structure and appears to be a major constituent of the postsynaptic density. GKAP colocalizes and coimmunoprecipitates with PSD-95 in vivo, and coclusters with PSD-95 and K⁺ channels/ NMDA receptors in heterologous cells. Given their apparent lack of guanylate kinase enzymatic activity, the fact that the GK domain can act as a site for protein– protein interaction has implications for the function of diverse GK-containing proteins (such as p55, ZO-1, and LIN-2/CASK).     

Brain-specific potential guanine nucleotide exchange factor for Arf, synArfGEF (Po), is localized to postsynaptic density

We cloned from a rat brain cDNA library a novel cDNA and named it a potential synaptic guanine nucleotide exchange factor (GEF) for Arf (synArfGEF (Po)) (GenBank Accession no. AB057643) based on its domain structure and localization. The cloned gene was 7410 bases long with a 3585-bp coding sequence encoding a protein of 1194 amino acids. The deduced protein contained a coiled-coil structure in the N-terminal portion followed by Sec7 and Plekstrin homology (PH) domains. Thus, the protein was a member of the Sec7 family of proteins, GEFs. Conservation of the ADP-ribosylation factor (Arf)-binding sequence suggested that the protein was a GEF for Arf. The gene was expressed specifically in the brain, where it exhibited region-specific expression. The protein was highly enriched in the postsynaptic density (PSD) fraction prepared from the rat forebrain. Uniquely, the protein interacted with PSD-95, SAP97 and Homer/Vesl 1/PSD-Zip45 via its C-terminal PDZ-binding motif and co-localized with these proteins in cultured cortical neurons. These results supported its localization in the PSD. The postsynaptic localization was also supported by immunohistochemical examination of the rat brain. The mRNA for the synArfGEF was also localized to dendrites, as well as somas, of neuronal cells. Thus, both the mRNA and the protein were localized in the postsynaptic compartments. These results suggest a postsynaptic role of synArfGEF in the brain.     

Synaptic accumulation of PSD-95 and synaptic function regulated by phosphorylation of serine-295 of PSD-95

The scaffold protein PSD-95 promotes the maturation and strengthening of excitatory synapses, functions that require proper localization of PSD-95 in the postsynaptic density (PSD). Here we report that phosphorylation of ser-295 enhances the synaptic accumulation of PSD-95 and the ability of PSD-95 to recruit surface AMPA receptors and potentiate excitatory postsynaptic currents. We present evidence that a Rac1-JNK1 signaling pathway mediates ser-295 phosphorylation and regulates synaptic content of PSD-95. Ser-295 phosphorylation is suppressed by chronic elevation, and increased by chronic silencing, of synaptic activity. Rapid dephosphorylation of ser-295 occurs in response to NMDA treatment that causes chemical long-term depression (LTD). Overexpression of a phosphomimicking mutant (S295D) of PSD-95 inhibited NMDA-induced AMPA receptor internalization and blocked the induction of LTD. The data suggest that synaptic strength can be regulated by phosphorylation-dephosphorylation of ser-295 of PSD-95 and that synaptic depression requires the dephosphorylation of ser-295.     

Novel ZIP kinase isoform lacks leucine zipper

Zipper-interacting protein kinase (ZIP kinase) has been thought to be involved in apoptosis and the C-terminal leucine zipper motif is important for its function. Recent studies have revealed that ZIP kinase also plays a role in regulating myosin phosphorylation. Here, we found novel ZIP kinase isoform in which the C-terminal non-kinase domain containing a leucine zipper is eliminated (hZIPK-S). hZIPK-S binds to myosin phosphatase targeting subunit 1(MYPT1) similar to the long isoform (hZIPK-L). In addition, we found that hZIPK-S as well as hZIPK-L bind to myosin. These results indicate that a leucine zipper is not critical for the binding of ZIP kinase to MYPT1 and myosin. Consistently, hZIPK-S localized with stress-fibers where they co-localized with myosin. The residues 278-311, the C-terminal side of the kinase domain common to the both isoforms, is involved in the binding to MYPT1, while the myosin binding domain is within the kinase domain. These results suggest that the newly found hZIPK-S as well as the long isoform play an important role in the regulation of myosin phosphorylation.     

Mechanism of DNA binding by the DnaB helicase of Escherichia coli: analysis of the roles of domain gamma in DNA binding.

We have analyzed the mechanism of single-stranded DNA (ssDNA) binding mediated by the C-terminal domain gamma of the DnaB helicase of Escherichia coli. Sequence analysis of this domain indicated a specific basic region, "RSRARR", and a leucine zipper motif that are likely involved in ssDNA binding. We have carried out deletion as well as in vitro mutagenesis of specific amino acid residues in this region in order to determine their function(s) in DNA binding. The functions of the RSRARR domain in DNA binding were analyzed by site-directed mutagenesis. DnaBMut1, with mutations R(328)A and R(329)A, had a significant decrease in the DNA dependence of ATPase activity and lost its DNA helicase activity completely, indicating the important roles of these residues in DNA binding and helicase activities. DnaBMut2, with mutations R(324)A and R(326)A, had significantly attenuated DNA binding as well as DNA-dependent ATPase and DNA helicase activities, indicating that these residues also play a role in DNA binding and helicase activities. The role(s) of the leucine zipper dimerization motif was (were) determined by deletion analysis. The DnaB Delta 1 mutant with a 55 amino acid C-terminal deletion, which left the leucine zipper and basic DNA binding regions intact, retained DNA binding as well as DNA helicase activities. However, the DnaB Delta 2 mutant with a 113 amino acid C-terminal deletion that included the leucine zipper dimerization motif, but not the RSRARR sequence, lost DNA binding, DNA helicase activities, and hexamer formation. The major findings of this study are (i) the leucine zipper dimerization domain, I(361)-L(389), is absolutely required for (a) dimerization and (b) ssDNA binding; (ii) the base-rich RSRARR sequence is required for DNA binding; (iii) three regions of domain gamma (gamma I, gamma II, and gamma III) differentially regulate the ATPase activity; (iv) there are likely three ssDNA binding sites per hexamer; and (v) a working model of DNA unwinding by the DnaB hexamer is proposed.     

Phosphorylation of stargazin by protein kinase A regulates its interaction with PSD-95.

Stargazin is the first transmembrane protein known to associate with AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) glutamate receptors (AMPARs) and regulate their synaptic targeting by two distinct mechanisms, specifically via delivery of AMPARs to the surface membrane and synaptic targeting of these receptors by binding to PSD-95/SAP-90 and related PDZ proteins. However, it is not known whether and how this stargazin-mediated synaptic targeting of AMPARs is regulated. Stargazin interacts with the PDZ domains of PSD-95 through the C-terminal PDZ-binding motif. The stargazin C terminus contains a consensus sequence for phosphorylation by cAMP-dependent protein kinase A (PKA). Phosphorylation site-specific stargazin antibodies reveal that the stargazin C terminus is phosphorylated at the Thr-321 residue in heterologous cells and in vivo. Stargazin phosphorylation is enhanced by the catalytic subunit of PKA. Mutations mimicking stargazin phosphorylation (T321E and T321D) lead to elimination of yeast two-hybrid interactions, in vitro coimmunoprecipitation, and coclustering between stargazin and PSD-95. Phosphorylated stargazin shows a selective loss of coimmunoprecipitation with PSD-95 in heterologous cells and limited enrichment in postsynaptic density fractions of rat brain. These results suggest that phosphorylation of the stargazin C terminus by PKA regulates its interaction with PSD-95 and synaptic targeting of AMPARs.     

Structure of the GH1 domain of guanylate kinase-associated protein from Rattus norvegicus

Guanylate-kinase-associated protein (GKAP) is a scaffolding protein that links NMDA receptor-PSD-95 to Shank–Homer complexes by protein–protein interactions at the synaptic junction. GKAP family proteins are characterized by the presence of a C-terminal conserved GKAP homology domain 1 (GH1) of unknown structure and function. In this study, crystal structure of the GH1 domain of GKAP from Rattus norvegicus was determined in fusion with an N-terminal maltose-binding protein at 2.0 Å resolution. The structure of GKAP GH1 displays a three-helix bundle connected by short flexible loops. The predicted helix α4 which was not visible in the crystal structure associates weakly with the helix α3 suggesting dynamic nature of the GH1 domain. The strict conservation of GH1 domain across GKAP family members and the lack of a catalytic active site required for enzyme activity imply that the GH1 domain might serve as a protein–protein interaction module for the synaptic protein clustering.     

Polarized targeting of peripheral membrane proteins in neurons

Differential targeting of neuronal proteins to axons and dendrites is essential for directional information flow within the brain, however, little is known about this protein-sorting process. Here, we investigate polarized targeting of lipid-anchored peripheral membrane proteins, postsynaptic density-95 (PSD-95) and growth-associated protein-43 (GAP-43). Whereas the N-terminal palmitoylated motif of PSD-95 is necessary but not sufficient for sorting to dendrites, the palmitoylation motif of GAP-43 is sufficient for axonal targeting and can redirect a PSD-95 chimera to axons. Systematic mutagenesis of the GAP-43 and PSD-95 palmitoylation motifs indicates that the spacing of the palmitoylated cysteines and the presence of nearby basic amino acids determine polarized targeting by these two motifs. Similarly, the axonal protein paralemmin contains a C-terminal palmitoylated domain, which resembles that of GAP-43 and also mediates axonal targeting. These axonally targeted palmitoylation motifs also mediate targeting to detergent-insoluble glycolipid-enriched complexes in heterologous cells, suggesting a possible role for specialized lipid domains in axonal sorting of peripheral membrane proteins.     

Structure of the enabled/VASP homology 1 domain-peptide complex: a key component in the spatial control of actin assembly.

The Enabled/VASP homology 1 (EVH1; also called WH1) domain is an interaction module found in several proteins implicated in actin-based cell motility. EVH1 domains bind the consensus proline-rich motif FPPPP and are required for targeting the actin assembly machinery to sites of cytoskeletal remodeling. The crystal structure of the mammalian Enabled (Mena) EVH1 domain complexed with a peptide ligand reveals a mechanism of recognition distinct from that used by other proline-binding modules. The EVH1 domain fold is unexpectedly similar to that of the pleckstrin homology domain, a membrane localization module. This finding demonstrates the functional plasticity of the pleckstrin homology fold as a binding scaffold and suggests that membrane association may play an auxiliary role in EVH1 targeting.     

Post-synaptic density perturbs insulin-induced Kv1.3 channel modulation via a clustering mechanism involving the SH3 domain

The olfactory bulb (OB) contains the highest concentration of the insulin receptor (IR) kinase in the central nervous system; however, its functional role and modulation in this region remains poorly understood. IR kinase contains a number of proline-rich motifs, making it an excellent candidate for modulation by SH₃ domain-containing adaptor proteins. Kv1.3, a voltage-gated Shaker potassium channel and tyrosine phosphorylation substrate of IR kinase, contains several proline-rich sequences and a canonical post-synaptic density 95 (PSD-95)/discs large/zO-1 domain (PDZ) recognition motif common to most Shaker family members. We sought to determine if a functional relationship existed between Kv1.3, IR kinase, and the SH₃/PDZ adaptor protein PSD-95. Through patch-clamp electrophysiology, immunochemistry, and co-immunoprecipitation, we found that while Kv1.3 and PSD-95 alone interact via the canonical C-terminal PDZ recognition motif of the channel, this molecular site of interaction acts to cluster the channels but the PSD-95 SH₃-guanylate kinase domain functionally modulates Kv1.3 activity via two proline-rich domains in its N- and C-terminal. Therefore, these data suggest that adaptor domains responsible for ion-channel clustering and functional modulation are not necessarily coupled. Moreover, IR kinase and Kv1.3 can only be co-immunoprecipitated in the presence of PSD-95 as the adapting linker. Functionally, insulin-dependent Kv1.3 phosphorylation that causes channel current suppression is blocked via interaction with the PSD-95 SH₃-guanylate kinase domain. Because all the three proteins co-localize in multiple lamina of the OB that are known to be rich in synaptic connections, membrane excitability and synaptic transmission at critical locations in the OB have the capacity to be finely regulated.     

Molecular dissociation of the role of PSD-95 in regulating synaptic strength and LTD

The postsynaptic density protein PSD-95 influences synaptic AMPA receptor (AMPAR) content and may play a critical role in LTD. Here we demonstrate that the effects of PSD-95 on AMPAR-mediated synaptic responses and LTD can be dissociated. Our findings suggest that N-terminal-domain-mediated dimerization is important for PSD-95's effect on basal synaptic AMPAR function, whereas the C-terminal SH(3)-GK domains are also necessary for localizing PSD-95 to synapses. We identify PSD-95 point mutants (Q15A, E17R) that maintain PSD-95's influence on basal AMPAR synaptic responses yet block LTD. These point mutants increase the proteolysis of PSD-95 within its N-terminal domain, resulting in a C-terminal fragment that functions as a dominant negative likely by scavenging critical signaling proteins required for LTD. Thus, the C-terminal portion of PSD-95 serves a dual function. It is required to localize PSD-95 at synapses and as a scaffold for signaling proteins that are required for LTD.     

ProSAP-interacting protein 1 (ProSAPiP1), a novel protein of the postsynaptic density that links the spine-associated Rap-Gap (SPAR) to the scaffolding protein ProSAP2/Shank3

ProSAPs/Shanks are a family of proteins that have a major scaffolding function for components of the postsynaptic density (PSD) of excitatory brain synapses. Members of the family harbor a variety of domains for protein-protein interactions, one of which is a unique PDZ domain that differs significantly from those of other proteins. We have identified a novel binding partner for this PDZ domain, termed ProSAPiP1, that is highly enriched in the PSD and shares significant sequence homology with the PSD protein PSD-Zip70. Both molecules code for a Fez1 domain that can be found in a total of four related proteins. ProSAPiP1 is widely expressed in rat brain and co-localizes with ProSAP2/Shank3 in excitatory spines and synapses. ProSAP2/Shank3 co-immunoprecipitates with ProSAPiP1 but not with PSD-Zip70. Both proteins, however, bind and recruit SPAR to synapses with a central coiled-coil region that harbors a leucine zipper motif. This region is also responsible for homo- and heteromultimerization of ProSAPiP1 and PSD-Zip70. Thus, ProSAPiP1 and PSD-Zip70 are founders of a novel family of scaffolding proteins, the "Fezzins," which adds further complexity to the organization of the PSD protein network.