Rapid and differential regulation of AMPA and kainate receptors at hippocampal mossy fibre synapses by PICK1 and GRIP

We identified four PDZ domain-containing proteins, syntenin, PICK1, GRIP, and PSD95, as interactors with the kainate receptor (KAR) subunits GluR5(2b,) GluR5(2c), and GluR6. Of these, we show that both GRIP and PICK1 interactions are required to maintain KAR-mediated synaptic function at mossy fiber-CA3 synapses. In addition, PKC alpha can phosphorylate ct-GluR5(2b) at residues S880 and S886, and PKC activity is required to maintain KAR-mediated synaptic responses. We propose that PICK1 targets PKC alpha to phosphorylate KARs, causing their stabilization at the synapse by an interaction with GRIP. Importantly, this mechanism is not involved in the constitutive recycling of AMPA receptors since blockade of PDZ interactions can simultaneously increase AMPAR- and decrease KAR-mediated synaptic transmission at the same population of synapses.     

The PDZ domain protein PICK1 and the sodium channel BNaC1 interact and localize at mechanosensory terminals of dorsal root ganglion neurons and dendrites of central neurons.

Members of the BNaC/ASIC family of ion channels have been implicated in mechanotransduction and nociception mediated by dorsal root ganglion (DRG) neurons. These ion channels are also expressed in the CNS. We identified the PDZ domain protein PICK1 as an interactor of BNaC1(ASIC2) in a yeast two-hybrid screen. We show by two-hybrid assays, glutathione S-transferase pull-down assays, and coimmunoprecipitations that the BNaC1-PICK1 interaction is specific, and that coexpression of both proteins leads to their clustering in intracellular compartments. The interaction between BNaC1 and PICK1 requires the PDZ domain of PICK1 and the last four amino acids of BNaC1. BNaC1 is similar to two other BNaC/ASIC family members, BNaC2 (ASIC1) and ASIC4, at its extreme C terminus, and we show that PICK1 also interacts with BNaC2. We found that PICK1, like BNaC1 and BNaC2, is expressed by DRG neurons and, like the BNaC1alpha isoform, is present at their peripheral mechanosensory endings. Both PICK1 and BNaC1alpha are also coexpressed by some pyramidal neurons of the cortex, by pyramidal neurons of the CA3 region of hippocampus, and by cerebellar Purkinje neurons, localizing to their dendrites and cell bodies. Therefore, PICK1 interacts with BNaC/ASIC channels and may regulate their subcellular distribution or function in both peripheral and central neurons.     

Syntenin-syndecan binding requires syndecan-synteny and the co-operation of both PDZ domains of syntenin

Syntenin is an adaptor-like molecule that binds to the cytoplasmic domains of all four vertebrate syndecans. Syntenin-syndecan binding involves the C-terminal part of syntenin that contains a tandem of PDZ domains. Here we provide evidence that each PDZ domain of syntenin can interact with a syndecan. Isolated or combined mutations of the carboxylate binding lysines in the inter-betaAbetaB loops and of the alphaB1 residues in either one or both the PDZ domains of syntenin all reduce syntenin-syndecan binding in yeast two-hybrid, blot-overlay, and surface plasmon resonance assays. PDZ2 mutations have more pronounced effects on binding than PDZ1 mutations, but complete abrogation of syntenin-syndecan binding requires the combination of both the lysine and the alphaB1 mutations in both the PDZ domains of syntenin. Isothermal calorimetric titration of syntenin with syndecan peptide reveals the presence of two binding sites in syntenin. Yet, unlike a tandem of two PDZ2 domains and a reconstituted PDZ1+PDZ2 tandem, a tandem of two PDZ1 domains and isolated PDZ1 or PDZ2 domains do not interact with syndecan bait. We conclude to a co-operative binding mode whereby neither of these two PDZ domains is sufficient by itself but where PDZ2 functions as a "major" or "high affinity" syndecan binding domain, and PDZ1 functions as an "accessory" or "low affinity" syndecan binding domain. The paired, but not the isolated PDZ domains of syntenin bind also strongly to the immobilized cytoplasmic domains of neurexin and B-class ephrins. By inference, these data suggest a model whereby recruitment of syntenin to membrane surfaces requires two compatible types of bait that are in "synteny" (occurring together in location) and engages both PDZ domains of syntenin. The synteny of compatible bait may result from the assemblies and co-assemblies of syndecans and other similarly suited partners in larger supramolecular complexes. In general, an intramolecular combination of PDZ domains that are weak, taken individually, would appear to be designed to detect rather than drive the formation of specific molecular assemblies.     

Molecular determinants for PICK1 synaptic aggregation and mGluR7a receptor coclustering: role of the PDZ, coiled-coil, and acidic domains

PSD-95/Disc-large/ZO-1 (PDZ) domain-containing proteins play a central role in synaptic organization by their involvement in neurotransmitter receptor clustering and signaling complex assembly. The protein interacting with protein kinase C (PICK1), a synaptic PDZ domain protein that also contains a coiled-coil and acidic domain, binds to several synaptic components including the metabotropic glutamate receptor mGluR7a. Coexpression of PICK1 and mGluR7a in heterologous cells induces coclustering of these two proteins. To examine the role of the different structural motifs of PICK1 in synaptic aggregation of PICK1 and mGluR7a coclustering, several PICK1 mutants were generated to analyze their distribution in transfected hippocampal cultured neurons and to test their ability to induce coclusters with mGluR7a when coexpressed in fibroblast cells. The PDZ and coiled-coil domains are both required, whereas the acidic region plays an inhibitory role in these processes. Our data suggest that synaptic aggregation and receptor coclustering depend on PICK1 binding to a target membrane receptor, e.g. mGluR7a, by a PDZ-mediated interaction and on PICK1 oligomerization through the coiled-coil domain. This study defined three structural signals within PICK1 regulating its synaptic localization and receptor coclustering activity, which could represent molecular substrates involved in synaptic development and plasticity.     

Cerebellar long-term depression requires PKC-regulated interactions between GluR2/3 and PDZ domain-containing proteins

Cerebellar LTD requires activation of PKC and is expressed, at least in part, as postsynaptic AMPA receptor internalization. Recently, it was shown that AMPA receptor internalization requires clathrin-mediated endocytosis and depends upon the carboxy-terminal region of GluR2/3. Phosphorylation of Ser-880 in this region by PKC differentially regulates the binding of the PDZ domain-containing proteins GRIP/ABP and PICK1. Peptides, corresponding to the phosphorylated and dephosphorylated GluR2 carboxy-terminal PDZ binding motif, were perfused in cerebellar Purkinje cells grown in culture. Both the dephospho form (which blocks binding of GRIP/ABP and PICK1) and the phospho form (which selectively blocks PICK1) attenuated LTD induction by glutamate/depolarization pairing, as did antibodies directed against the PDZ domain of PICK1. These findings indicate that expression of cerebellar LTD requires PKC-regulated interactions between the carboxy-terminal of GluR2/3 and PDZ domain-containing proteins.     

Presynaptic clustering of mGluR7a requires the PICK1 PDZ domain binding site

Aggregation of neurotransmitter receptors at pre- and postsynaptic structures is crucial for efficient neuronal communication. In contrast to the wealth of information about postsynaptic specializations, little is known about the molecular organization of presynaptic membrane proteins. We show here that the metabotropic glutamate receptor mGluR7a, which localizes specifically to presynaptic active zones, interacts in vitro and in vivo with PICK1. Coexpression in heterologous systems induces coclustering dependent upon the extreme C terminus of mGluR7a and the PDZ domain of PICK1. mGluR7a and PICK1 localize to excitatory synapses in hippocampal neurons. Furthermore, whereas transfected mGluR7a clusters at presynaptic sites, mGluR7adelta3 lacking the PICK1 binding site targets to axons but does not cluster. These results suggest that PICK1 is a component of the presynaptic machinery involved in mGluR7a aggregation and in modulation of glutamate neurotransmission.     

Ggamma13 interacts with PDZ domain-containing proteins

The G protein gamma13 subunit (Ggamma13) is expressed in taste and retinal and neuronal tissues and plays a key role in taste transduction. We identified PSD95, Veli-2, and other PDZ domain-containing proteins as binding partners for Ggamma13 by yeast two-hybrid and pull-down assays. In two-hybrid assays, Ggamma13 interacted specifically with the third PDZ domain of PSD95, the sole PDZ domain of Veli-2, and the third PDZ domain of SAP97, a PSD95-related protein. Ggamma13 did not interact with the other PDZ domains of PSD95. Coexpression of Ggamma13 with its Gbeta1 partner did not interfere with these two-hybrid interactions. The physical interaction of Ggamma13 with PSD95 in the cellular milieu was confirmed in pull-down assays following heterologous expression in HEK293 cells. The interaction of Ggamma13 with the PDZ domain of PSD95 was via the C-terminal CAAX tail of Ggamma13 (where AA indicates the aliphatic amino acid); alanine substitution of the CTAL sequence at the C terminus of Ggamma13 abolished its interactions with PSD95 in two-hybrid and pull-down assays. Veli-2 and SAP97 were identified in taste tissue and in Ggamma13-expressing taste cells. Coimmunoprecipitation of Ggamma13 and PSD95 from brain and of Ggamma13 and SAP97 from taste tissue indicates that Ggamma13 interacts with these proteins endogenously. This is the first demonstration that PDZ domain proteins interact with heterotrimeric G proteins via the CAAX tail of Ggamma subunits. The interaction of Ggamma13 with PDZ domain-containing proteins may provide a means to target particular Gbetagamma subunits to specific subcellular locations and/or macromolecular complexes involved in signaling pathways.     

PICK1 interacts with ABP/GRIP to regulate AMPA receptor trafficking

PICK1 and ABP/GRIP bind to the AMPA receptor (AMPAR) GluR2 subunit C terminus. Transfer of the receptor from ABP/GRIP to PICK1, facilitated by GluR2 S880 phosphorylation, may initiate receptor trafficking. Here we report protein interactions that regulate these steps. The PICK1 BAR domain interacts intermolecularly with the ABP/GRIP linker II region and intramolecularly with the PICK1 PDZ domain. Binding of PKCalpha or GluR2 to the PICK1 PDZ domain disrupts the intramolecular interaction and facilitates the PICK1 BAR domain association with ABP/GRIP. Interference with the PICK1-ABP/GRIP interaction impairs S880 phosphorylation of GluR2 by PKC and decreases the constitutive surface expression of GluR2, the NMDA-induced endocytosis of GluR2, and recycling of internalized GluR2. We suggest that the PICK1 interaction with ABP/GRIP is a critical step in controlling GluR2 trafficking.     

Phospholipase Cdelta4 associates with glutamate receptor interacting protein 1 in testis

We reported previously that phospholipase C (PLC) delta4 is required for calcium mobilization in the zona pellucida-induced acrosome reaction in sperm. Here we focused on the function of the C2 domain of PLCdelta4 and report that glutamate receptor-interacting protein1 (GRIP1) was identified as a binding protein of the PLCdelta4-C2 domain on yeast two-hybrid screening. Physiological interaction of GRIP1 with PLCdelta4 in mouse testis was confirmed by immunoprecipitation with anti-PLCdelta4 antibodies and the association seemed to correlate with the maturation stage of sperm. We also determined that a PDZ-binding motif at the C-terminus of the PLCdelta4-C2 domain is responsible for GRIP1 binding, whereas the sixth or seventh PDZ domain of GRIP1 is essential and sufficient for association with the PLCdelta4-C2 domain. These results indicate that PLCdelta4 binds via its C2 domain to the PDZ6 or PDZ7 domain of GRIP1, and that this association may play a role in spermatogenesis.     

PDZ binding to the BAR domain of PICK1 is elucidated by coarse-grained molecular dynamics

A key regulator of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor traffic, PICK1 is known to interact with over 40 other proteins, including receptors, transporters and ionic channels, and to be active mostly as a homodimer. The current lack of a complete PICK1 structure determined at atomic resolution hinders the elucidation of its functional mechanisms. Here, we identify interactions between the component PDZ and BAR domains of PICK1 by calculating possible binding sites for the PDZ domain of PICK1 (PICK1-PDZ) to the homology-modeled, crescent-shaped dimer of the PICK1-BAR domain using multiplexed replica-exchange molecular dynamics (MREMD) and canonical molecular dynamics simulations with the coarse-grained UNRES force field. The MREMD results show that the preferred binding site for the single PDZ domain is the concave cavity of the BAR dimer. A second possible binding site is near the N-terminus of the BAR domain that is linked directly to the PDZ domain. Subsequent short canonical molecular dynamics simulations used to determine how the PICK1-PDZ domain moves to the preferred binding site on the BAR domain of PICK1 revealed that initial hydrophobic interactions drive the progress of the simulated binding. Thus, the concave face of the BAR dimer accommodates the PDZ domain first by weak hydrophobic interactions and then the PDZ domain slides to the center of the concave face, where more favorable hydrophobic interactions take over.     

PDZ tandem of human syntenin: crystal structure and functional properties

Syntenin, a 33 kDa protein, interacts with several cell membrane receptors and with merlin, the product of the causal gene for neurofibromatosis type II. We report a crystal structure of the functional fragment of human syntenin containing two canonical PDZ domains, as well as binding studies for full-length syntenin, the PDZ tandem, and isolated PDZ domains. We show that the functional properties of syntenin are a result of independent interactions with target peptides, and that each domain is able to bind peptides belonging to two different classes: PDZ1 binds peptides from classes I and III, while PDZ2 interacts with classes I and II. The independent binding of merlin by PDZ1 and syndecan-4 by PDZ2 provides direct evidence for the coupling of syndecan-mediated signaling to actin regulation by merlin.     

Interaction of the PDZ domain of human PICK1 with class I ADP-ribosylation factors

We have cloned the cDNA encoding human PICK1 (protein interacting with C kinase 1), a PDZ domain-containing protein of 415 amino acids, and also identified the Drosophila homologue by search of the databank. Northern blot analysis shows a single mRNA of about 2.0 kb ubiquitously expressed in human tissues. Although PICK1 proteins harbor a region homologous to arfaptin1 and arfaptin2, two proteins that bind to the ARF (ADP-ribosylation factor), this region of PICK1 does not interact with ARFs in the yeast two-hybrid system. On the other hand, the PDZ domain of PICK1 is capable of interacting with constitutively active, GTP-bound forms of ARF1 and ARF3, but neither with those of ARF5/6 nor with the GDP-bound ARFs. The PICK1-ARF interaction is abrogated by introduction of mutations in the PDZ domain or by deletion of the extreme C-terminus of ARF1. Thus, PICK1 specifically interacts with ARF1/3 in the GTP-bound state, suggesting that PICK1 participates in ARF1/3-mediated cellular processes.     

A single PDZ domain protein interacts with the Menkes copper ATPase, ATP7A. A new protein implicated in copper homeostasis

The homeostatic regulation of essential elements such as copper requires many proteins whose activities are often mediated and tightly coordinated through protein-protein interactions. This regulation ensures that cells receive enough copper without intracellular concentrations reaching toxic levels. To date, only a small number of proteins implicated in copper homeostasis have been identified, and little is known of the protein-protein interactions required for this process. To identify other proteins important for copper homeostasis, while also elucidating the protein-protein interactions that are integral to the process, we have utilized a known copper protein, the copper ATPase ATP7A, as a bait in a yeast two-hybrid screen of a human cDNA library to search for interacting partners. One of the ATP7A-interacting proteins identified is a novel protein with a single PDZ domain. This protein was recently identified to interact with the plasma membrane calcium ATPase b-splice variants. We propose a change in name for this protein from PISP (plasma membrane calcium ATPase-interacting single-PDZ protein) to AIPP1 (ATPase-interacting PDZ protein) and suggest that it represents the protein that interacts with the class I PDZ binding motif identified at the ATP7A C terminus. The interaction in mammalian cells was confirmed and an additional splice variant of AIPP1 was identified. This study represents an essential step forward in identifying the proteins and elucidating the network of protein-protein interactions involved in maintaining copper homeostasis and validates the use of the yeast two-hybrid approach for this purpose.     

The binding affinities of proteins interacting with the PDZ domain of PICK1

Protein interacting with C kinase (PICK1) is well conserved throughout evolution and plays a critical role in synaptic plasticity by regulating the trafficking and posttranslational modification of its interacting proteins. PICK1 contains a single PSD95/DlgA/Zo-1 (PDZ) protein-protein interaction domain, which is promiscuous and shown to interact with over 60 proteins, most of which play roles in neuronal function. Several reports have suggested the role of PICK1 in disorders such as epilepsy, pain, brain trauma and stroke, drug abuse and dependence, schizophrenia and psychosis. Importantly, lead compounds that block PICK1 interactions are also now becoming available. Here, a new modeling approach was developed to investigate binding affinities of PDZ interactions. Using these methods, the binding affinities of all major PICK1 interacting proteins are reported and the effects of PICK1 mutations on these interactions are described. These modeling methods have important implications in defining the binding properties of proteins interacting with PICK1 as well as the general structural requirements of PDZ interactions. The study also provides modeling methods to support in the drug design of ligands for PDZ domains, which may further aid in development of the family of PDZ domains as a drug target.     

Classification of PDZ domains.

A diverse family of PDZ domains has been identified, but the rules that govern their ligand specificity are not clear. Here we propose a novel classification of PDZ domains based on the nature of amino acids in the two critical positions in the PDZ domain fold. Using these principles, we classified PDZ domains present in the SMART database. Using yeast two-hybrid, in vitro pull-down and plasmon surface resonance assays, we demonstrated that in agreement with their position in the proposed classification the Mint1-1, hINADL-5, and PAR6 PDZ domains display similar dual ligand specificity. The proposed classification helps to organize PDZ domain containing proteins.     

Corequirement of PICK1 binding and PKC phosphorylation for stable surface expression of the metabotropic glutamate receptor mGluR7

The presynaptic metabotropic glutamate receptor (mGluR) mGluR7 modulates excitatory neurotransmission by regulating neurotransmitter release and plays a critical role in certain forms of synaptic plasticity. Although the dynamic regulation of mGluR7 surface expression governs a form of metaplasticity in the hippocampus, little is known about the molecular mechanisms regulating mGluR7 trafficking. We now show that mGluR7 surface expression is stabilized by both PKC phosphorylation and by receptor binding to the PDZ domain-containing protein PICK1. Phosphorylation of mGluR7 on serine 862 (S862) inhibits CaM binding, thereby increasing mGluR7 surface expression and receptor binding to PICK1. Furthermore, in mice lacking PICK1, PKC-dependent increases in mGluR7 phosphorylation and surface expression are diminished, and mGluR7-dependent plasticity at mossy fiber-interneuron hippocampal synapses is impaired. These data support a model in which PICK1 binding and PKC phosphorylation act together to stabilize mGluR7 on the cell surface in vivo.     

A four PDZ domain-containing splice variant form of GRIP1 is localized in GABAergic and glutamatergic synapses in the brain

We have isolated, from a rat brain cDNA library, a clone corresponding to a 2779-bp cDNA encoding a novel splice form of the glutamate receptor interacting protein-1 (GRIP1). We call this 696-amino acid splice form GRIP1c 4-7 to differentiate it from longer splice forms of GRIP1a/b containing seven PDZ domains. The four PDZ domains of GRIP1c 4-7 are identical to PDZ domains 4-7 of GRIP1a/b. GRIP1c 4-7 also contains 35 amino acids at the N terminus and 12 amino acids at the C terminus that are different from GRIP1a/b. In transfected HEK293 cells, a majority of GRIP1c 4-7 was associated with the plasma membrane. GRIP1c 4-7 interacted with GluR2/3 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor. In low density hippocampal cultures, GRIP1c 4-7 clusters colocalized with GABAergic (where GABA is gamma-aminobutyric acid) and glutamatergic synapses, although a higher percentage of GRIP1c 4-7 clusters colocalized with gamma-aminobutyric acid, type A, receptor (GABA(A)R) clusters than with alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor clusters. Transfection of hippocampal neurons with hemagglutinin-tagged GRIP1c 4-7 showed that it could target to the postsynaptic complex of GABAergic synapses colocalizing with GABA(A)R clusters. GRIP1c 4-7-specific antibodies, which did not recognize previously described splice forms of GRIP1, recognized a 75-kDa protein that is enriched in a postsynaptic density fraction isolated from rat brain. EM immunocytochemistry experiments showed that in intact brain GRIP1c 4-7 concentrates at postsynaptic complexes of both type I glutamatergic and type II GABAergic synapses although it is also presynaptically localized. These results indicate that GRIP1c 4-7 plays a role not only in glutamatergic synapses but also in GABAergic synapses.     

Schwannomin isoform-1 interacts with syntenin via PDZ domains.

The neurofibromatosis type 2 gene (NF2) is involved in the pathogenesis of benign tumors of the human nervous system. The NF2 protein, called schwannomin or merlin, is inactivated in virtually all schwannomas and meningiomas. The molecular mechanisms by which schwannomin functions as a tumor suppressor is unknown but believed to involve plasma membrane-cytoskeletal interactions. Two major alternatively spliced isoforms of schwannomin differing in their C termini have been reported. Using the yeast two-hybrid system, we have identified syntenin as a binding partner for schwannomin isoform-1 (sch-1). Syntenin is an adapter protein that couples transmembrane proteoglycans to cytoskeletal components and is involved in intracellular vesicle transport. The C terminus 25 amino acids of sch-1 and the two PDZ domains of syntenin mediate their binding, and mutations introduced within the VAFFEEL region of sch-1 defined a sequence crucial for syntenin recognition. We have showed that the two proteins interacted in vitro and in vivo and localized underneath the plasma membrane. Fibroblast cells expressing heterologous antisense syntenin display alterations in the subcellular distribution of sch-1. Together, these results provide the first functional clue to the existence of schwannomin isoforms and could unravel novel pathways for the transport and subcellular localization of schwannomin in vivo.     

PDZ7 of glutamate receptor interacting protein binds to its target via a novel hydrophobic surface area

Glutamate receptor interacting protein 1 (GRIP1) is a scaffold protein composed of seven PDZ (Postsynaptic synaptic density-95/Discs large/Zona occludens-1) domains. The protein plays important roles in the synaptic targeting of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The interaction between GRIP1 PDZ7 and a Ras guanine nucleotide exchange factor, GRASP-1, regulates synaptic distribution of AMPA receptors. Here, we describe the three-dimensional structure of GRIP1 PDZ7 determined by NMR spectroscopy. GRIP1 PDZ7 contains a closed carboxyl group-binding pocket and a narrow alphaB/betaB-groove that is not likely to bind to classical PDZ ligands. Unexpectedly, GRIP1 PDZ7 contains a large solvent-exposed hydrophobic surface at a site distinct from the conventional ligand-binding alphaB/betaB-groove. NMR titration experiments show that GRIP1 PDZ7 binds to GRASP-1 via this hydrophobic surface. Our data uncover a novel PDZ domain-mediated protein interaction mode that may be responsible for multimerization of other PDZ domain-containing scaffold proteins.     

Interaction partners of the PDZ domain of erbin

In order to identify proteins that bind to the PDZ domain of Erbin, we tested the C-termini of several proteins in a yeast two-hybrid assay. ErbB2, APC, beta-catenin, c-Rel and HTLV-1 Tax were identified as ligands of the PDZ domain of Erbin. The interactions were verified by co-immunoprecipitation experiments. These findings demonstrate the promiscuity of the PDZ domain of Erbin.