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 AMPA receptors. The interaction between GRIP1 PDZ7 and a Ras guanine nucleotide exchange factor, GRASP-1, regulates synaptic distribution of AMPA receptors. The three-dimensional structure of GRIP1 PDZ7 determined by NMR spectroscopy is reported. 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. These data uncover a novel PDZ domain-mediated protein interaction mode that may be responsible for multimerization of other PDZ domain-containing scaffold proteins (Feng, 2002).
The interaction of the glutamate receptor subunits 2 and 3 (GluR2/3: see Drosophila Glutamate receptor IIA and Glutamate receptor IIB) with multi-PDZ domain glutamate receptor-interacting protein (GRIP) is important for the synaptic trafficking and clustering of the receptors. Binding of GluR2/3 to GRIP requires both the fourth and fifth PDZ domains (PDZ4 and PDZ5) to be covalently linked, although only one PDZ domain is directly involved in binding to the receptor tail. To elucidate the molecular basis of this mode of PDZ domain-mediated target recognition, the solution structures of the PDZ45 tandem and the isolated PDZ4 of GRIP were solved. The two PDZ domains form a compact structure with a fixed interdomain orientation. The interdomain packing and the stable folding of both PDZ domains require a short stretch of amino acids N-terminal to PDZ4 and a conserved linker connecting PDZ4 and PDZ5. PDZ4 contains a deformed aB-bB groove that is unlikely to bind to carboxyl peptides. Instead, the domain stabilizes the structure of PDZ5 (Feng, 2003).
PDZ domains bind to short segments within target proteins in a sequence-specific fashion. GRIP/ABP family proteins contain six to seven PDZ domains and interact via the sixth PDZ domain (class II) with the C termini of various proteins including liprin-alpha. In addition, the PDZ456 domain mediates the formation of homo- and hetero-multimers of GRIP proteins. To better understand the structural basis of peptide recognition by a class II PDZ domain and PDZ-mediated multimerization, the crystal structures of the GRIP1 PDZ6 domain alone and in complex with a synthetic C-terminal octapeptide of human liprin-alpha was determined at resolutions of 1.5 and 1.8 Å, respectively. Remarkably, unlike other class II PDZ domains, Ile-736 at alphaB5 rather than conserved Leu-732 at alphaB1 makes a direct hydrophobic contact with the side chain of the Tyr at the -2 position of the ligand. Moreover, the peptide-bound structure of PDZ6 shows a slight reorientation of helix alphaB, indicating that the second hydrophobic pocket undergoes a conformational adaptation to accommodate the bulkiness of the Tyr side chain, and forms an antiparallel dimer through an interface located at a site distal to the peptide-binding groove. This configuration may enable formation of GRIP multimers and efficient clustering of GRIP-binding proteins (Im, 2003).
A clone has been isolated from a rat brain cDNA library corresponding to a 2779-bp cDNA encoding a novel splice form of the glutamate receptor interacting protein-1 (GRIP1). This 696-amino acid has been termed 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 interacts with GluR2/3 subunits of the AMPA 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 AMPA receptor clusters. Transfection of hippocampal neurons with hemagglutinin-tagged GRIP1c 4-7 showed that it targets 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 show 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 (Charych, 2004).
The mechanisms by which glutamate receptors are concentrated in brain excitatory synapses are believed to involve interactions between receptor subunits and postsynaptic anchoring or scaffolding proteins. Recently GRIP, a protein containing seven PDZ domains, was identified as an AMPA receptor binding protein and implicated in the synaptic targeting of AMPA receptors. GRIP mRNA is also expressed in some tissues outside of the brain, including testis and kidney. Specific antibodies were raised to study GRIP protein. On Western blots, GRIP protein appears as a heterogeneous band (approximately 130 kilodaltons) that is expressed in widespread brain regions and throughout postnatal development. Biochemical studies reveal that GRIP is largely membrane associated and enriched in the postsynaptic density (PSD), though not as highly concentrated in the PSD as is PSD-95. By immunohistochemistry, GRIP is distributed in a somatodendritic pattern in neurons of adult rat brain, with especially prominent expression in a subset of interneurons (Wyszynski, 1998).
The PDZ domain-containing proteins, such as PSD-95 and GRIP, have been suggested to be involved in the targeting of glutamate receptors, a process that plays a critical role in the efficiency of synaptic transmission and plasticity. To address the molecular mechanisms underlying AMPA receptor synaptic localization, several GRIP-associated proteins (GRASPs) have been identified that bind to distinct PDZ domains within GRIP. GRASP-1 is a neuronal rasGEF associated with GRIP and AMPA receptors in vivo. Overexpression of GRASP-1 in cultured neurons specifically reduces the synaptic targeting of AMPA receptors. In addition, the subcellular distribution of both AMPA receptors and GRASP-1 is rapidly regulated by the activation of NMDA receptors. These results suggest that GRASP-1 may regulate neuronal ras signaling and contribute to the regulation of AMPA receptor distribution by NMDA receptor activity (Ye, 2000).
LTP and LTD have been proposed to be mediated, in part, by changes in AMPA receptor function. Increases in AMPA receptor responses have been observed during the expression of LTP. Recently, it has been shown that a high proportion of synapses in hippocampal CA1 region contain only NMDA receptors and acquire AMPA receptors only after the induction of LTP. This emergence of AMPA receptor current seems due to the appearance of synaptic AMPA receptors. Moreover, NMDA receptor-dependent LTD in cultured neurons has recently been observed to correlate with a decrease in the levels of synaptic AMPA receptors. Previous studies have suggested that AMPA receptor-associated proteins, such as GRIP, are involved in the synaptic targeting of AMPA receptors. In this study, GRASP-1 has been added to this complex and evidence is provided that GRASP-1 may also be important in regulation of AMPA receptor function and may play a role in AMPA receptor synaptic targeting. Overexpression of GRASP-1 in neurons downregulates synaptic AMPA receptor clusters, while it has no effect on synaptic NMDA receptor synaptic targeting. Both the rasGEF catalytic domain and the C-terminal 'regulatory' domain were required for this activity. Activation of NMDA receptors dramatically induces the redistribution of both GRASP-1 and AMPA receptors from punctate membrane structures to a more diffuse pattern. Together with the GRASP-1 overexpression data, these results suggest that the overall spatial distribution of GRASP-1, as well as the absolute levels, may be important for AMPA receptor targeting. These results suggest that GRASP-1 and possibly ras signaling may play a role in the regulation of AMPA receptor synaptic targeting and its regulation by NMDA receptor activity (Ye, 2000).
Excitatory synaptic currents in the mammalian brain are typically mediated by the neurotransmitter glutamate, acting at AMPA receptors. Immunocytochemistry has been used to investigate the distribution of AMPA receptor-binding protein (ABP) in the cerebral neocortex. ABP is most prominent in pyramidal neurons, although it was also present (at lower levels) in interneurons. ABP and its putative binding partners, the GluR2/3 subunits of the AMPA receptor, exhibit prominent cellular colocalization. Under appropriate processing conditions, colocalization was documented in puncta, many of which were recognized as dendritic spines. However, a sizable minority of GluR2/3-positive puncta were immunonegative for ABP. Because glutamate receptor-interacting protein (GRIP) may also anchor GluR2, the relative distribution of ABP and GRIP was studied. There was extensive colocalization of these two antigens at the cellular level, although GRIP, unlike ABP, was strongest in nonpyramidal neurons. Different parts of a single dendrite could stain selectively for ABP or GRIP. To further characterize this heterogeneity, punctate staining of neuropil was investigated using synaptophysin and the membrane tracer DiA to identify probable synapses. Some puncta were comparably positive for both ABP and GRIP, but the majority were strongly positive for one antigen and only weakly positive or immunonegative for the other. This heterogeneity could be seen even within adjacent spines of a single dendrite. These data suggest that ABP may act as a scaffold for AMPA receptors either in concert with or independently from GRIP (Burette, 2001).
Long-term changes in excitatory synapse strength are thought to reflect changes in synaptic abundance of AMPA receptors mediated by receptor trafficking. AMPA receptor-binding protein (ABP; GRIP2) and glutamate receptor-interacting protein (GRIP) are two similar PDZ (postsynaptic density 95/Discs large/zona occludens 1) proteins that interact with glutamate receptors 2 and 3 (GluR2 and GluR3) subunits. Both proteins have proposed roles during long-term potentiation and long-term depression in the delivery and anchorage of AMPA receptors at synapses. A variant of ABP-L (seven PDZ form of ABP) called pABP-L is palmitoylated at a cysteine residue at position 11 within a novel 18 amino acid N-terminal leader sequence encoded through differential splicing. In cultured hippocampal neurons, nonpalmitoylated ABP-L localizes with internal GluR2 pools expressed from a Sindbis virus vector, whereas pABP-L is membrane targeted and associates with surface-localized GluR2 receptors at the plasma membrane in spines. Mutation of Cys-11 to alanine blocks the palmitoylation of pABP-L and targets the protein to intracellular clusters, confirming that targeting the protein to spines is dependent on palmitoylation. Non-palmitoylated GRIP is primarily intracellular, but a chimera with the pABP-L N-terminal palmitoylation sequence linked to the body of the GRIP protein is targeted to spines. It is suggested that pABP-L and ABP-L provide, respectively, synaptic and intracellular sites for the anchorage of AMPA receptors during receptor trafficking to and from the synapse (DeSouza, 2002).
Postsynaptic molecules with PDZ domains (PDZ proteins) interact with various glutamate receptors and regulate their subcellular trafficking and stability. In rat neocortical development, the protein expression of AMPA-type glutamate receptor GluR1 lags behind its mRNA expression and rather parallels an increase in PDZ protein levels. One of the neurotrophins, brain-derived neurotrophic factor (BDNF), appears to contribute to this process, regulating the PDZ protein expression. In neocortical cultures, BDNF treatment upregulates SAP97, GRIP1, and Pick1 PDZ proteins. Conversely, BDNF gene targeting downregulates these same PDZ molecules. The BDNF-triggered increases in PDZ proteins results in the elevation of their total association with the AMPA receptors GluR1 and GluR2/3, which leads to the increase in AMPA receptor proteins. When Sindbis viruses carrying GluR1 or GluR2 C-terminal decoys disrupted their interactions, GluR2 C-terminal decoys inhibited both BDNF-triggered GluR1 and GluR2/3 increases, whereas GluR1 C-terminal decoys blocked only the BDNF-triggered GluR1 increase. In agreement, coexpression of SAP97 and GluR1 in nonneuronal HEK293 cells increases both proteins compared with their single transfection, implying mutual stabilization. This work reveals a novel function of BDNF in postsynaptic development by regulating the PDZ protein expression (Jourdi, 2003).
In cells, molecular motors operate in polarized sorting of molecules, although the steering mechanisms of motors remain elusive. In neurons, the kinesin motor conducts vesicular transport such as the transport of synaptic vesicle components to axons and of neurotransmitter receptors to dendrites, indicating that vesicles may have to drive the motor for the direction to be correct. An AMPA receptor subunit--GluR2-interacting protein (GRIP1)--can directly interact and steer kinesin heavy chains to dendrites as a motor for AMPA receptors. As would be expected if this complex is functional, both gene targeting and dominant negative experiments of heavy chains of mouse kinesin showed abnormal localization of GRIP1. Moreover, expression of the kinesin-binding domain of GRIP1 resulted in accumulation of the endogenous kinesin predominantly in the somatodendritic area. This pattern was different from that generated by the overexpression of the kinesin-binding scaffold protein JSAP1 (JNK/SAPK-associated protein-1, also known as Mapk8ip3), which occurred predominantly in the somatoaxon area. These results indicate that directly binding proteins can determine the traffic direction of a motor protein (Setou, 2002).
Glutamate receptor interacting protein (GRIP) is a member of the PDZ domain-containing protein family that is localized in the postsynaptic density area. This protein has been reported to interact specifically with the C-termini of AMPA-selective glutamate receptor channel subunits, GluRalpha2 and GluRalpha3 through its PDZ domains. To clarify the physiological functions of GRIP, mouse GRIP1 was cloned; there are three sites for alternative splicing and two putative translational start codons. Metabolic labeling of COS-7 cells expressing two N-terminal GRIP1 proteins demonstrate that these proteins differed in their pattern of palmitoylation. These findings suggested that the molecular diversity of GRIP1 underlies the localization and functional heterogeneity of this protein (Yamazaki, 2001).
Glutamate receptor-interacting protein 1 (GRIP1) is an adaptor protein composed of seven PDZ (postsynaptic density-95/Discs large/zona occludens-1) domains, capable of mediating diverse protein-protein interactions. GRIP1 has been implicated in the regulation of neuronal synaptic function, but its physiologic roles have not been defined in vivo. Elimination of murine GRIP1 results in embryonic lethality. GRIP1-/- embryos develop abnormalities of the dermo-epidermal junction, resulting in extensive skin blistering around day 12 of embryonic life. Ultra-structural characterization of the blisters (or bullae) revealed cleavage of the dermo-epidermal junction below the lamina densa, an alteration reminiscent of the dystrophic form of human epidermolysis bullosa. Blisters were also observed in the lateral ventricle of the brain and in the meninges covering the cerebral cortex. These genetic data suggest that the GRIP1 scaffolding protein is required for the formation and integrity of the dermo-epidermal junction and reveal the importance of PDZ domains in the organization of supramolecular structures essential for mammalian embryonic development (Bladt, 2002).
During embryogenesis, the epidermis consists of basal epithelial cells and periderm, a layer of thin, endothelium-like cells that develops between E9 and E11. To resist mechanical shearing forces, the epidermis is anchored to the dermis at the dermo-epidermal junction. Basal keratinocytes are strongly anchored to the papillary dermis through a complex system of attachment units organized inside and around the basement membrane zone (BMZ), comprising plasma membrane, lamina lucida, and lamina densa (LD). The hemidesmosomes are electron-dense cytoplasmic plaques connected intracellularly with keratin intermediate filaments and extracellularly through anchoring filaments to the anchoring fibrils (Afb) in the LD. Afb extend from the LD to the anchoring plaque (AP) in the dermis. A number of large oligomeric proteins, including alpha6ß4 integrin, laminin 5, and collagens VII and XVII form these anchoring complexes and have been implicated in the pathogenesis of clinical variants of epidermolysis bullosa (EB) (Bladt, 2002).
Dystrophic EB (DEB), the type of disorder apparently observed in GRIP1-/- embryos, is characterized by tissue cleavage below the LD. In humans, DEB comprises two dominantly and four recessively inherited disorders, associated with mutations of the collagen VII gene, COL7A1. Targeted deletion of collagen VII in the mouse also results in DEB-type skin lesions and death within 2 weeks of birth. In Col7A1-/- newborn skin adhesion of the epidermis and dermis occurs, but is very fragile and exhibits low resistance to external shearing forces. In contrast, in the absence of GRIP1, the cohesion between dermis and epidermis seems to be entirely abrogated, and cleavage of the two layers occurs in an environment, which is highly protected from mechanical stress by the amniotic fluid. There is presently no evidence for GRIP1 mutations in inherited human blistering disorders, but the role of human GRIP1 in skin formation warrants study (Bladt, 2002).
In addition to the epidermal blistering, defects in the brain, characterized by the formation of bullae protruding into the lateral ventricle and at the level of the meninges covering the cerebral cortex, were observed. Interestingly loss of basement membrane components has been previously associated with brain defects. Absence of Hsp47 , a stress-inducible glycoprotein, which transiently binds to newly synthesized procollagen, results in abnormal orientation of the neuroepithelium with invasion of the underlying mesenchyme. This defect is probably caused by altered collagen I and IV synthesis and loss of the basement membrane. Deletion of ß1-integrin in neurons and glia causes abnormal remodeling of the meningeal basement membrane. The structural similarity between the epidermal-dermal junction and the basement membrane of meninges and the neuroepithelium of the lateral ventricle supports the hypothesis that skin blistering and intracranial bullae in GRIP1 mutants may have a common pathogenetic mechanism. Despite the expression of GRIP1 in the olfactory epithelium and retina, these tissues did not exhibit obvious abnormalities. A compensatory role of GRIP2 and/or background-dependent factors may have a protective effect on these epithelial structures (Bladt, 2002).
These findings raise a number of questions as to the in vivo function of GRIP1. It is intriguing that the lack of an intracellular protein results in detachment of the dermal-epidermal junction below the LD. Furthermore, preliminary observations that GRIP1-/- embryos have altered collagen type VII immunostaining and reduced plasma membrane electron density suggest a profound structural disorganization of the BMZ. GRIP1 could participate in multiple ways in the organization of the BMZ. First, GRIP1 may be involved in the scaffolding or localization of adhesion complexes and anchoring molecules responsible for epidermal-dermal cohesion. In addition, GRIP1 is implicated in vesicular trafficking and could potentially regulate secretion and/or processing of extracellular matrix proteins. In any event, the phenotype of GRIP1-/- embryos strongly suggests that the architecture of the epidermal-dermal junction requires PDZ domain-mediated interactions, likely to organize junctional components into a functional structure (Bladt, 2002).
The importance of GRIP1 to the integrity of the dermo-epidermal junction is not at odds with a role in the nervous system. Since these findings indicate an essential role of GRIP1 in organizing the basal domain of epithelial cells and previous reports have shown its involvement in synaptic activity, it is tempting to speculate that GRIP1 may have functional similarities to Caenorhabditis elegans LIN-10. This PDZ domain-containing protein is required for both basolateral localization of the LET-23 receptor tyrosine kinase in vulval precursor epithelial cells and postsynaptic localization of the glutamate receptor GLR-1. It will be of interest to explore the extent to which the basal domain of polarized epithelial cells is functionally analogous to the dendritic compartment of neurons (Bladt, 2002).
Cell adhesion to extracellular matrix (ECM) proteins is crucial for the structural integrity of tissues and epithelial-mesenchymal interactions mediating organ morphogenesis. Loss of GRIP1 leads to the formation of subepidermal hemorrhagic blisters, renal agenesis, syndactyly or polydactyly and permanent fusion of eyelids (cryptophthalmos). Similar malformations are characteristic of individuals with Fraser syndrome and animal models of this human genetic disorder, such as mice carrying the blebbed mutation (bl) in the gene encoding the Fras1 ECM protein. GRIP1 can physically interact with Fras1 and is required for the localization of Fras1 to the basal side of cells. In one animal model of Fraser syndrome, the eye-blebs (eb) mouse, Grip1 is disrupted by a deletion of two coding exons. These data indicate that GRIP1 is required for normal cell-matrix interactions during early embryonic development and that inactivation of Grip1 causes Fraser syndrome-like defects in mice (Takamiya, 2004).
AMPA glutamate receptors mediate the majority of rapid excitatory synaptic transmission in the central nervous system and play a role in the synaptic plasticity underlying learning and memory. AMPA receptors are heteromeric complexes of four homologous subunits (GluR1-4) that differentially combine to form a variety of AMPA receptor subtypes. These subunits are thought to have a large extracellular amino-terminal domain, three transmembrane domains and an intracellular carboxy-terminal domain. AMPA receptors are localized at excitatory synapses and are not found on adjacent inhibitory synapses enriched in GABA(A) receptors. The targeting of neurotransmitter receptors, such as AMPA receptors, and ion channels to synapses is essential for efficient transmission. A protein motif called a PDZ domain is important in the targeting of a variety of membrane proteins to cell-cell junctions including synapses. A synaptic PDZ domain-containing protein GRIP (glutamate receptor interacting protein) has been identified that specifically interacts with the C termini of AMPA receptors. GRIP is a new member of the PDZ domain-containing protein family that has seven PDZ domains and no catalytic domain. GRIP appears to serve as an adapter protein that links AMPA receptors to other proteins and may be critical for the clustering of AMPA receptors at excitatory synapses in the brain (Dong, 1997).
AMPA receptor-binding protein (ABP) is a postsynaptic density (PSD) protein related to glutamate receptor-interacting protein (GRIP). ABP has two sets of three PDZ domains, which bind the GluR2/3 AMPA receptor subunits. ABP exhibits widespread CNS expression and is found at the postsynaptic membrane. The protein interactions of the ABP/GRIP family differ from the PSD-95 family, which bind N-methyl-D-aspartate (NMDA) receptors. ABP binds to the GluR2/3 C-terminal VKI-COOH motif via class II hydrophobic PDZ interactions, distinct from the class I PSD-95-NMDA receptor interaction. ABP and GRIP also form homo- and hetero-multimers through PDZ-PDZ interactions but do not bind PSD-95. It is suggested that the ABP/GRIP and PSD-95 families form distinct scaffolds that anchor, respectively, AMPA and NMDA receptors (Srivastava, 1998).
The NMDA and AMPA classes of ionotropic glutamate receptors are concentrated at postsynaptic sites in excitatory synapses. NMDA receptors interact via their NR2 subunits with PSD-95/SAP90 family proteins, whereas AMPA receptors bind via their GluR2/3 subunits to glutamate receptor-interacting protein (GRIP), AMPA receptor-binding protein (ABP), and protein interacting with C kinase 1 (PICK1). A novel cDNA (termed ABP-L/GRIP2) is described that is virtually identical to ABP except for additional GRIP-like sequences at the N-terminal and C-terminal ends. Like GRIP (here termed GRIP1), ABP-L/GRIP2 contains a seventh PDZ domain at its C terminus. Using antibodies that recognize both these proteins, the subcellular localization of GRIP1 and ABP-L/GRIP2 (collectively termed GRIP) and their biochemical association with AMPA receptors are reported. GRIP is present at excitatory synapses and also at nonsynaptic membranes and within intracellular compartments. The association of native GRIP and AMPA receptors was confirmed biochemically by coimmunoprecipitation from rat brain extracts. A majority of detergent-extractable GluR2/3 is complexed with GRIP in the brain. However, only approximately half of GRIP is associated with AMPA receptors. Unexpectedly, immunocytochemistry of cultured hippocampal neurons and rat brain at the light microscopic level shows enrichment of GRIP in GABAergic neurons and in GABAergic nerve terminals. Thus GRIP is associated with inhibitory as well as excitatory synapses. Collectively, these findings support a role for GRIP in the synaptic anchoring of AMPA receptors but also suggest that GRIP has additional functions unrelated to the binding of AMPA receptors (Wyszynski, 1999).
The molecular mechanisms underlying the targeting and localization of glutamate receptors at postsynaptic sites is poorly understood. A PDZ domain-containing protein, glutamate receptor-interacting protein 1 (GRIP1), has been identified that specifically binds to the C termini of AMPA receptor subunits and may be involved in the synaptic targeting of these receptors. The cloning of GRIP2, a homolog of GRIP1, is reported along with the characterization of the GRIP1 and GRIP2 proteins in the rat CNS. Recently, a GluR2/3 binding protein homologous to GRIP1, AMPA receptor-binding protein (ABP), has been described (Srivastava, 1998). ABP is apparently a short splice variant of GRIP2 that lacks the N terminus and the seventh PDZ domain of GRIP2. Similar to GRIP1, GRIP2 contains seven PDZ domains that are very homologous to GRIP1 within the PDZ domains (64%-93% identity) but has little sequence similarity in the linker regions between the PDZ domains. GRIP1 and GRIP2 are ~130 kDa proteins that are highly enriched in brain. GRIP1 and GRIP2 are widely expressed in brain, with the highest levels found in the cerebral cortex, hippocampus, and olfactory bulb. Biochemical studies show that GRIP1 and GRIP2 are enriched in synaptic plasma membrane and postsynaptic density fractions. GRIP1 is expressed early in embryonic development before the expression of AMPA receptors and peaks in expression at postnatal day 8-10. In contrast, GRIP2 is expressed relatively late in development and parallels the expression of AMPA receptors. Immunohistochemistry using the GRIP1 antibodies demonstrates that GRIP1 is expressed in neurons in a somatodendritic staining pattern. At the ultrastructural level GRIP1 is enriched in dendritic spines near the postsynaptic density and is expressed in dendritic shafts and in peri-Golgi regions in the neuronal soma. GRIP1 appears to be clustered at both glutamatergic and GABAergic synapses. These results suggest that GRIP1 and GRIP2 are AMPA receptor binding proteins potentially involved in the targeting of AMPA receptors to synapses. GRIP1 also may play functional roles at both excitatory and inhibitory synapses, as well as in early neuronal development (Dong, 1999b).
Interaction with the multi-PDZ protein GRIP is required for the synaptic targeting of AMPA receptors. GRIP binds to the liprin-alpha/SYD2 family of proteins that interacts with LAR receptor protein tyrosine phosphatases (LAR-RPTPs) that are implicated in presynaptic development. In neurons, liprin-alpha and LAR-RPTP are enriched at synapses and coimmunoprecipitate with GRIP and AMPA receptors. Dominant-negative constructs that interfere with the GRIP-liprin interaction disrupt the surface expression and dendritic clustering of AMPA receptors in cultured neurons. Thus, by mediating the targeting of liprin/GRIP-associated proteins, liprin-alpha is important for postsynaptic as well as presynaptic maturation (Wyszynski, 2002).
Four PDZ domain-containing proteins, syntenin, PICK1, GRIP, and PSD95, have been identified as interactors with the kainate receptor (KAR) subunits GluR52b, GluR52c, and GluR6. Of these, it is shown that both GRIP and PICK1 interactions are required to maintain KAR-mediated synaptic function at mossy fiber-CA3 synapses. In addition, PKCalpha can phosphorylate ct-GluR52b at residues S880 and S886, and PKC activity is required to maintain KAR-mediated synaptic responses. It is proposed that PICK1 targets PKCalpha 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 (Hirbec, 2003).
The finding that KARs and AMPARs can bind to a common pool of PDZ proteins suggests that these proteins may play important general roles in the regulation of glutamatergic synapses. Based on the present findings and previous work on AMPARs, it is possible to speculate on the molecular mechanisms that mediate the differential regulation of AMPARs and KARs by these PDZ proteins. In this scheme, AMPARs are secured in intracellular pools via association of the GluR2 subunit with GRIP and/or ABP. These 'gripped' receptors are immobile over the time course of the electrophysiology experiments. PICK1 exchanges for GRIP and targets PKCalpha, which then phosphorylates S880 of GluR2, thereby preventing the rebinding of GRIP. The S880-phosphorylated AMPARs are mobile and available for surface expression. It is proposed that KARs are also 'gripped' by GRIP, but in this case, PICK1-targetted, PKC-dependent phosphorylation stabilizes the GRIP interaction with GluR5/6 and anchors the receptors at the postsynaptic membrane. These data are entirely consistent with the observations that blockade of either GRIP or PICK1 binding, or inhibition of PKC, results in a rapid decrease in KAR-mediated synaptic currents. It is speculated that, whereas phosphorylation of S880 of GluR2 prevents GRIP binding, phosphorylation of S880 and/or S886 of GluR52b (and/or equivalent residues of GluR6) stabilizes GRIP binding and anchors the receptors at the synapse (Hirbec, 2003).
These differences in the molecular consequences of PKC-mediated phosphorylation of AMPARs and KARs can explain the differential regulation in opposite directions of the functional synaptic responses. The results showing that, at the same population of synapses, disruption of PDZ protein interactions results in an increase in EPSCA and a simultaneous decrease in EPSCK suggests that these proteins may act to regulate the relative proportions of AMPARs and KARs at synapses. Physiologically, given the distinct biophysical and functional profiles of AMPARs and KARs, the dynamic regulation of these interactions will play important roles in the modulation of basal glutamatergic synaptic transmission. Furthermore, it has been reported previously that some forms of developmental and activity-dependent synaptic plasticity involve a switch from functionally expressed KARs to AMPARs. The differential effects of PDZ-interacting proteins demonstrated here on these two receptor types provide an attractive molecular mechanism to account for these developmental and activity-dependent changes in the AMPAR and KAR complement at synapses (Hirbec, 2003).
Liprin-alpha is a multidomain protein that interacts with the LAR family of receptor protein tyrosine phosphatases and the GRIP/ABP family of AMPA receptor-interacting proteins. Previous studies have indicated that liprin-alpha regulates the development of presynaptic active zones and that the association of liprin-alpha with GRIP is required for postsynaptic targeting of AMPA receptors. However, the underlying molecular mechanisms are not well understood. Liprin-alpha directly interacts with GIT1, a multidomain protein with GTPase-activating protein activity for the ADP-ribosylation factor family of small GTPases known to regulate protein trafficking and the actin cytoskeleton. Electron microscopic analysis indicates that GIT1 distributes to the region of postsynaptic density (PSD) as well as presynaptic active zones. GIT1 is enriched in PSD fractions and forms a complex with liprin-alpha, GRIP, and AMPA receptors in brain. Expression of dominant-negative constructs interfering with the GIT1-liprin-alpha interaction leads to a selective and marked reduction in the dendritic and surface clustering of AMPA receptors in cultured neurons. These results suggest that the GIT1-liprin-alpha interaction is required for AMPA receptor targeting and that GIT1 may play an important role in the organization of presynaptic and postsynaptic multiprotein complexes (Ko, 2003).
The proteoglycan NG2 is expressed by immature glial cells in the developing and adult central nervous system. Using the COOH-terminal region of NG2 as bait in a yeast two-hybrid screen, the glutamate receptor interaction protein GRIP1, a multi-PDZ domain protein, was identified as an interacting partner. NG2 exhibits a PDZ binding motif at the extreme COOH terminus that binds to the seventh PDZ domain of GRIP1. In addition to the published expression in neurons, GRIP1 is expressed by immature glial cells. GRIP1 is known to bind to the GluRB subunit of the AMPA glutamate receptor expressed by subpopulations of neurons and immature glial cells. In cultures of primary oligodendrocytes, cells coexpress GluRB and NG2. A complex of NG2, GRIP1, and GluRB can be precipitated from transfected mammalian cells and from cultures of primary oligodendrocytes. Furthermore, NG2 and GRIP can be coprecipitated from developing brain tissue. These data suggest that GRIP1 acts as a scaffolding molecule clustering NG2 and AMPA receptors in immature glia. In view of the presence of synaptic contacts between neurons and NG2-positive glial cells in the hippocampus and the close association of NG2-expressing glial cells with axons, a role is suggested for the NG2.AMPA receptor complex in glial-neuronal recognition and signaling (Stegmuller, 2003).
At many excitatory central synapses, activity produces a lasting change in the synaptic response by modifying postsynaptic AMPA receptors (AMPARs). Although much is known about proteins involved in the trafficking of Ca2+-impermeable (GluR2-containing) AMPARs, little is known about protein partners that regulate subunit trafficking and plasticity of Ca2+-permeable (GluR2-lacking) AMPARs. At cerebellar parallel fiber-stellate cell synapses, activity triggers a novel type of plasticity: Ca2+ influx through GluR2-lacking synaptic AMPARs drives incorporation of GluR2-containing AMPARs, generating rapid, lasting changes in excitatory postsynaptic current properties. This study examined how GRIP and protein interacting with C-kinase-1 (PICK) regulate subunit trafficking and plasticity. It was found that repetitive synaptic activity triggers loss of synaptic GluR2-lacking AMPARs by selectively disrupting their interaction with GRIP and that PICK drives activity-dependent delivery of GluR2-containing receptors. This dynamic regulation of AMPARs provides a feedback mechanism for controlling Ca2+ permeability of synaptic receptors (Liu, 2005).
Several proteins have been shown to interact specifically with the C termini of the GluR2 and GluR3 AMPA receptor subunits. These include three PDZ proteins, ABP (AMPA Receptor Binding protein), GRIP (Glutamate Receptor Interacting Protein), and PICK1 (Protein Interacting with C Kinase). The two splice forms of ABP that contain either 6 or 7 PDZ domains, and the 7 PDZ domain GRIP are members of a novel sequence-related protein family. GRIP PDZ4-5 domains and ABP PDZ5 domain show the highest affinity for the GluR2/3 C terminus. The remaining PDZ domains of GRIP and ABP are likely to mediate additional interactions, possibly anchoring the AMPA receptor to cytoskeletal proteins or coupling the receptor to intracellular enzymes. PICK1, which was cloned as a PKC-interacting protein, contains a single N-terminal PDZ domain. When coexpressed with GluR2 in heterologous cells, PICK1 induces GluR2 surface clustering and intracellular redistribution (Osten, 2000 and references therein).
The roles of GRIP, ABP, and PICK1 in GluR2 AMPA receptor trafficking have been studied. An epitope-tagged MycGluR2 subunit, when expressed in hippocampal cultured neurons, is specifically targeted to the synaptic surface. With the mutant MycGluR2delta1-10, which lacks the PDZ binding site, the overall dendritic intracellular transport and the synaptic surface targeting are not affected. However, over time, MycGluR2delta1-10 accumulates at synapses significantly less than MycGluR2. Notably, a single residue substitution, S880A, which blocks binding to ABP/GRIP but not to PICK1, reduces synaptic accumulation to the same extent as the PDZ site truncation. It is concluded that the association of GluR2 with ABP and/or GRIP but not PICK1 is essential for maintaining the synaptic surface accumulation of the receptor, possibly by limiting its endocytotic rate (Osten, 2000).
AMPA receptor stability and movement at synapses are important factors controlling synaptic strength. The roles have been studied of proteins N-ethylmaleimide-sensitive fusion protein (NSF), glutamate receptor AMPAR binding protein (ABP)-interacting protein (GRIP)/(ABP), and protein interacting with C-kinase-1 (PICK1) that interact with the GluR2 subunit in the control of the surface expression and cycling of AMPA receptors. Epitope-tagged GluR2 formed functional receptors that exhibited targeting to synaptic sites. Constructs in which binding to NSF, PDZ proteins (GRIP/ABP and PICK1), or GRIP/ABP alone was eliminated each exhibited normal surface targeting and constitutive cycling. The lack of NSF binding, however, resulted in receptors that were endocytosed to a greater extent than wild-type receptors in response to application of AMPA or NMDA. Conversely, the behavior of the GluR2 mutants incapable of binding to GRIP/ABP suggests that these PDZ proteins play a role in the stabilization of an intracellular pool of AMPA receptors that have been internalized on stimulation, thus inhibiting their recycling to the synaptic membrane. These results provide further evidence for distinct functional roles of GluR2-interacting proteins in AMPA receptors trafficking (Braithwaite, 2002).
Transmembrane ephrinB proteins have important functions during embryonic patterning as ligands for Eph receptor tyrosine kinases and presumably as signal-transducing receptor-like molecules. Consistent with 'reverse' signaling, ephrinB1 is localized in sphingo-lipid/cholesterol-enriched raft microdomains, platforms for the localized concentration and activation of signaling molecules. Glutamate receptor-interacting protein (GRIP) and a highly related protein, termed GRIP2, are recruited into these rafts through association with the C-terminal PDZ target site of ephrinB1. Stimulation of ephrinB1 with soluble EphB2 receptor ectodomain causes the formation of large raft patches that also contain GRIP proteins. Moreover, a GRIP-associated serine/threonine kinase activity is recruited into ephrinB1-GRIP complexes. These findings suggest that GRIP proteins provide a scaffold for the assembly of a multiprotein signaling complex downstream of ephrinB ligands (Bruckner, 1999).
Ephrin B proteins function as ligands for B class Eph receptor tyrosine kinases and are postulated to possess an intrinsic signaling function. The sequence at the carboxyl terminus of B-type ephrins contains a putative PDZ binding site, providing a possible mechanism through which transmembrane ephrins might interact with cytoplasmic proteins. To test this notion, a day 10.5 mouse embryonic expression library was screened with a biotinylated peptide corresponding to the carboxyl terminus of ephrin B3. Three of the positive cDNAs encode polypeptides with multiple PDZ domains, representing fragments of the molecule GRIP, the protein syntenin, and PHIP, a novel PDZ domain-containing protein related to Caenorhabditis elegans PAR-3. In addition, the binding specificities of PDZ domains previously predicted by an oriented library approach identified the tyrosine phosphatase FAP-1 as a potential binding partner for B ephrins. In vitro studies have demonstrated that the fifth PDZ domain of FAP-1 and full-length syntenin bind ephrin B1 via the carboxyl-terminal motif. Lastly, syntenin and ephrin B1 could be co-immunoprecipitated from transfected COS-1 cells, suggesting that PDZ domain binding of B ephrins can occur in cells. These results indicate that the carboxyl-terminal motif of B ephrins provides a binding site for specific PDZ domain-containing proteins, which might localize the transmembrane ligands for interactions with Eph receptors or participate in signaling within ephrin B-expressing cells (Lin, 1999).
The site of induction of long-term potentiation (LTP) at mossy fiber-CA3 synapses in the hippocampus is unresolved, with data supporting both pre- and post-synaptic mechanisms. Mossy fiber LTP is reduced by perfusion of postsynaptic neurons with peptides and antibodies that interfere with binding of EphB receptor tyrosine kinases (EphRs) to the PDZ protein GRIP. Mossy fiber LTP was also reduced by extracellular application of soluble forms of B-ephrins, which are normally membrane-anchored presynaptic ligands for the EphB receptors. The application of soluble ligands for presynaptic ephrins increased basal excitatory transmission and occluded both tetanus and forskolin-induced synaptic potentiation. These findings suggest that PDZ interactions in the postsynaptic neuron and trans-synaptic interactions between postsynaptic EphB receptors and presynaptic B-ephrins are necessary for the induction of mossy fiber LTP (Contractor, 2002).
The function of the multi-PDZ domain scaffold protein GRIP1 (glutamate receptor interacting protein 1) in neurons is unclear. To explore the function of GRIP1 in hippocampal neurons, RNA interference (RNAi) was used to knock down the expression of GRIP1. Knockdown of GRIP1 by small interfering RNA (siRNA) in cultured hippocampal neurons causes a loss of dendrites, associated with mislocalization of the GRIP-interacting proteins GIuR2 (AMPA receptor subunit), EphB2 (receptor tyrosine kinase) and KIF5 (also known as kinesin 1; microtubule motor). The loss of dendrites by GRIP1-siRNA was rescued by overexpression of the extracellular domain of EphB2, and was phenocopied by overexpression of the intracellular domain of EphB2 and extracellular application of ephrinB-Fc fusion proteins. Neurons from EphB1-EphB2-EphB3 triple knockout mice showed abnormal dendrite morphogenesis. Disruption of the KIF5-GRIP1 interaction inhibited EphB2 trafficking and strongly impaired dendritic growth. These results indicate an important role for GRIP1 in dendrite morphogenesis by serving as an adaptor protein for kinesin-dependent transport of EphB receptors to dendrites (Hoogenraad, 2005).
Activation of the calcium-dependent protease calpain has been proposed to be a key step in synaptic plasticity in the hippocampus. However, the exact pathway through which calpain mediates or modulates changes in synaptic function remains to be clarified. Glutamate receptor-interacting protein (GRIP) is a substrate of calpain; calpain-mediated GRIP degradation was demonstrated using three different approaches: (1) purified calpain I digestion of synaptic membranes, (2) calcium treatment of frozen-thawed brain sections, and (3) NMDA-stimulated organotypic hippocampal slice cultures. More importantly, calpain activation resulted in the disruption of GRIP binding to the GluR2 subunit of AMPA receptors. Because GRIP has been proposed to function as an AMPA receptor-targeting and synaptic-stabilizing protein, as well as a synaptic-organizing molecule, calpain-mediated degradation of GRIP and disruption of AMPA receptor anchoring are likely to play important roles in the structural and functional reorganization accompanying synaptic modifications in long-term potentiation and long-term depression (Lu, 2001).
The DLX homeodomain proteins control development of the basal ganglia and branchial arches. To identify co-factors that regulate DLX function the yeast two-hybrid assay was used; a DLX interacting protein (DIP2) was found that binds to the N-terminal region of DLX2 via a PDZ domain. DIP2 appears to be an alternatively spliced form of GRIP1, a protein known to bind AMPA glutamate receptors via PDZ domains, and was thus named GRIP1b. Evidence is provided that GRIP1b can function as a transcriptional co-activator of DLX2 and DLX5. Glutamate receptors inhibit this co-activation. These results suggest that some PDZ proteins may regulate transcription via their interactions with homeodomain proteins. Furthermore, these results suggest a link between glutamate receptors, PDZ proteins and the DLX transcription factors, all of which are co-expressed in the developing basal ganglia (Yu, 2001).
Matrix metalloproteinases (MMPs) have been proposed to remodel the extracellular environment of neurons. The metalloproteinase membrane-type 5 MMP (MT5-MMP) binds to AMPA receptor binding protein (ABP) and GRIP (glutamate receptor interaction protein), two related postsynaptic density PDZ domain proteins that target AMPA receptors to synapses. The MT5-MMP C terminus binds ABP PDZ5 and the two proteins coimmunoprecipitate and colocalize in heterologous cells and neurons. MT5-MMP localizes in filopodia at the tips of growth cones in young cultured embryonic hippocampal neurons, and at synapses in mature (21 DIV) neurons. Its enrichment in synaptosomes also indicates a synaptic localization in the mature brain. Deletion of the PDZ binding site impairs membrane trafficking of MT5-MMP, whereas exogenous ABP splice forms that are associated either with the plasma membrane or with the cytosol, respectively, colocalize with MT5-MMP in synaptic spines or recruit MT5-MMP to intracellular compartments. Endogenous MT5-MMP is found in cultured neurons and brain lysates in a proenzyme form that is activated by furin and degraded by auto-proteolysis. Cadherins function as MT5-MMP substrates. These results suggest that ABP directs MT5-MMP proteolytic activity to growth cones and synaptic sites in neurons, where it may regulate axon pathfinding or synapse remodeling through proteolysis of cadherins or other ECM or cell adhesion molecules (Monea, 2006).
Cerebellar LTD requires activation of PKC and is expressed, at least in part, as postsynaptic AMPA receptor internalization. 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) attenuates LTD induction by glutamate/depolarization pairing, as do 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 (Xia, 2000).
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