Interaction between SAP97 and PSD-95, two Maguk proteins involved in synaptic trafficking of AMPA receptors

Synapse-associated protein 97 (SAP97) and postsynaptic density 95 (PSD-95) are closely related membrane-associated guanylate kinase homologs (Maguks) implicated in the synaptic targeting and anchoring of alpha-amino-5-methyl-3-hydroxy-4-isoxazolepropionic acid (AMPA)-selective glutamate receptors. Prompted by accumulating evidence for an oligomeric nature of Maguks, we examined the potential of SAP97 and PSD-95 to form heteromeric complexes. SAP97 and PSD-95 coimmunoprecipitated from rat brain detergent extracts and subsequent glutathione S-transferase pull-down and immunoprecipitation experiments showed that the interaction is mediated by binding of the N-terminal segment of SAP97 (SAP97(NTD)) to the Src homology 3 domain of PSD-95 (PSD-95(SH3)). In cultured hippocampal neurons, expression of green fluorescent protein-tagged PSD-95 triggered accumulation of SAP97 in synaptic spines, which was totally inhibited by coexpression of PSD-95(SH3). Furthermore, overexpression of green fluorescent protein-PSD-95 induced dendritic clustering of GluR-A subunit-containing AMPA receptors, which was strongly inhibited by cotransfection with SAP97(NTD) and PSD-95(SH3) constructs. Our results demonstrated a direct interaction between SAP97 and PSD-95 and suggested that this association may play a functional role in the trafficking and clustering of AMPA receptors.     

Ligand binding is a critical requirement for plasma membrane expression of heteromeric kainate receptors

Intracellular trafficking of ionotropic glutamate receptors is controlled by multiple discrete determinants in receptor subunits. Most such determinants have been localized to the cytoplasmic carboxyl-terminal domain, but other domains in the subunit proteins can play roles in modulating receptor surface expression. Here we demonstrate that formation of an intact glutamate binding site also acts as an additional quality-control check for surface expression of homomeric and heteromeric kainate receptors. A key ligand-binding residue in the KA2 subunit, threonine 675, was mutated to either alanine or glutamate, which eliminated affinity for the receptor ligands kainate and glutamate. We found that plasma membrane expression of heteromeric GluR6/KA2(T675A) or GluR6/KA2(T675E) kainate receptors was markedly reduced compared with wild-type GluR6/KA2 receptors in transfected HEK 293 and COS-7 cells and in cultured neurons. Surface expression of homomeric KA2 receptors lacking a retention/retrieval determinant (KA2-R/A) was also reduced upon mutation of Thr-675 and elimination of the ligand binding site. KA2 Thr-675 mutant subunits were able to co-assemble with GluR5 and GluR6 subunits and were degraded at the same rate as wild-type KA2 subunit protein. These results suggest that glutamate binding and associated conformational changes are prerequisites for forward trafficking of intracellular kainate receptors following multimeric assembly.     

Tyrosine phosphorylation of ionotropic glutamate receptors by Fyn or Src differentially modulates their susceptibility to calpain and enhances their binding to spectrin and PSD-95

Both tyrosine phosphorylation and calpain-mediated truncation of ionotropic glutamate receptors are important mechanisms for synaptic plasticity. Previous work from our laboratory has shown that calpain activation results in truncation of the C-terminal domains of several glutamate receptor subunits. To test whether and how tyrosine phosphorylation of glutamate ionotropic receptor subunits modulates calpain susceptibility, synaptic membranes were phosphorylated by Fyn or Src, two members of the Src family tyrosine kinases. Tyrosine phosphorylation of synaptic membranes by Src significantly reduced calpain-mediated truncation of both NR2A and NR2B subunits of NMDA receptors, but not of GluR1 subunits of AMPA receptors. In contrast, phosphorylation with Fyn significantly protected calpain-mediated truncation of GluR1 subunits of AMPA receptors, but enhanced calpain-mediated truncation of NR2A subunits of NMDA receptors. Similar results were observed with NR2A and NR2B C-terminal domain fusion proteins phosphorylated by Fyn or Src before incubation with calpain and calcium. In addition, phosphorylation of NR2A and NR2B C-terminal fusion proteins by Fyn or Src enhanced their binding to spectrin and PSD-95. Thus, tyrosine phosphorylation impairs or facilitates calpain-mediated truncation of glutamate receptor subunits, depending on which tyrosine kinase is activated. Such mechanisms could serve to regulate receptor integrity and location, in addition to modulating channel properties.     

Quaternary structure, protein dynamics, and synaptic function of SAP97 controlled by L27 domain interactions

Single-particle electron microscopy (EM) combined with biochemical measurements revealed the molecular shape of SAP97 and a monomer-dimer transition that depended on the N-terminal L27 domain. Overexpression of SAP97 drove GluR1 to synapses, potentiated AMPA receptor (AMPAR) excitatory postsynaptic currents (EPSCs), and occluded LTP. Synaptic potentiation and GluR1 delivery were dissociable by L27 domain mutants that inhibit multimerization of SAP97. Loss of potentiation was correlated with faster turnover of monomeric SAP97 mutants in dendritic spines. We propose that L27-mediated interactions of SAP97 with itself or other proteins regulate the synaptic delivery of AMPARs. RNAi knockdown of endogenous PSD-95 depleted surface GluR1 and impaired AMPA EPSCs. In contrast, RNAi knockdown of endogenous SAP97 reduced surface expression of both GluR1 and GluR2 and inhibited both AMPA and NMDA EPSCs. Thus SAP97 has a broader role than its close relative, PSD-95, in the maintenance of synaptic function.     

Calcium/calmodulin-dependent protein kinase II phosphorylation drives synapse-associated protein 97 into spines

Synapse-associated protein 97 (SAP97) has been involved in the correct delivery and clustering of glutamate ionotropic receptors to the postsynaptic compartment. Here we demonstrate that synaptic trafficking of SAP97 itself was modulated by calcium/calmodulin-dependent protein kinase II (CaMKII) in cultured hippocampal neurons. CaMKII activation led to increased targeting of SAP97 into dendritic spines, whereas CaMKII inhibition was responsible for SAP97 high colocalization in the cell soma with the endoplasmic reticulum protein disulfide-isomerase. No effect was detected for other members of the membrane-associated guanylate kinase protein family, such as SAP102 and PSD-95. Transfection of activated alphaCaMKII T286D dramatically increased concentration of both endogenous and transfected SAP97 at postsynaptic terminals. In vitro CaMKII phosphorylation of the SAP97 N-terminal fusion protein and metabolic labeling of transfected COS7 cells indicated SAP97-Ser-39 as a CaMKII phosphosite in the SAP97 protein sequence. Moreover, transfection in hippocampal neurons of SAP97 mutants that blocked or mimicked Ser-39 phosphorylation had effects similar to those observed upon inhibiting or constitutively activating CaMKII. Further, CaMKII-dependent SAP97-Ser-39 phosphorylation determined a redistribution of the glutamate receptor subunit (GluR1) of the AMPA receptor. In conclusion, our data show that CaMKII-dependent SAP97-Ser-39 phosphorylation regulates the association of SAP97 with the postsynaptic complex, thus providing a fine molecular mechanism responsible for the synaptic delivery of SAP97 interacting proteins, i.e. ionotropic glutamate receptor subunits.     

Regulation of Kainate Receptor Subunit mRNA by Stress and Corticosteroids in the Rat Hippocampus

Kainate receptors are a class of ionotropic glutamate receptors that have a role in the modulation of glutamate release and synaptic plasticity in the hippocampal formation. Previous studies have implicated corticosteroids in the regulation of these receptors and recent clinical work has shown that polymorphisms in kainate receptor subunit genes are associated with susceptibility to major depression and response to anti-depressant treatment. In the present study we sought to examine the effects of chronic stress and corticosteroid treatments upon the expression of the mRNA of kainate receptor subunits GluR5-7 and KA1-2. Our results show that, after 7 days, adrenalectomy results in increased expression of hippocampal KA1, GluR6 and GluR7 mRNAs, an effect which is reversed by treatment with corticosterone in the case of KA1 and GluR7 and by aldosterone treatment in the case of GluR6. 21 days of chronic restraint stress (CRS) elevated the expression of the KA1 subunit, but had no effect on the expression of the other subunits. Similarly, 21 days of treatment with a moderate dose of corticosterone also increased KA1 mRNA in the dentate gyrus, whereas a high corticosterone dose has no effect. Our results suggest an interaction between hippocampal kainate receptor composition and the hypothalamic-pituitary-adrenal (HPA) axis and show a selective chronic stress induced modulation of the KA1 subunit in the dentate gyrus and CA3 that has implications for stress-induced adaptive structural plasticity.     

The PDZ1 domain of SAP90. Characterization of structure and binding.

The structural features of the PDZ1 domain of the synapse-associated protein SAP90 have been characterized by NMR. A comparison with the structures of the PDZ2 and PDZ3 domains of SAP90 illustrates significant differences, which may account for the unique binding properties of these homologous domains. Within the postsynaptic density, SAP90 functions as a molecular scaffold with a number of the protein-protein interactions mediated through the PDZ1 domain. Here, using fluorescence anisotropy and NMR chemical shift analysis, we have characterized the association of PDZ1 to the C-terminal peptides of the GluR6 subunit of the kainate receptor, voltage-gated K(+) channel Kv1.4, and microtubule-associate protein CRIPT, all of which are known to associate with SAP90. The latter two, which possess the consensus sequence for binding to PDZ domains (T/S-X-V-oh), have low micromolar binding affinities (1.5-15 microm). The C terminus of GluR6, RLPGKETMA-oh, lacking the consensus sequence, binds to PDZ1 of SAP90 with an affinity of 160 microm. The NMR data illustrate that although all three peptides occupy the binding groove capped by the GLGF loop of PDZ1, specific differences are present, consistent with the variation in binding affinities.     

Interaction of NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase protein, with calmodulin and PSD-95/SAP90. A possible regulatory role in molecular clustering at synaptic sites

NE-dlg/SAP102, a neuronal and endocrine tissue-specific membrane-associated guanylate kinase family protein, is known to bind to C-terminal ends of N-methyl-D-aspartate receptor 2B (NR2B) through its PDZ (PSD-95/Dlg/ZO-1) domains. NE-dlg/SAP102 and NR2B colocalize at synaptic sites in cultured rat hippocampal neurons, and their expressions increase in parallel with the onset of synaptogenesis. We have identified that NE-dlg/SAP102 interacts with calmodulin in a Ca2+-dependent manner. The binding site for calmodulin has been determined to lie at the putative basic alpha-helix region located around the src homology 3 (SH3) domain of NE-dlg/SAP102. Using a surface plasmon resonance measurement system, we detected specific binding of recombinant NE-dlg/SAP102 to the immobilized calmodulin with a Kd value of 44 nM. However, the binding of Ca2+/calmodulin to NE-dlg/SAP102 did not modulate the interaction between PDZ domains of NE-dlg/SAP102 and the C-terminal end of rat NR2B. We have also identified that the region near the calmodulin binding site of NE-dlg/SAP102 interacts with the GUK-like domain of PSD-95/SAP90 by two-hybrid screening. Pull down assay revealed that NE-dlg/SAP102 can interact with PSD-95/SAP90 in the presence of both Ca2+ and calmodulin. These findings suggest that the Ca2+/calmodulin modulates interaction of neuronal membrane-associated guanylate kinase proteins and regulates clustering of neurotransmitter receptors at central synapses.     

Interaction of the tyrosine kinase Pyk2 with the N-methyl-D-aspartate receptor complex via the Src homology 3 domains of PSD-95 and SAP102

The protein-tyrosine kinase Pyk2/CAKbeta/CADTK is a key activator of Src in many cells. At hippocampal synapses, induction of long term potentiation requires the Pyk2/Src signaling pathway, which up-regulates the activity of N-methyl-d-aspartate-type glutamate receptors. Because localization of protein kinases close to their substrates is crucial for effective phosphorylation, we investigated how Pyk2 might be recruited to the N-methyl-d-aspartate receptor complex. This interaction is mediated by PSD-95 and its homolog SAP102. Both proteins colocalize with Pyk2 at postsynaptic dendritic spines in the cerebral cortex. The proline-rich regions in the C-terminal half of Pyk2 bind to the SH3 domain of PSD-95 and SAP102. The SH3 and guanylate kinase homology (GK) domain of PSD-95 and SAP102 interact intramolecularly, but the physiological significance of this interaction has been unclear. We show that Pyk2 effectively binds to the Src homology 3 (SH3) domain of SAP102 only when the GK domain is removed from the SH3 domain. Characterization of PSD-95 and SAP102 as adaptor proteins for Pyk2 fills a critical gap in the understanding of the spatial organization of the Pyk2-Src signaling pathway at the postsynaptic site and reveals a physiological function of the intramolecular SH3-GK domain interaction in SAP102.     

Intracellular trafficking of KA2 kainate receptors mediated by interactions with coatomer protein complex I (COPI) and 14-3-3 chaperone systems

Assembly and trafficking of neurotransmitter receptors are processes contingent upon interactions between intracellular chaperone systems and discrete determinants in the receptor proteins. Kainate receptor subunits, which form ionotropic glutamate receptors with diverse roles in the central nervous system, contain a variety of trafficking determinants that promote either membrane expression or intracellular sequestration. In this report, we identify the coatomer protein complex I (COPI) vesicle coat as a critical mechanism for retention of the kainate receptor subunit KA2 in the endoplasmic reticulum. COPI subunits immunoprecipitated with KA2 subunits from both cerebellum and COS-7 cells, and beta-COP protein interacted directly with immobilized KA2 peptides containing the arginine-rich retention/retrieval determinant. Association between COPI proteins and KA2 subunits was significantly reduced upon alanine substitution of this signal in the cytoplasmic tail of KA2. Temperature-sensitive degradation of COPI complex proteins was correlated with an increase in plasma membrane localization of the homologous KA2 receptor. Assembly of heteromeric GluR6a/KA2 receptors markedly reduced association of KA2 and COPI. Finally, the reduction in COPI binding was correlated with an increased association with 14-3-3 proteins, which mediate forward trafficking of other integral signaling proteins. These interactions therefore represent a critical early checkpoint for biosynthesis of functional KARs.     

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

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

Glutamate-induced cobalt uptake elicited by kainate receptors in rat taste bud cells

Glutamate-induced cobalt uptake reveals non-N-methyl-D-aspartate (non-NMDA) glutamate receptors (GluRs) in rat taste bud cells. However, it is not known which type of non-NMDA glutamate receptors is involved. We used a cobalt staining technique combined with pharmacological tests for kainate or alpha-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) receptors and/or immunohistochemistry against subunits of GluRs to examine the presence of non-NMDA receptors in rat foliate tastebud cells. Cobalt uptake into taste cells was elicited by treating taste buds with glutamate, kainate or SYM 2081, a kainate receptor agonist. Treating taste buds with AMPA or fluorowillardiine did not stimulate significant cobalt uptake. Moreover, 6-cyano-7-nitro-quinoxaline-2, 3-dione significantly reduced cobalt staining elicited by glutamate or kainate receptor agonists, but SYM 2206, an AMPA receptor antagonist, did not. Immunohistochemistry against subunits of GluRs reveals GluR6 and KA1-like immunoreactivity. Moreover, most glutamate-induced cobalt-stained cells showed GluR6 and KA1-like immunoreactivity. These results suggest that glutamate-induced cobalt uptake in taste cells occurs mainly via kainate type GluRs.     

Differential interaction of NMDA receptor subtypes with the post-synaptic density-95 family of membrane associated guanylate kinase proteins

NMDA receptors are a subclass of ionotropic glutamate receptors. They are trafficked and/or clustered at synapses by the post-synaptic density (PSD)-95 membrane associated guanylate kinase (MAGUK) family of scaffolding proteins that associate with NMDA receptor NR2 subunits via their C-terminal glutamate serine (aspartate/glutamate) valine motifs. We have carried out a systematic study investigating in a heterologous expression system, the association of the four major NMDA receptor subtypes with the PSD-95 family of MAGUK proteins, chapsyn-110, PSD-95, synapse associated protein (SAP) 97 and SAP102. We report that although each PSD-95 MAGUK was shown to co-immunoprecipitate with NR1/NR2A, NR1/NR2B, NR1/NR2C and NR1/NR2D receptor subtypes, they elicited differential effects with regard to the enhancement of total NR2 subunit expression which then results in an increased cell surface expression of NMDA receptor subtypes. PSD-95 and chapsyn-110 enhanced NR2A and NR2B total expression which resulted in increased NR1/NR2A and NR1/NR2B receptor cell surface expression whereas SAP97 and SAP102 had no effect on total or cell surface expression of these subtypes. PSD-95, chapsyn-110, SAP97 and SAP102 had no effect on either total NR2C and NR2D subunit expression or cell surface NR1/NR2C and NR1/NR2D expression. A comparison of PSD-95α, PSD-95β and PSD-95α^C3S,C5S showed that PSD-95-enhanced cell surface expression of NR1/NR2A receptors was dependent upon the PSD-95 N-terminal C3,C5 cysteines. These observations support differential interaction of NMDA receptor subtypes with different PSD-95 MAGUK scaffolding proteins. This has implications for the stabilisation, turnover and compartmentalisation of NMDA receptor subtypes in neurones during development and in the mature brain.     

Intramolecular interactions regulate SAP97 binding to GKAP

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

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

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

Identification of N-methyl-D-aspartic acid (NMDA) receptor subtype-specific binding sites that mediate direct interactions with scaffold protein PSD-95

N-methyl-D-aspartate (NMDA) neurotransmitter receptors and the postsynaptic density-95 (PSD-95) membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins are integral components of post-synaptic macromolecular signaling complexes that serve to propagate glutamate responses intracellularly. Classically, NMDA receptor NR2 subunits associate with PSD-95 MAGUKs via a conserved ES(E/D)V amino acid sequence located at their C termini. We previously challenged this dogma to demonstrate a second non-ES(E/D)V PSD-95-binding site in both NMDA receptor NR2A and NR2B subunits. Here, using a combination of co-immunoprecipitations from transfected mammalian cells, yeast two-hybrid interaction assays, and glutathione S-transferase (GST) pulldown assays, we show that NR2A subunits interact directly with PSD-95 via the C-terminal ESDV motif and additionally via an Src homology 3 domain-binding motif that associates with the Src homology 3 domain of PSD-95. Peptide inhibition of co-immunoprecipitations of NR2A and PSD-95 demonstrates that both the ESDV and non-ESDV sites are required for association in native brain tissue. Furthermore, we refine the non-ESDV site within NR2B to residues 1149-1157. These findings provide a molecular basis for the differential association of NMDA receptor subtypes with PSD-95 MAGUK scaffold proteins. These selective interactions may contribute to the organization, lateral mobility, and ultimately the function of NMDA receptor subtypes at synapses. Furthermore, they provide a more general molecular mechanism by which the scaffold, PSD-95, may discriminate between potential interacting partner proteins.     

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).     

Selective binding of synapse-associated protein 97 to GluR-A alpha-amino-5-hydroxy-3-methyl-4-isoxazole propionate receptor subunit is determined by a novel sequence motif

A family of four closely related PDZ domain-containing membrane-associated guanylate kinase homologues (MAGUKs) is involved in the regulation of the amount and functional state of ionotropic glutamate receptors in excitatory synapses. To understand the mechanisms that determine the specificity of these interactions, we examined the structural basis of the highly selective association between the ionotropic GluR subunit GluR-A and synapse-associated protein 97 (SAP97). The C terminus of GluR-A bound to the PDZ domains of SAP97, but not to those of three related MAGUKs, PSD-93, PSD-95, and SAP102. Experiments with single PDZ domains indicated that the strongest contribution was by the second PDZ domain. Unexpectedly, mutation analysis of the GluR-A C terminus revealed that a tripeptide sequence SSG at position -9 to -11 plays an essential role in this binding, in addition to a C-terminal type I PDZ binding motif (leucine at C terminus and threonine at the -2 position). Analysis of the in vitro MAGUK-binding properties of a GluR-D mutant with a one-residue deletion at the C terminus provides further support for the view that an SSG sequence located N-terminally from a type I PDZ binding motif can mediate selective binding to SAP97 and suggest the existence of a novel variation of the PDZ domain-peptide interaction.     

Differential activation of individual subunits in heteromeric kainate receptors

Neuronal kainate receptors are assembled from subunits with dissimilar specificities for agonists and antagonists. The composite biophysical behavior of heteromeric kainate receptors is determined by intersubunit interactions whose nature is unclear. Here we use dysiherbaine, a selective kainate receptor agonist, to show that GluR5 subunits assembled in heteromeric GluR5/KA-2 kainate receptor complexes can gate current without concomitant activation of their partner KA-2 subunits. A long-lasting interaction between dysiherbaine and GluR5 subunits elicits a tonic current from GluR5/KA-2 receptors; subsequent cooperative gating of KA-2 subunits can be elicited by both agonists, such as glutamate, and some classically defined antagonists, such as CNQX. This study demonstrates that each type of subunit within a heteromeric kainate receptor contributes a distinct conductance upon activation by agonist binding, and therefore provides insight into the biophysical function of ionotropic glutamate receptors.