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David S Bredt

Publications and source records attributed to David S Bredt.

50 records · Page 3Linked to original sources
PubMed2003

Interaction of transmembrane AMPA receptor regulatory proteins with multiple membrane associated guanylate kinases.

Surface expression of AMPA type glutamate receptors requires stargazin or a related transmembrane AMPA receptor regulatory protein (TARP). Furthermore, interaction of the cytosolic tail of TARPs with PDZ domains of PSD-95 targets AMPA receptors to postsynaptic densities. Here, we screened for additional proteins that might interact with the cytosolic domain of TARPs. Screening a rat brain cDNA library with the yeast two-hybrid system yielded six PDZ proteins that can bind tail of TARPs. These PDZ proteins include the four neuronal membrane associated guanylate kinases, PSD-95/SAP-90, PSD-93/Chapsyn-110, SAP-97/hDLG and SAP-102; the multi-PDZ protein, MUPP1; and the mitochondrial PDZ protein, OMP-25. Although all of these proteins can bind to TARPs in vitro, only two of these, PSD-95 and PSD-93 associate with TARPs in brain. We also found that all three PDZ domains from PSD-95 associate with the TARP C-termini with similar affinities. This work identifies biochemical promiscuity for interaction of the TARP C-termini with PDZ domains in vitro, but also shows that only specific synaptic PDZ proteins associate with TARPs in brain.

Animals↗
PubMed2003

Assembly and plasticity of the glutamatergic postsynaptic specialization.

Glutamate mediates most excitatory synaptic transmission in the brain. Synaptic strength at glutamatergic synapses shows a remarkable degree of use-dependent plasticity and such modifications may represent a physiological correlate to learning and memory. Glutamate receptors and downstream enzymes are organized at synapses by cytoskeletal proteins containing multiple protein-interacting domains. Recent studies demonstrate that these 'scaffolding' proteins within the postsynaptic specialization have the capacity to promote synaptic maturation, influence synapse size, and modulate glutamate receptor function.

Animals↗
PubMed2003

Stargazin differentially controls the trafficking of alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate and kainate receptors.

Synaptic plasticity at excitatory synapses in the brain is largely achieved by rapid changes in the number of synaptic alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) receptors. Stargazin, a membrane protein that interacts with AMPA receptors, is believed to play a pivotal role in trafficking AMPA receptors to the plasma membrane and targeting them to the synapse. However, it is unclear whether the trafficking of kainate receptors, which are structurally very similar to AMPA receptors, is also dependent on stargazin. Here we show that in both cerebellar granule cells and in Xenopus laevis oocytes expression system, surface delivery of kainate receptor is independent of stargazin. These results suggest that stargazin action is highly selective for AMPA receptors.

Animals↗
PubMed2002

Functional analysis of the nucleotide binding domain of membrane-associated guanylate kinases.

Membrane-associated guanylate kinases (MAGUKs) regulate cellular adhesion and signal transduction at sites of cell-cell contact. MAGUKs are composed of modular protein-protein interaction motifs including L27, PDZ, Src homology (SH) 3, and guanylate kinase domains that aggregate adhesion molecules and receptors. Genetic analyses reveal that lethal mutations of MAGUKs often occur in the guanylate kinase domain, indicating a critical role for this domain. Here, we explored whether GMP binding to the guanylate kinase domain regulates MAGUK function. Surprisingly, and in contrast to previously published studies, we failed to detect GMP binding to the MAGUKs postsynaptic density-95 (PSD-95) and CASK. Two amino acid residues in the GMP binding pocket that differ between MAGUKs and authentic guanylate kinase explain this lack of binding, as swapping these residues largely prevent GMP binding to yeast guanylate kinase. Conversely, these mutations restore GMP binding but not catalytic activity to PSD-95. Protein ligands for the PSD-95 guanylate kinase domain, guanylate kinase-associated protein (GKAP) and MAP1A, appear not to interact with the canonical GMP binding pocket, and GMP binding does not influence the intramolecular SH3/guanylate kinase (GK) interaction within PSD-95. These studies indicate that MAGUK proteins have lost affinity for GMP but may have retained the guanylate kinase structure to accommodate a related regulatory ligand.

Amino Acid Motifs↗
PubMed2002

Identification of a monogenic locus (jams1) causing juvenile audiogenic seizures in mice.

Epilepsy is a debilitating disease with a strong genetic component. Positional cloning has identified a few genes for rare monogenic epilepsy syndromes; however, the genetics of common human epilepsies are too complex to be analyzed easily by current techniques. Mouse models of epilepsy can further this analysis by eliminating genetic background heterogeneity and enabling the production of sufficient numbers of offspring. Here, we report that Black Swiss mice have a heretofore unrecognized specific susceptibility to audiogenic seizures. These seizures are characterized by wild running, loss of righting reflex, and tonic flexion and extension, and are followed by a postictal period. The susceptibility to these seizures is developmentally regulated, peaking at 21 d of age and nearly disappearing by adulthood. Interestingly, both the susceptibility to seizures and their developmental regulation appear unrelated to hearing thresholds in the Black Swiss strain and backcrossed progeny. Genetic mapping and linkage analysis of hybrid mice localize the seizure gene, jams1 (juvenile audiogenic monogenic seizures), to a 1.6 +/- 0.5 centimorgan (cM) region on mouse chromosome 10, delimited by the gene basigin (Bsg) and marker D10Mit140. Interestingly, the majority of the critical region is syntenic to a region on human chromosome 19p13.3 implicated in a familial form of juvenile febrile convulsions. Cloning the gene for audiogenic seizures in these mice may provide important insight into the fundamental mechanisms for developmentally regulated human epilepsy syndromes.

Acoustic Stimulation↗
PubMed2002

Direct interactions between PSD-95 and stargazin control synaptic AMPA receptor number.

Excitatory synapses in the brain exhibit a remarkable degree of functional plasticity, which largely reflects changes in the number of synaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). However, mechanisms involved in recruiting AMPARs to synapses are unknown. Here we use hippocampal slice cultures and biolistic gene transfections to study the targeting of AMPARs to synapses. We show that AMPARs are localized to synapses through direct binding of the first two PDZ domains of synaptic PSD-95 (postsynaptic density protein of 95 kDa) to the AMPAR-associated protein, stargazin. Increasing the level of synaptic PSD-95 recruits new AMPARs to synapses without changing the number of surface AMPARs. At the same time, we show that stargazin overexpression drastically increases the number of extra-synaptic AMPARs, but fails to alter synaptic currents if synaptic PSD-95 levels are kept constant. Finally, we make compensatory mutations to both PSD-95 and stargazin to demonstrate the central role of direct interactions between them in determining the number of synaptic AMPARs.

Animals↗
PubMed2002

A combination of three distinct trafficking signals mediates axonal targeting and presynaptic clustering of GAD65.

The signals involved in axonal trafficking and presynaptic clustering are poorly defined. Here we show that targeting of the gamma-aminobutyric acid-synthesizing enzyme glutamate decarboxylase 65 (GAD65) to presynaptic clusters is mediated by its palmitoylated 60-aa NH(2)-terminal domain and that this region can target other soluble proteins and their associated partners to presynaptic termini. A Golgi localization signal in aa 1-23 followed by a membrane anchoring signal upstream of the palmitoylation motif are required for this process and mediate targeting of GAD65 to the cytosolic leaflet of Golgi membranes, an obligatory first step in axonal sorting. Palmitoylation of a third trafficking signal downstream of the membrane anchoring signal is not required for Golgi targeting. However, palmitoylation of cysteines 30 and 45 is critical for post-Golgi trafficking of GAD65 to presynaptic sites and for its relative dendritic exclusion. Reduction of cellular cholesterol levels resulted in the inhibition of presynaptic clustering of palmitoylated GAD65, suggesting that the selective targeting of the protein to presynaptic termini is dependent on sorting to cholesterol-rich membrane microdomains. The palmitoylated NH(2)-terminal region of GAD65 is the first identified protein region that can target other proteins to presynaptic clusters.

Animals↗
PubMed2002

Postsynaptic targeting of alternative postsynaptic density-95 isoforms by distinct mechanisms.

Members of the postsynaptic density-95 (PSD95)/synapse-associated protein-90 (SAP90) family of scaffolding proteins contain a common set of modular protein interaction motifs including PDZ (postsynaptic density-95/Discs large/zona occludens-1), Src homology 3, and guanylate kinase domains, which regulate signaling and plasticity at excitatory synapses. We report that N-terminal alternative splicing of PSD95 generates an isoform, PSD95beta that contains an additional "L27" motif, which is also present in SAP97. Using yeast two hybrid and coimmunoprecipitation assays, we demonstrate that this N-terminal L27 domain of PSD-95beta, binds to an L27 domain in the membrane-associated guanylate kinase calcium/calmodulin-dependent serine kinase, and to Hrs, an endosomal ATPase that regulates vesicular trafficking. By transfecting heterologous cells and hippocampal neurons, we find that interactions with the L27 domain regulate synaptic clustering of PSD95beta. Disrupting Hrs-regulated early endosomal sorting in hippocampal neurons selectively blocks synaptic clustering of PSD95beta but does not interfere with trafficking of the palmitoylated isoform, PSD95alpha. These studies identify molecular and functional heterogeneity in synaptic PSD95 complexes and reveal critical roles for L27 domain interactions and Hrs regulated vesicular trafficking in postsynaptic protein clustering.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Phosphorylation of the postsynaptic density-95 (PSD-95)/discs large/zona occludens-1 binding site of stargazin regulates binding to PSD-95 and synaptic targeting of AMPA receptors.

Dynamic regulation of AMPA-type receptors at the synapse is proposed to play a critical role in alterations of the synaptic strength seen in cellular models of learning and memory such as long-term potentiation in the hippocampus. Stargazin, previously identified as an AMPA receptor (AMPAR)-interacting protein, is critical for surface expression and synaptic targeting of AMPARs. Stargazin interacts with postsynaptic density-95/discs large/zona occludens-1 (PDZ) proteins via a C-terminal PDZ binding motif. Interestingly, the C terminal of stargazin also predicts phosphorylation at a threonine residue critical for PDZ protein binding. Because protein phosphorylation regulates synaptic plasticity, we characterized this site and the effects of stargazin phosphorylation on AMPAR function. In vitro peptide phosphorylation assays and Western blot analysis with phospho-stargazin-specific antibodies indicate that the critical threonine within the stargazin PDZ binding site is phosphorylated by protein kinase A. This phosphorylation disrupts stargazin interaction and clustering with postsynaptic density-95 (PSD-95) in transfected COS-7 cells. Furthermore, a stargazin construct with a Thr-to-Glu mutation that mimics phosphorylation fails to cluster at synaptic spines and downregulates synaptic AMPAR function in cultured hippocampal neurons. These data suggest that phosphorylation of the stargazin PDZ ligand can disrupt stargazin interaction with PSD-95 and thereby regulate synaptic AMPAR function.

Animals↗
PubMed2002

Synaptic strength regulated by palmitate cycling on PSD-95.

Dynamic regulation of AMPA-type glutamate receptors represents a primary mechanism for controlling synaptic strength, though mechanisms for this process are poorly understood. The palmitoylated postsynaptic density protein, PSD-95, regulates synaptic plasticity and associates with the AMPA receptor trafficking protein, stargazin. Here, we identify palmitate cycling on PSD-95 at the synapse and find that palmitate turnover on PSD-95 is regulated by glutamate receptor activity. Acutely blocking palmitoylation disperses synaptic clusters of PSD-95 and causes a selective loss of synaptic AMPA receptors. We also find that rapid glutamate-mediated AMPA receptor internalization requires depalmitoylation of PSD-95. In a nonneuronal model system, clustering of PSD-95, stargazin, and AMPA receptors is also regulated by ongoing palmitoylation of PSD-95 at the plasma membrane. These studies suggest that palmitate cycling on PSD-95 can regulate synaptic strength and regulates aspects of activity-dependent plasticity.

Animals↗
PubMed2002

Synaptic glutamate receptor clustering in mice lacking the SH3 and GK domains of SAP97.

Postsynaptic targeting of the Drosophila tumour suppressor discs-large (Dlg) critically depends on its SH3 and GK domains. Here, we asked whether these domains are also involved in subcellular targeting of the mammalian Dlg homolog SAP97 and its interacting partners in CNS cortical neurons by analysing a recently described mouse mutant lacking the SH3 and GK domains of SAP97. Both wildtype and truncated SAP97 were predominantly expressed in perinuclear regions, in a pattern suggesting association with the endoplasmic reticulum. Weaker immunoreactivity was found in neurites colocalizing with both dendritic and axonal markers. As SAP97 has been implicated in the early intracellular processing of the glutamate receptor GluR1, we studied biochemical maturation and subcellular localization of GluR1 in the mutants. Both the glycosylation pattern and synaptic clustering of GluR1 were indistinguishable from wildtype mice. Synaptic clustering of the guanylate kinase domain interacting protein GKAP was also intact. Our data demonstrate that truncation of the SH3 and GK domains of SAP97 in mice does neither change its subcellular distribution nor does it disrupt synaptic structure or protein clustering, as opposed to severe missorting of the respective mutant Dlg protein in Drosophila.

Adaptor Proteins, Signal Transducing↗
PubMed1998

PDZ proteins bind, cluster, and synaptically colocalize with Eph receptors and their ephrin ligands.

Localizing cell surface receptors to specific subcellular positions can be critical for their proper functioning, as most notably demonstrated at neuronal synapses. PDZ proteins apparently play critical roles in such protein localizations. Receptor tyrosine kinases have not been previously shown to interact with PDZ proteins in vertebrates. We report that Eph receptors and their membrane-linked ligands all contain PDZ recognition motifs and can bind and be clustered by PDZ proteins. In addition, we find that Eph receptors and ligands colocalize with PDZ proteins at synapses. Thus, PDZ proteins may play critical roles in localizing vertebrate receptor tyrosine kinases and/or their ligands and may be particularly important for Eph function in guidance or patterning or at the synapse.

Amino Acid Sequence↗