Biomedical subjects

E D Gundelfinger

Publications and source records attributed to E D Gundelfinger.

At least 37 records · Page 2Linked to original sources

PubMed2000

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.

Adaptor Proteins, Signal Transducing↗

PubMed2000

The calcium-independent receptor for alpha-latrotoxin from human and rodent brains interacts with members of the ProSAP/SSTRIP/Shank family of multidomain proteins.

Subtypes of the calcium-independent receptors for alpha-latrotoxin (CIRL1-3) define a distinct subgroup within the large family of the seven-transmembrane region cell surface receptors. The physiological function of CIRLs is unknown because neither extracellular ligands nor intracellular coupling proteins (G-proteins) have been identified. Using yeast two-hybrid screening, we identified a novel interaction between the C termini of CIRL1 and -2 and the PSD-95/discs large/ZO-1 (PDZ) domain of a recently discovered multidomain protein family (ProSAP/SSTRIP/Shank) present in human and rat brain. In vitro, CIRL1 and CIRL2 interacted strongly with the PDZ domain of ProSAP1. The specificity of this interaction has been verified by in vivo experiments using solubilized rat brain membrane fractions and ProSAP1 antibodies; only CIRL1, but not CIRL2, was co-immunoprecipitated with ProSAP1. In situ hybridization revealed that ProSAP1 and CIRL1 are co-expressed in the cortex, hippocampus, and cerebellum. Colocalization was also observed at the subcellular level, as both CIRL1 and ProSAP1 are enriched in the postsynaptic density fraction from rat brain. Expression of all three CIRL isoforms is highly regulated during postnatal brain development, with CIRL3 exhibiting its highest expression levels immediately after birth, followed by CIRL2 and finally CIRL1 in aged rats.

Amino Acid Sequence↗

PubMed2000

Neuronal nicotinic acetylcholine receptors of Drosophila melanogaster: the alpha-subunit dalpha3 and the beta-type subunit ARD co-assemble within the same receptor complex.

Dalpha3 is a functional alpha-subunit of Drosophila melanogaster nicotinic acetylcholine receptors (nAChRs). Here, we produced Dalpha3-specific antibodies to study which other nAChR subunits can co-assemble with Dalpha3 in receptor complexes of the Drosophila nervous system. Immunohistochemical studies revealed that Dalpha3 is co-distributed with the beta-subunit ARD in synaptic neuropil regions of the optic lobe. Both subunits can be co-purified by alpha-bungarotoxin affinity chromatography. Dalpha3 antibodies co-immunoprecipitate Dalpha3 and ARD proteins and, vice versa, anti-ARD antibodies co-precipitate ARD and Dalpha3. These data demonstrate that one type of fly nAChRs includes these two subunits as integral components.

Animals↗

PubMed2000

Synaptic targeting and localization of discs-large is a stepwise process controlled by different domains of the protein.

BACKGROUND: Membrane-associated guanylate kinases (MAGUKs) assemble ion channels, cell-adhesion molecules and components of second messenger cascades into synapses, and are therefore potentially important for co-ordinating synaptic strength and structure. Here, we have examined the targeting of the Drosophila MAGUK Discs-large (DLG) to larval neuromuscular junctions. RESULTS: During development, DLG was first found associated with the muscle subcortical compartment and plasma membrane, and later was recruited to the postsynaptic membrane. Using a transgenic approach, we studied how mutations in various domains of the DLGprotein affect DLG targeting. Deletion of the HOOK region-the region between the Src homology 3 (SH3) domain and the guanylate-kinase-like (GUK) domain-prevented association of DLG with the subcortical network and rendered the protein largely diffuse. Loss of the first two PDZ domains led to the formation of large clusters throughout the plasma membrane, with scant targeting to the neuromuscular junction. Proper trafficking of DLG missing the GUK domain depended on the presence of endogenous DLG. CONCLUSIONS: Postsynaptic targeting of DLG requires a HOOK-dependent association with extrasynaptic compartments, and interactions mediated by the first two PDZ domains. The GUK domain routes DLG between compartments, possibly by interacting with recently identified cytoskeletal-binding partners.

Animals↗

PubMed2000

Membrane association of presynaptic cytomatrix protein bassoon.

Components of the specialized cytomatrix at active zones of presynaptic nerve terminals are thought to be involved in organizing synaptic events such as immobilisation or translocation of synaptic vesicles and assemblingactive zone components. The 420-kDa non-transmembraneprotein Bassoon is a specific componentof the presynaptic cytomatrix that shares features with both cytoskeleton-associated and peripheral-membrane proteins. Using immunogold electron microscopy we show here that synapse associated Bassoon is distributed in a subregion of active zones. Using a biochemical assay we show that a fraction of Bassoon is membrane associated. Electron microscopy performed on the same biochemical fraction further revealed that Bassoon is associated with vesicular structures. Together these data suggest that at least a fraction of Bassoon is associated with a membraneous compartment in neurons.

Animals↗

PubMed2000

The synaptic glycoprotein neuroplastin is involved in long-term potentiation at hippocampal CA1 synapses.

Neuroplastin-65 and -55 (previously known as gp65 and gp55) are glycoproteins of the Ig superfamily that are enriched in rat forebrain synaptic membrane preparations. Whereas the two-Ig domain isoform neuroplastin-55 is expressed in many tissues, the three-Ig domain isoform neuroplastin-65 is brain-specific and enriched in postsynaptic density (PSD) protein preparations. Here, we have assessed the function of neuroplastin in long-term synaptic plasticity. Immunocytochemical studies with neuroplastin-65-specific antibodies differentially stain distinct synaptic neuropil regions of the rat hippocampus with most prominent immunoreactivity in the CA1 region and the proximal molecular layer of the dentate gyrus. Kainate-induced seizures cause a significant enhancement of neuroplastin-65 association with PSDs. Similarly, long-term potentiation (LTP) of CA1 synapses in hippocampal slices enhanced the association of neuroplastin-65 with a detergent-insoluble PSD-enriched protein fraction. Several antibodies against the neuroplastins, including one specific for neuroplastin-65, inhibited the maintenance of LTP. A similar effect was observed when recombinant fusion protein containing the three extracellular Ig domains of neuroplastin-65 was applied to hippocampal slices before LTP induction. Microsphere binding experiments using neuroplastin-F(c) chimeric proteins show that constructs containing Ig1-3 or Ig1 domains, but not Ig2-3 domains mediate homophilic adhesion. These data suggest that neuroplastin plays an essential role in implementing long-term changes in synaptic activity, possibly by means of a homophilic adhesion mechanism.

Animals↗

PubMed2000

Temporal appearance of the presynaptic cytomatrix protein bassoon during synaptogenesis.

Bassoon is a 420-kDa presynaptic cytomatrix protein potentially involved in the structural organization of neurotransmitter release sites. In this study, we have investigated a possible role for Bassoon in synaptogenesis and in defining synaptic vesicle recycling sites. We find that it is expressed at early stages of neuronal differentiation in which it is selectively sorted into axons. As synaptogenesis begins, Bassoon clusters appear along dendritic profiles simultaneously with synaptotagmin I, sites of synaptic vesicle recycling, and the acquisition of functional excitatory and inhibitory synapses. A role for Bassoon in the assembly of excitatory and inhibitory synapses is supported by the colocalization of Bassoon clusters with clusters of GKAP and AMPA receptors as well as GABA(A) receptors. These data indicate that the recruitment of Bassoon is an early step in the formation of synaptic junctions.

Animals↗

PubMed2000

Molecular organization of excitatory chemical synapses in the mammalian brain.

Chemical synapses are highly specialized cell-cell junctions designed for efficient signaling between nerve cells. Distinct cytoskeletal matrices are assembled at either side of the synaptic junction. The presynaptic cytomatrix at the active zone (CAZ) defines and organizes the site of neurotransmitter release from presynaptic nerve terminals. The postsynaptic density (PSD) tethers neurotransmitter receptors and the postsynaptic signal transduction machinery. Recent progress in the identification and characterization of novel CAZ and PSD components has revealed new insights into the molecular organization and assembly mechanisms of the synaptic neurotransmission apparatus. On the presynaptic side, Bassoon and Piccolo, two related giant proteins, are crucially involved in scaffolding the CAZ. On the postsynaptic side, two families of multidomain adaptor proteins, the MAGuKs (membrane-associated guanylate kinase homologs) and the ProSAP (proline-rich synapse-associated protein, also termed Shank) family members are thought to be major organizing molecules of the PSD.

Animals↗

PubMed2000

The neuronal EF-hand calcium-binding protein visinin-like protein-3 is expressed in cerebellar Purkinje cells and shows a calcium-dependent membrane association.

Visinin-like protein-3 is a member of the family of intracellular neuronal calcium sensors belonging to the superfamily of EF-hand proteins. Members of this family are involved in the calcium-dependent regulation of signal transduction cascades. To gain insights into the characteristics of visinin-like protein-3, we have generated specific antibodies against visinin-like protein-3 and determined the developmental and tissue distribution of the protein and its exact cellular and subcellular localization. Expression of visinin-like protein-3 protein appeared late in development mainly in the cerebellum. It is strongly expressed in cerebellar Purkinje cells. The protein expression results were further confirmed by in situ hybridization and compared with hippocalcin messenger RNA localization. Native cerebellar visinin-like protein-3 was shown to bind calcium and to associate in a calcium-dependent manner with membrane fractions during subcellular fractionation. Recombinant wild-type visinin-like protein-3 was shown to be N-terminally myristoylated in transfected cells. The membrane association was strongly reduced for the non-myristoylated mutant of visinin-like protein-3 in transfected cells. These results suggest that visinin-like protein-3, which is mainly expressed in Purkinje cells in vivo, shows a calcium-dependent association with cell membranes which is mediated by a calcium-myristoyl switch.

Aging↗

PubMed2000

Piccolo, a presynaptic zinc finger protein structurally related to bassoon.

Piccolo is a novel component of the presynaptic cytoskeletal matrix (PCM) assembled at the active zone of neurotransmitter release. Analysis of its primary structure reveals that Piccolo is a multidomain zinc finger protein structurally related to Bassoon, another PCM protein. Both proteins were found to be shared components of glutamatergic and GABAergic CNS synapses but not of the cholinergic neuromuscular junction. The Piccolo zinc fingers were found to interact with the dual prenylated rab3A and VAMP2/Synaptobrevin II receptor PRA1. We show that PRA1 is a synaptic vesicle-associated protein that is colocalized with Piccolo in nerve terminals of hippocampal primary neurons. These data suggest that Piccolo plays a role in the trafficking of synaptic vesicles (SVs) at the active zone.

Amino Acid Sequence↗

PubMed2000

Ca2+-induced Ca2+ release supports the relay mode of activity in thalamocortical cells.

Ca2+ ions play an important role during rhythmic bursting of thalamocortical neurons within sleep. The function of Ca2+ during the tonic relay mode of these neurons during wakefulness is less clear. Here, we report that tonic activity in thalamocortical cells results in an increase in the intracellular Ca2+ concentration and subsequent release of Ca2+ from intracellular stores mediated via ryanodine receptors (RyRs). Blockade of Ca2+ release shifted the regular firing of single action potentials toward the generation of spike clusters. Regular spike firing and intracellular Ca2+ release thus appear to be functionally coupled in a positive feedback manner, thereby supporting the relay mode of thalamocortical cells during wakefulness. Regulatory influences may be coupled to this system via the cyclic ADP ribose pathway.

Action Potentials↗

PubMed2000

Molecular determinants of presynaptic active zones.

The presynaptic cytoskeletal matrix (cytomatrix) assembled at active zones has been implicated in defining neurotransmitter release sites. Munc13, Rim, Bassoon and Piccolo/Aczonin are recently identified presynaptic cytomatrix proteins. These multidomain proteins are thought to organize the exocytotic and endocytotic machinery precisely at active zones.

Animals↗

PubMed2000

A comparison of synaptic protein localization in hippocampal mossy fiber terminals and neurosecretory endings of the neurohypophysis using the cryo-immunogold technique.

In central synapses synaptic vesicle docking and exocytosis occurs at morphologically specialized sites (active zones) and requires the interaction of specific proteins in the formation of a SNARE complex. In contrast, neurosecretory terminals lack active zones. Using the cryo-immunogold technique we analyzed the localization of synaptic vesicle proteins and of proteins of the docking complex at active zones. This was compared to the localization of the identical proteins in neurosecretory terminals. In addition we compared the vesicular and granular localization of the proteins investigated. Synaptic vesicles in rat hippocampal mossy fiber synapses and microvesicles in the neurosecretory terminals of the neurohypophysis contained in common the proteins VAMP II (a v-SNARE), SV2, rab3A, and N-type Ca(2+) channels. Only minor immunolabeling for these proteins was observed at neurosecretory granules. These results support the notion of a close functional identity of microvesicles from neurosecretory endings of the neurohypophysis and of synaptic vesicles. The vesicular pool of N-type Ca(2+) channels may serve their stimulation-induced translocation into the plasma membrane. We find increased labeling for VAMP II, SNAP-25, N-type Ca(2+) channels and of rab3A at the active zones of mossy fiber synapses. Labeling at release sites is by far highest for Bassoon, a high molecular weight protein of the active zone. The labeling pattern implies an association of Bassoon with presynaptic dense projections. Bassoon is absent from neurosecretory terminals and VAMP II, SNAP-25, rab3A, and N-type Ca(2+) channels reveal a scattered distribution over the plasma membrane. The competence of the presynaptic active zone for selective vesicle docking may not primarily result from its contents in SNARE proteins but rather from the preformation of presynaptic dense projections as structural guides for vesicle exocytosis.

Animals↗

PubMed2000

Neuronal nicotinic acetylcholine receptors from Drosophila: two different types of alpha subunits coassemble within the same receptor complex.

Although neuronal nicotinic acetylcholine receptors from insects have been reconstituted in vitro more than a decade ago, our knowledge about the subunit composition of native receptors as well as their functional properties still remains limited. Immunohistochemical evidence has suggested that two alpha subunits, alpha-like subunit (ALS) and Drosophila alpha2 subunit (Dalpha2), are colocalized in the synaptic neuropil of the Drosophila CNS and therefore may be subunits of the same receptor complex. To gain further understanding of the composition of these nicotinic receptors, we have examined the possibility that a receptor may imbed more than one alpha subunit using immunoprecipitations and electrophysiological investigations. Immunoprecipitation experiments of fly head extracts revealed that ALS-specific antibodies coprecipitate Dalpha2, and vice versa, and thereby suggest that these two alpha subunits must be contained within the same receptor complex, a result that is supported by investigations of reconstituted receptors in Xenopus oocytes. Discrimination between binary (ALS/beta2 or Dalpha2/beta2) and ternary (ALS/Dalpha2/beta2) receptor complexes was made on the basis of their dose-response curve to acetylcholine as well as their sensitivity to alpha-bungarotoxin or dihydro-beta-erythroidine. These data demonstrate that the presence of the two alpha subunits within a single receptor complex confers new receptor properties that cannot be predicted from knowledge of the binary receptor's properties.

Acetylcholine↗

PubMed1999

Proline-rich synapse-associated proteins ProSAP1 and ProSAP2 interact with synaptic proteins of the SAPAP/GKAP family.

We have recently isolated a novel proline-rich synapse-associated protein-1 (ProSAP1) that is highly enriched in postsynaptic density (PSD). A closely related multidomain protein, ProSAP2, shares a highly conserved PDZ (PSD-95/discs-large/ZO-1) domain (80% identity), a ppI domain that mediates the interaction with cortactin, and a C-terminal SAM (sterile alpha-motif) domain. In addition, ProSAP2 codes for five ankyrin repeats and a SH3 (Src homology 3) domain. Transcripts for both proteins are coexpressed in many regions of rat brain, but show a distinct expression pattern in the cerebellum. Using the PDZ domains of ProSAP1 and 2 as bait in the yeast two-hybrid system, we isolated several clones of the SAPAP/GKAP (SAP90/PSD-95-associated protein/guanylate kinase-associated protein) family. The association of the proteins was verified by coimmunoprecipitation and cotransfection in HEK cells. Therefore, proteins of the ProSAP family represent a novel link between SAP90/PSD-95 bound membrane receptors and the cytoskeleton at glutamatergic synapses of the central nervous system.

Amino Acid Sequence↗

PubMed1999

Proline-rich synapse-associated protein-1/cortactin binding protein 1 (ProSAP1/CortBP1) is a PDZ-domain protein highly enriched in the postsynaptic density.

The postsynaptic density (PSD) is crucially involved in the structural and functional organization of the postsynaptic neurotransmitter reception apparatus. Using antisera against rat brain synaptic junctional protein preparations, we isolated cDNAs coding for proline-rich synapse-associated protein-1 (ProSAP1), a PDZ-domain protein. This protein was found to be identical to the recently described cortactin-binding protein-1 (CortBP1). Homology screening identified a related protein, ProSAP2. Specific antisera raised against a C-terminal fusion construct and a central part of ProSAP1 detect a cluster of immunoreactive bands of 180 kDa in the particulate fraction of rat brain homogenates that copurify with the PSD fraction. Transcripts and immunoreactivity are widely distributed in the brain and are upregulated during the period of synapse formation in the brain. In addition, two short N-terminal insertions are detected; they are differentially regulated during brain development. Confocal microscopy of hippocampal neurons showed that ProSAP1 is predominantly localized in synapses, and immunoelectron microscopy in situ revealed a strong association with PSDs of hippocampal excitatory synapses. The accumulation of ProSAP1 at synaptic structures was analyzed in the developing cerebral cortex. During early postnatal development, strong immunoreactivity is detectable in neurites and somata, whereas from postnatal day 10 (P10) onward a punctate staining is observed. At the ultrastructural level, the immunoreactivity accumulates at developing PSDs starting from P8. Both interaction with the actin-binding protein cortactin and early appearance at postsynaptic sites suggest that ProSAP1/CortBP1 may be involved in the assembly of the PSD during neuronal differentiation.

Aging↗

PubMed1999

Presynaptic cytomatrix protein bassoon is localized at both excitatory and inhibitory synapses of rat brain.

Bassoon is a 420-kDa protein specifically localized at the active zone of presynaptic nerve terminals. It is thought to be involved in the structural organization of the neurotransmitter release site. We studied the distribution of Bassoon transcripts and protein in rat brain and assessed which types of presynaptic terminals contain the protein. As shown by in situ hybridization, Bassoon transcripts are widely distributed in the brain and occur primarily in excitatory neurons. In addition, examples of gamma-aminobutyric acid (GABA)-ergic neurons expressing Bassoon are detected. At the light microscopic level, Bassoon immunoreactivity is found in synaptic neuropil regions throughout the brain, with the strongest expression in the hippocampus, the cerebellar cortex, and the olfactory bulb. Immunoelectron microscopy showed that Bassoon immunoreactivity is found in both asymmetric type 1 and symmetric type 2 synapses. Immunopositive asymmetric synapses include mossy fiber boutons and various spine and shaft synapses in the hippocampus and mossy fiber terminals and parallel fiber terminals in the cerebellum. Bassoon-containing symmetric synapses are observed, e.g., between basket and granule cells in the hippocampus, between Golgi cells and granule cells, and between basket cells and Purkinje cells in the cerebellum. Within synaptic terminals, Bassoon appears highly concentrated at sites opposite to postsynaptic densities. In cultured hippocampal neurons, Bassoon was found to colocalize with GABA(A) and glutamate (GluR1) receptors. These data indicate that Bassoon is a component of the presynaptic apparatus of both excitatory glutamatergic and inhibitory GABAergic synapses.

Animals↗

PubMed1999

The presynaptic cytomatrix protein Bassoon: sequence and chromosomal localization of the human BSN gene.

Bassoon is a novel 420-kDa protein recently identified as a component of the cytoskeleton at presynaptic neurotransmitter release sites. Analysis of the rat and mouse sequences revealed a polyglutamine stretch in the C-terminal part of the protein. Since it is known for some proteins that abnormal amplification of such polyglutamine regions can cause late-onset neurodegeneration, we cloned and localized the human BASSOON gene (BSN). Phage clones spanning most of the open reading frame and the 3' untranslated region were isolated from a human genomic library and used for chromosomal localization of BSN to chromosome 3p21 by FISH. The localization was confirmed by PCR on rodent/human somatic cell hybrids; it is consistent with the localization of the murine Bsn gene at chromosome 9F. Sequencing revealed a polyglutamine stretch of only five residues in human, and PCR amplifications from 50 individuals showed no obvious length polymorphism in this region. Analysis of the primary structure of Bassoon and comparison to previous database entries provide evidence for a newly emerging protein family.

Amino Acid Sequence↗