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H Betz

Publications and source records attributed to H Betz.

At least 55 records · Page 3Linked to original sources
PubMed1999

Dominant-negative alleles of 14-3-3 proteins cause defects in actin organization and vesicle targeting in the yeast Saccharomyces cerevisiae.

14-3-3 Proteins are thought to function as adapters in signaling complexes [1,2], thereby participating in cellular processes including vesicle trafficking and exocytosis [3,4]. To delineate further the function of 14-3-3 proteins during vesicle trafficking, we generated dominant-negative alleles of the two 14-3-3 homologues, Bmh1p and Bmh2p, in budding yeast and analyzed their phenotype in respect to exocytosis. Cells overexpressing the carboxy-terminal region of Bmh2p failed to polarize vesicular transport although bulk exocytosis remained unaffected and showed a disrupted actin cytoskeleton. Our data suggest that 14-3-3 proteins may act primarily on the actin cytoskeleton to regulate vesicle targeting.

14-3-3 Proteins↗
PubMed1999

Calmodulin dependence of presynaptic metabotropic glutamate receptor signaling.

Glutamatergic neurotransmission is controlled by presynaptic metabotropic glutamate receptors (mGluRs). A subdomain in the intracellular carboxyl-terminal tail of group III mGluRs binds calmodulin and heterotrimeric guanosine triphosphate-binding protein (G protein) betagamma subunits in a mutually exclusive manner. Mutations interfering with calmodulin binding and calmodulin antagonists inhibit G protein-mediated modulation of ionic currents by mGluR 7. Calmodulin antagonists also prevent inhibition of excitatory neurotransmission via presynaptic mGluRs. These results reveal a novel mechanism of presynaptic modulation in which Ca(2+)-calmodulin is required to release G protein betagamma subunits from the C-tail of group III mGluRs in order to mediate glutamatergic autoinhibition.

Amino Acid Sequence↗
PubMed1999

Loss of postsynaptic GABA(A) receptor clustering in gephyrin-deficient mice.

The tubulin-binding protein gephyrin, which anchors the inhibitory glycine receptor (GlyR) at postsynaptic sites, decorates GABAergic postsynaptic membranes in various brain regions, and postsynaptic gephyrin clusters are absent from cortical cultures of mice deficient for the GABA(A) receptor gamma2 subunit. Here, we investigated the postsynaptic clustering of GABA(A) receptors in gephyrin knock-out (geph -/-) mice. Both in brain sections and cultured hippocampal neurons derived from geph -/- mice, synaptic GABA(A) receptor clusters containing either the gamma2 or the alpha2 subunit were absent, whereas glutamate receptor subunits were normally localized at postsynaptic sites. Western blot analysis and electrophysiological recording revealed that normal levels of functional GABA(A) receptors are expressed in geph -/- neurons, however the pool size of intracellular GABA(A) receptors appeared increased in the mutant cells. Thus, gephyrin is required for the synaptic localization of GlyRs and GABA(A) receptors containing the gamma2 and/or alpha2 subunits but not for the targeting of these receptors to the neuronal plasma membrane. In addition, gephyrin may be important for efficient membrane insertion and/or metabolic stabilization of inhibitory receptors at developing postsynaptic sites.

Animals↗
PubMed1999

Molecular determinants of glycine receptor subunit assembly.

The inhibitory glycine receptor (GlyR) is a pentameric transmembrane protein composed of homologous alpha and beta subunits. Single expression of alpha subunits generates functional homo-oligomeric GlyRs, whereas the beta subunit requires a co-expressed alpha subunit to assemble into hetero-oligomeric channels of invariant stoichiometry (alpha(3)beta(2)). Here, we identified eight amino acid residues within the N-terminal region of the alpha1 subunit that are required for the formation of homo-oligomeric GlyR channels. We show that oligomerization and N-glycosylation of the alpha1 subunit are required for transit from the endoplasmic reticulum to the Golgi apparatus and later compartments, and that addition of simple carbohydrate side chains occurs prior to GlyR subunit assembly. Our data are consistent with both intersubunit surface and conformational differences determining the different assembly behaviour of GlyR alpha and beta subunits.

Amino Acid Sequence↗
PubMed1999

Interaction of RAFT1 with gephyrin required for rapamycin-sensitive signaling.

RAFT1 (rapamycin and FKBP12 target 1; also called FRAP or mTOR) is a member of the ATM (ataxia telangiectasia mutated)-related family of proteins and functions as the in vivo mediator of the effects of the immunosuppressant rapamycin and as an important regulator of messenger RNA translation. In mammalian cells RAFT1 interacted with gephyrin, a widely expressed protein necessary for the clustering of glycine receptors at the cell membrane of neurons. RAFT1 mutants that could not associate with gephyrin failed to signal to downstream molecules, including the p70 ribosomal S6 kinase and the eIF-4E binding protein, 4E-BP1. The interaction with gephyrin ascribes a function to the large amino-terminal region of an ATM-related protein and reveals a role in signal transduction for the clustering protein gephyrin.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Structure and functions of inhibitory and excitatory glycine receptors.

The strychnine-sensitive glycine receptor (GlyR) is a pentameric chloride channel protein that exists in several developmentally and regionally regulated isoforms in the CNS. These result from the differential expression of four genes encoding different variants (alpha 1-alpha 4) of the ligand-binding subunit of the GlyR. Their assembly with the structural beta subunit is governed by "assembly cassettes" within the extracellular domains of these proteins and creates chloride channels of distinct conductance properties. GlyR gating is potentiated by Zn2+, a metal ion co-released with different neurotransmitters. Site-directed mutagenesis has unraveled major determinants of agonist binding and Zn2+ potentiation. During development, glycine receptors mediate excitation that results in Ca2+ influx and neurotransmitter release. Ca2+ influx triggered by the activation of embryonic GlyRs is required for the synaptic localization of the GlyR and its anchoring protein gepyhrin. In the adult, mutations in GlyR-subunit genes result in motor disorders. The spastic and spasmodic phenotypes in mouse as well as human hereditary startle disease will be discussed.

Animals↗
PubMed1999

Incorporation of a gephyrin-binding motif targets NMDA receptors to gephyrin-rich domains in HEK 293 cells.

The peripheral membrane protein gephyrin is essential for the postsynaptic localization of inhibitory glycine receptors (GlyRs). Binding of gephyrin to the GlyR beta subunit is mediated by a sequence motif located in the intracellular loop region connecting transmembrane segments 3 and 4. Here, insertion of this binding motif is shown to alter the subcellular distribution of an excitatory neurotransmitter receptor in transfected mammalian cells. Upon coexpression with gephyrin, a mutant N-methyl-D-aspartate (NMDA) receptor containing NMDA receptor 1 (NR1) subunits which harboured a gephyrin-binding motif within its cytoplasmic tail region, was targeted to intracellular gephyrin-rich domains, as previously observed for the GlyR beta subunit. Our data indicate that a gephyrin-binding motif located in a cytoplasmic domain of an integral membrane protein suffices for routing to gephyrin-rich domains.

Carrier Proteins↗
PubMed1999

Dynamic interaction between soluble tubulin and C-terminal domains of N-methyl-D-aspartate receptor subunits.

The cytoplasmic C-terminal domains (CTs) of the NR1 and NR2 subunits of the NMDA receptor have been implicated in its anchoring to the subsynaptic cytoskeleton. Here, we used affinity chromatography with glutathione S-transferase-NR1-CT and -NR2B-CT fusion proteins to identify novel binding partner(s) of these NMDA receptor subunits. Upon incubation with rat brain cytosolic protein fraction, both NR1-CT and NR2B-CT, but not glutathione S-transferase, specifically bound tubulin. The respective fusion proteins also bound tubulin purified from brain, suggesting a direct interaction between the two binding partners. In tubulin polymerization assays, NR1-CT and NR2B-CT significantly decreased the rate of microtubule formation without destabilizing preformed microtubules. Moreover, only minor fractions of either fusion protein coprecipitated with the newly formed microtubules. Consistent with these findings, ultrastructural analysis of the newly formed microtubules revealed a limited association only with the CTs of the NR1 and NR2B. These data suggest a direct interaction of the NMDA receptor channel subunit CTs and tubulin dimers or soluble forms of tubulin. The efficient modulation of microtubule dynamics by the NR1 and NR2 cytoplasmic domains suggests a functional interaction of the receptor and the subsynaptic cytoskeletal network that may play a role during morphological adaptations, as observed during synaptogenesis and in adult CNS plasticity.

Animals↗
PubMed1999

Hydrophobic interactions mediate binding of the glycine receptor beta-subunit to gephyrin.

Glycine receptors (GlyRs) are ligand-gated chloride channel proteins composed of alpha- and beta-subunits. GlyRs are located to and anchored at postsynaptic sites by the receptor-associated protein gephyrin. Previous work from our laboratory has identified a core motif for gephyrin binding in the cytoplasmic loop of the GlyR beta-subunit. Here, we localized amino acid residues implicated in gephyrin binding by site-directed mutagenesis. In a novel transfection assay, a green fluorescent protein-gephyrin binding motif fusion protein was used to monitor the consequences of amino acid substitutions for beta-subunit interaction with gephyrin. Only multiple, but not single, replacements of hydrophobic side chains abolished the interaction between the two proteins. Our data are consistent with gephyrin binding being mediated by the hydrophobic side of an imperfect amphipathic helix.

Amino Acid Sequence↗
PubMed1999

Selective blocking effects of tropisetron and atropine on recombinant glycine receptors.

Some serotonin 5-HT3 receptor ligands of tropeine structure have been recently shown to modulate ionophore function and binding of glycine receptors. This led us to study the effects of the tropeines tropisetron and atropine on recombinant human glycine receptors transiently expressed in Xenopus oocytes by using whole-cell voltage-clamp electrophysiology. Glycine currents were inhibited by atropine in an apparently competitive manner and with considerable selectivity of the tropeines for alpha2 versus alpha1 subunits. Coexpression of beta with alpha subunits and replacement of the N-terminal region of the alpha1 subunits by the corresponding beta segment resulted in similar increases in the inhibitory potencies. Our data suggest common sites of the tropeines for inhibition on the N-terminal region of glycine receptors. The point mutations R271K and R271L of the alpha1 subunit decreased, whereas a T112A substitution increased, the inhibition constants (Ki) of the tropeines. These changes in the Ki values of the tropeines were associated with opposite changes in the EC50 of glycine. Selectivities for the tropeines versus glycine (EC50/Ki) varied within three orders of magnitude. These results, when expressed in terms of free energy changes, can be interpreted according to a two-state receptor model.

Animals↗
PubMed1999

A novel domain of the inhibitory glycine receptor determining antagonist efficacies: further evidence for partial agonism resulting from self-inhibition.

Different amino side chains in the N-terminal extracellular region of the inhibitory glycine receptor (GlyR) have been shown to be crucial for ligand recognition. Here we describe a novel domain of the GlyRalpha1 subunit that constitutes an important determinant of antagonist activity. The antagonists strychnine, nipecotic acid, and isobutyric acid displayed reduced potencies at recombinant GlyRs formed from alpha1 subunits, in which lysine 104, phenylalanine 108, or threonine 112 were replaced by alanine. Agonist affinities, in contrast, were slightly increased at these mutant receptors. Taurine and beta-aminoisobutyric acid, which are partial agonists at the wild-type GlyR, behaved as full agonists at the mutant GlyRs and failed to inhibit glycine-induced currents. This is consistent with apolar residues at positions 104, 108, and 112 of the alpha1 subunit reducing the antagonistic, but not the agonistic, binding of beta-amino acids. Our data support a model in which the partial agonism of beta-amino acids results from their self-inhibitory activity.

Amino Acid Substitution↗
PubMed1998

Dual requirement for gephyrin in glycine receptor clustering and molybdoenzyme activity.

Glycine receptors are anchored at inhibitory chemical synapses by a cytoplasmic protein, gephyrin. Molecular cloning revealed the similarity of gephyrin to prokaryotic and invertebrate proteins essential for synthesizing a cofactor required for activity of molybdoenzymes. Gene targeting in mice showed that gephyrin is required both for synaptic clustering of glycine receptors in spinal cord and for molybdoenzyme activity in nonneural tissues. The mutant phenotype resembled that of humans with hereditary molybdenum cofactor deficiency and hyperekplexia (a failure of inhibitory neurotransmission), suggesting that gephyrin function may be impaired in both diseases.

Animals↗
PubMed1998

Differentiation of glycine antagonist sites of N-methyl-D-aspartate receptor subtypes. Preferential interaction of CGP 61594 with NR1/2B receptors.

The binding site for the co-agonist glycine on N-methyl-D-aspartate (NMDA) receptors has been mapped to the NR1 subunit whereas binding of the principal agonist glutamate is mediated by the NR2 subunits. Using the novel glycine site antagonist and photoaffinity label CGP 61594, distinct contributions of the NR2 subunit variants to the glycine antagonist binding domains of NMDA receptor subtypes are demonstrated. High affinity sites for CGP 61594 were exclusively displayed by NR1/2B receptors, as shown by their co-distribution with the NR2B subunit, by subunit-selective immunoprecipitation and by functional analysis of NR1/2B receptors expressed in Xenopus oocytes (inhibitory potency, IC50 = 45 +/- 11 nM). Other NMDA receptor subtypes are clearly distinguished by reduced inhibitory potencies for CGP 61594, being low for NR1/2A and NR1/2D receptors (IC50 = 430 +/- 105 nM and 340 +/- 61 nM, respectively) and intermediate for NR1/2C receptors (IC50 = 164 +/- 27 nM). Glycine antagonist sites with low and intermediate affinity for [3H]CGP 61594 were detected also in situ by radioligand binding in brain areas predominantly expressing the NR2A and NR2C subunits, respectively. Thus, [3H]CGP 61594 is the first antagonist radioligand that reliably distinguishes the glycine site of NMDA receptor subtypes. [3H]CGP 61594 is a promising tool to identify the NR2 subunit domains that contribute to differential glycine antagonist sites of NMDA receptor subtypes.

Allosteric Regulation↗
PubMed1998

Glycine-receptor activation is required for receptor clustering in spinal neurons.

The ability of nerve cells to receive up to several thousands of synaptic inputs from other neurons provides the anatomical basis for information processing in the vertebrate brain. The formation of functional synapses involves selective clustering of neurotransmitter receptors at presumptive postsynaptic regions of the neuronal plasma membrane. Receptor-associated proteins are believed to be crucial for this process. In spinal neurons, synaptic targeting of the inhibitory glycine receptor (GlyR) depends on the expression of the anchoring protein gephyrin. Here we show that the competitive GlyR antagonist strychnine and L-type Ca2+-channel blockers inhibit the accumulation of GlyR and gephyrin at postsynaptic membrane areas in cultured rat spinal neurons. Our data are consistent with a model in which GlyR activation that results in Ca2+ influx is required for the clustering of gephyrin and GlyR at developing postsynaptic sites. Similar activity-driven mechanisms may be of general importance in synaptogenesis.

Animals↗
PubMed1998

A neuronal Sec1 homolog regulates neurotransmitter release at the squid giant synapse.

Sec1-related proteins are essential for membrane fusion at distinct stages of the constitutive and regulated secretory pathways in eukaryotic cells. Studies of neuronal isoforms of the Sec1 protein family have yielded evidence for both positive and negative regulatory functions of these proteins in neurotransmitter release. Here, we have identified a squid neuronal homolog (s-Sec1) of Sec1 proteins and examined its function in neurotransmitter release at the squid giant synapse. Microinjection of s-Sec1 into the presynaptic terminal of the giant synapse inhibited evoked neurotransmitter release, but this effect was prevented by coinjecting the cytoplasmic domain of squid syntaxin (s-syntaxin), one of the binding partners of s-Sec1. A 24 amino acid peptide fragment of s-Sec1, which inhibited the binding of s-Sec1 to s-syntaxin in vitro, completely blocked release, suggesting an essential function of the s-Sec1/s-syntaxin interaction in transmitter release. Electron microscopy showed that injection of s-Sec1 did not change the spatial distribution of synaptic vesicles at presynaptic release sites ("active zones"), whereas the inhibitory peptide increased the number of docked vesicles. These distinct morphological effects lead us to conclude that Sec1 proteins function at different stages of synaptic vesicle exocytosis, and that an interaction of s-Sec1 with syntaxin-at a stage blocked by the peptide-is necessary for docked vesicles to fuse.

Animals↗
PubMed1998

Evidence for a tetrameric structure of recombinant NMDA receptors.

The amino acids L-glutamate and glycine are essential agonists of the excitatory NMDA receptor, a subtype of the ionotropic glutamate receptor family. The native NMDA receptor is composed of two types of homologous membrane-spanning subunits, NR1 and NR2. Here, the numbers of glycine-binding NR1 and glutamate-binding NR2 subunits in the NMDA receptor hetero-oligomer were determined by coexpressing the wild-type (wt) NR1 with the low-affinity mutant NR1(Q387K), and the wt NR2B with the low-affinity mutant NR2BE387A, subunits in Xenopus oocytes. In both cases, analysis of the resulting dose-response curves revealed three independent components of glycine and glutamate sensitivity. These correspond to the respective wild-type and mutant affinities and an additional intermediate hybrid affinity, indicating the existence of three discrete receptor populations. Binomial analysis of these data indicates the presence of two glycine and two glutamate binding subunits in the functional receptor. In addition, we analyzed the inhibitory effects of the negative dominant NR1(R505K) and NR2BR493K mutants on maximal inducible whole-cell currents of wt NR1/NR2B receptors. The inhibition profiles obtained on expression of increasing amounts of these mutant proteins again were fitted best by assuming an incorporation of two NR1 and two NR2 subunits into the receptor hetero-oligomer. Our data are consistent with NMDA receptors being tetrameric proteins that are composed of four homologous subunits.

Animals↗
PubMed1998

Regulation of neurotransmitter release kinetics by NSF.

NSF (N-ethylmaleimide-sensitive factor) is an adenosine triphosphatase (ATPase) that contributes to a protein complex essential for membrane fusion. The synaptic function of this protein was investigated by injecting, into the giant presynaptic terminal of squid, peptides that inhibit the ATPase activity of NSF stimulated by the soluble NSF attachment protein (SNAP). These peptides reduced the amount and slowed the kinetics of neurotransmitter release as a result of actions that required vesicle turnover and occurred at a step subsequent to vesicle docking. These results define NSF as an essential participant in synaptic vesicle exocytosis that regulates the kinetics of neurotransmitter release and, thereby, the integrative properties of synapses.

Adenosine Triphosphatases↗