Biomedical subjects

H Betz

Publications and source records attributed to H Betz.

At least 37 records · Page 2Linked to original sources

PubMed2001

Inhibition of neurite extension by overexpression of individual domains of LIM kinase 1.

Lin-11, Isl-1 and Mec-3 (LIM) kinases are serine/threonine kinases that phosphorylate cofilin, an actin depolymerizing protein. LIM kinases have a highly modular structure composed of two N-terminal LIM domains (LIM 1/2), a PSD-95, Dlg and ZO-1 (PDZ) domain and a C-terminal protein kinase domain. Here, we overexpressed individual domains of mouse LIM kinase 1 (LIMK1) in PC12 cells and investigated their effects on neurite outgrowth. Although none of the LIMK1 domains had an effect on spontaneous neurite outgrowth, the N-terminal LIM 1/2 domains strongly inhibited differentiation of PC12 cells after stimulation with both nerve growth factor (NGF) and the Rho-kinase inhibitor Y-27632. In contrast, the overexpressed PDZ domain reduced neurite outgrowth only when differentiation had been induced by Y-27632, but not by NGF. Our data suggest that the different non-catalytic N-terminal domains of LIMK1 contribute to the regulation of neurite extension by using distinct signal transduction pathways.

Amides↗

PubMed2001

Identification of a gephyrin-binding motif in the GDP/GTP exchange factor collybistin.

The brain-specific GDP/GTP exchange factor collybistin interacts with the receptor-anchoring protein gephyrin and activates the Rho-like GTPase Cdc42, which is known to regulate actin cytoskeleton dynamics. Alternative splicing creates two collybistin variants, I and II. In coexpression experiments, collybistin II has been shown to induce the formation of submembraneous gephyrin aggregates which cluster with hetero-oligomeric glycine receptors (GlyRs). Here we identified residues critical for interaction with gephyrin in the linker region between the SH3 and the DH domains of collybistin. Respective collybistin deletion mutants failed to bind gephyrin upon coexpression in heterologous cells, in GST pull-down assays and in the yeast two-hybrid system. Site-directed mutagenesis revealed polar amino acid residues as essential determinants of gephyrin binding. Furthermore, in vitro gephyrin bound simultaneously to both collybistin and the GlyR beta-subunit binding motif. Our data are consistent with collybistin-gephyrin interactions occuring during inhibitory postsynaptic membrane formation.

Amino Acid Motifs↗

PubMed2000

Reduced synaptic clustering of GABA and glycine receptors in the retina of the gephyrin null mutant mouse.

Clustering of neurotransmitter receptors in postsynaptic densities involves proteins that aggregate the receptors and link them to the cytoskeleton. In the case of glycine and GABA(A) receptors, gephyrin has been shown to serve this function. However, it is unknown whether gephyrin is involved in the clustering of all glycine and GABA(A) receptors or whether it interacts only with specific isoforms. This was studied in the retinae of mice, whose gephyrin gene was disrupted, with immunocytochemistry and antibodies that recognize specific subunits of glycine and GABA(A) receptors. Because homozygous (geph -/-) mutants die around birth, an organotypic culture system of the mouse retina was established to study the clustering of gephyrin and the receptors in vitro. We found that all gephyrin and all glycine receptor clusters (hot spots) were abolished in the geph (-/-) mouse retina. In the case of GABA(A) receptors, there was a significant reduction of clusters incorporating the gamma2, alpha2, and alpha3 subunits; however, a substantial number of hot spots was still present in geph (-/-) mutant retinae. This shows that gephyrin interacts with all glycine receptor isoforms but with only certain forms of GABA(A) receptors. In heterozygous geph (+/-) mutants, no reduction of hot spots was observed in the retina in vivo, but a significant reduction was found in the organotypic cultures. This suggests that mechanisms may exist in vivo that allow for the compensation of a partial gephyrin deficit.

Animals↗

PubMed2000

The metabotropic GABAB receptor directly interacts with the activating transcription factor 4.

G protein-coupled receptors regulate gene expression by cellular signaling cascades that target transcription factors and their recognition by specific DNA sequences. In the central nervous system, heteromeric metabotropic gamma-aminobutyric acid type B (GABA(B)) receptors through adenylyl cyclase regulate cAMP levels, which may control transcription factor binding to the cAMP response element. Using yeast-two hybrid screens of rat brain libraries, we now demonstrate that GABA(B) receptors are engaged in a direct and specific interaction with the activating transcription factor 4 (ATF-4), a member of the cAMP response element-binding protein /ATF family. As confirmed by pull-down assays, ATF-4 associates via its conserved basic leucine zipper domain with the C termini of both GABA(B) receptor (GABA(B)R) 1 and GABA(B)R2 at a site which serves to assemble these receptor subunits in heterodimeric complexes. Confocal fluorescence microscopy shows that GABA(B)R and ATF-4 are strongly coclustered in the soma and at the dendritic membrane surface of both cultured hippocampal neurons as well as retinal amacrine cells in vivo. In oocyte coexpression assays short term signaling of GABA(B)Rs via G proteins was only marginally affected by the presence of the transcription factor, but ATF-4 was moderately stimulated in response to receptor activation in in vivo reporter assays. Thus, inhibitory metabotropic GABA(B)Rs may regulate activity-dependent gene expression via a direct interaction with ATF-4.

Activating Transcription Factor 4↗

PubMed2000

Ulip6, a novel unc-33 and dihydropyrimidinase related protein highly expressed in developing rat brain.

Here, we report the identification of Ulip6, a novel unc-33 and dihydropyrimidinase related protein that belongs to the Ulip/CRMP protein family. Ulip6 was found in a yeast two-hybrid screen using the neuronal glycine transporter GlyT2 as bait. The rat and human Ulip6 sequences are highly homologous and most closely related to the liver enzyme dihydropyrimidinase (Ulip5). Northern and Western analysis of rat tissues revealed that the distribution of the Ulip6 mRNA and protein resembles those of brain-type Ulip proteins. Like Ulip1-4, Ulip6 is highly expressed in embryonic and early postnatal brain and spinal cord. These findings are consistent with Ulip6 having a function in neuronal differentiation and/or axon growth.

Amidohydrolases↗

PubMed2000

Diversity and phylogeny of gephyrin: tissue-specific splice variants, gene structure, and sequence similarities to molybdenum cofactor-synthesizing and cytoskeleton-associated proteins.

Gephyrin is essential for both the postsynaptic localization of inhibitory neurotransmitter receptors in the central nervous system and the biosynthesis of the molybdenum cofactor (Moco) in different peripheral organs. Several alternatively spliced gephyrin transcripts have been identified in rat brain that differ in their 5' coding regions. Here, we describe gephyrin splice variants that are differentially expressed in non-neuronal tissues and different regions of the adult mouse brain. Analysis of the murine gephyrin gene indicates a highly mosaic organization, with eight of its 29 exons corresponding to the alternatively spliced regions identified by cDNA sequencing. The N- and C-terminal domains of gephyrin encoded by exons 3-7 and 16-29, respectively, display sequence similarities to bacterial, invertebrate, and plant proteins involved in Moco biosynthesis, whereas the central exons 8, 13, and 14 encode motifs that may mediate oligomerization and tubulin binding. Our data are consistent with gephyrin having evolved from a Moco biosynthetic protein by insertion of protein interaction sequences.

Alternative Splicing↗

PubMed2000

The gamma-aminobutyric acid type A receptor (GABAAR)-associated protein GABARAP interacts with gephyrin but is not involved in receptor anchoring at the synapse.

gamma-Aminobutyric acid type A receptors (GABA(A)Rs) are ligand-gated chloride channels that exist in numerous distinct subunit combinations. At postsynaptic membrane specializations, different GABA(A)R isoforms colocalize with the tubulin-binding protein gephyrin. However, direct interactions of GABA(A)R subunits with gephyrin have not been reported. Recently, the GABA(A)R-associated protein GABARAP was found to bind to the gamma2 subunit of GABA(A)Rs. Here we show that GABARAP interacts with gephyrin in both biochemical assays and transfected cells. Confocal analysis of neurons derived from wild-type and gephyrin-knockout mice revealed that GABARAP is highly enriched in intracellular compartments, but not at gephyrin-positive postsynaptic membrane specializations. Our data indicate that GABARAP-gephyrin interactions are not important for postsynaptic GABA(A)R anchoring but may be implicated in receptor sorting and/or targeting mechanisms. Consistent with this idea, a close homolog of GABARAP, p16, has been found to function as a late-acting intra-Golgi transport factor.

Adaptor Proteins, Signal Transducing↗

PubMed2000

The X-ray crystal structure of neuronal Sec1 from squid sheds new light on the role of this protein in exocytosis.

BACKGROUND: Sec1-like molecules have been implicated in a variety of eukaryotic vesicle transport processes including neurotransmitter release by exocytosis. They regulate vesicle transport by binding to a t-SNARE from the syntaxin family. This process is thought to prevent SNARE complex formation, a protein complex required for membrane fusion. Whereas Sec1 molecules are essential for neurotransmitter release and other secretory events, their interaction with syntaxin molecules seems to represent a negative regulatory step in secretion. RESULTS: Here we report the X-ray crystal structure of a neuronal Sec1 homologue from squid, s-Sec1, at 2.4 A resolution. Neuronal s-Sec1 is a modular protein that folds into a V-shaped three-domain assembly. Peptide and mutagenesis studies are discussed with respect to the mechanism of Sec1 regulation. Comparison of the structure of squid s-Sec1 with the previously determined structure of rat neuronal Sec1 (n-Sec1) bound to syntaxin-1a indicates conformational rearrangements in domain III induced by syntaxin binding. CONCLUSIONS: The crystal structure of s-Sec1 provides the molecular scaffold for a number of molecular interactions that have been reported to affect Sec1 function. The structural differences observed between s-Sec1 and the structure of a rat n-Sec1-syntaxin-1a complex suggest that local conformational changes are sufficient to release syntaxin-1a from neuronal Sec1, an active process that is thought to involve additional effector molecule(s).

Amino Acid Sequence↗

PubMed2000

Receptors, gephyrin and gephyrin-associated proteins: novel insights into the assembly of inhibitory postsynaptic membrane specializations.

The synaptic localization of ion channel receptors is essential for efficient synaptic trans-mission and the precise regulation of diverse neuronal functions, such as signal integration and synaptic plasticity. Emerging evidence points to an important role of cytoskeleton-associated proteins that assemble receptors and components of the subsynaptic machinery at postsynaptic membrane specializations. This article reviews interactions of inhibitory postsynaptic neurotransmitter receptors with the receptor anchoring protein gephyrin and intracellular components involved in downstream signalling and/or control of signal transduction processes. The presently available data suggest a central synaptic organizer function for gephyrin in inhibitory postsynaptic membrane assembly and stabilization.

Animals↗

PubMed2000

Subunit-dependent inhibition of recombinant rodent N-methyl-D-aspartate receptors by a HIV-1 glycoprotein 120 derived peptide.

Considerable evidence suggests that low (picomolar) concentrations of the HIV-1 envelope glycoprotein gp120 induce neuronal cell death by stimulating the release of microglial toxins, which in turn activate N-methyl-D-aspartate (NMDA) receptors. Conversely, high (micromolar) concentrations of gp120 have been reported to directly inhibit NMDA receptor-mediated currents and do not induce neurotoxicity. Here we show that micromolar concentrations of a synthetic peptide corresponding to the V3-loop of gp120 (V3-pep) inhibited agonist responses of recombinant heteromeric rodent NMDA receptors expressed in Xenopus laevis oocytes by decreasing their apparent glycine affinity. Different combinations of NMDA receptor subunits displayed differential sensitivities to inhibition by V3-pep, with a potency rank order of NR1/2B > NR1/2D > NR1/2C > or = NR1/2A. Our observations may provide an explanation for the reduced neurotoxicity of high doses of gp120 in cell cultures and may be useful for the pharmacological discrimination of NMDA receptor subtypes.

Amino Acid Sequence↗

PubMed2000

Kinetic and mutational analysis of Zn2+ modulation of recombinant human inhibitory glycine receptors.

1. The effects of Zn2+ on glycine receptor (GlyR) currents were analysed in Xenopus oocytes and human embryonic kidney cells expressing homomeric human wild-type and mutant alpha1 subunit GlyRs. 2. Low concentrations (10 microM) of extracellular Zn2+ converted the partial agonist taurine into a high-efficacy agonist. Concentration-response analysis showed that the EC50 for taurine decreased whereas the Hill coefficient increased under these conditions. In contrast, 50-500 microM Zn2+ showed an increased EC50 value and reduced maximal inducible taurine currents. The potency of competitive antagonists was not affected in the presence of Zn2+. 3. Single-channel recording from outside-out patches revealed different kinetics of glycine- and taurine-gated currents. With both agonists, Zn2+ altered the open probability of the alpha1 GlyR without changing its unitary conductance. Low Zn2+ concentrations (5 microM) increased both the opening frequency and mean burst duration, whereas higher Zn2+ concentrations (> 50 microM) reduced GlyR open probability mainly by decreasing the open frequency and the relative contribution of the longest burst of the single-channel events. 4. Site-directed mutagenesis of the GlyR alpha1 subunit identified aspartate 80 and threonine 112 as important determinants of Zn2+ potentiation and inhibition, respectively, without affecting potentiation by ethanol. 5. Our data support the view that Zn2+ modulates different steps of the receptor binding and gating cycle via specific allosteric high- and low-affinity binding sites in the extracellular N-terminal region of the GlyR alpha1 subunit.

Algorithms↗

PubMed2000

Clustering of inhibitory neurotransmitter receptors at developing postsynaptic sites: the membrane activation model.

Recent studies indicate an important role of cytoskeleton-associated and lipid-anchored proteins in the formation of inhibitory postsynaptic membrane specializations. Membrane apposition of the tubulin-binding protein gephyrin is essential for the recruitment of inhibitory glycine receptors and GABAA receptors to developing postsynaptic sites. Newly disclosed interactions between gephyrin, exchange factors for G proteins of the Rho and Rac families, the translational regulator RAFT1, and actin-binding proteins like profilin might integrate activity-dependent and trophic-factor-mediated signals at developing postsynaptic sites. A model of inhibitory neurotransmitter receptor clustering, is proposed, in which this process is initiated by receptor-driven activation of phosphatidylinositol 3-kinase.

Animals↗

PubMed2000

Collybistin, a newly identified brain-specific GEF, induces submembrane clustering of gephyrin.

The formation of postsynaptic GABAA and glycine receptor clusters requires the receptor-associated peripheral membrane protein gephyrin. Here we describe two splice variants of a novel gephyrin-binding protein, termed collybistin I and II, which belong to the family of dbl-like GDP/GTP exchange factors (GEFs). Co-expression of collybistin II with gephyrin induced the formation of submembrane gephyrin aggregates that accumulate hetero-oligomeric glycine receptors. Our data suggest that collybistin II regulates the membrane deposition of gephyrin by activating a GTPase of the Rho/Rac family. Therefore, this protein may be an important determinant of inhibitory postsynaptic membrane formation and plasticity.

Alternative Splicing↗

PubMed2000

Transient neuromotor phenotype in transgenic spastic mice expressing low levels of glycine receptor beta-subunit: an animal model of startle disease.

Startle disease or hereditary hyperekplexia has been shown to result from mutations in the alpha1-subunit gene of the inhibitory glycine receptor (GlyR). In hyperekplexia patients, neuromotor symptoms generally become apparent at birth, improve with age, and often disappear in adulthood. Loss-of-function mutations of GlyR alpha or beta-subunits in mice show rather severe neuromotor phenotypes. Here, we generated mutant mice with a transient neuromotor deficiency by introducing a GlyR beta transgene into the spastic mouse (spa/spa), a recessive mutant carrying a transposon insertion within the GlyR beta-subunit gene. In spa/spa TG456 mice, one of three strains generated with this construct, which expressed very low levels of GlyR beta transgene-dependent mRNA and protein, the spastic phenotype was found to depend upon the transgene copy number. Notably, mice carrying two copies of the transgene showed an age-dependent sensitivity to tremor induction, which peaked at approximately 3-4 weeks postnatally. This closely resembles the development of symptoms in human hyperekplexia patients, where motor coordination significantly improves after adolescence. The spa/spa TG456 line thus may serve as an animal model of human startle disease.

Animals↗

PubMed2000

Glycine receptors containing the alpha4 subunit in the embryonic sympathetic nervous system, spinal cord and male genital ridge.

Inhibitory glycine receptors (GlyRs) are known to mediate postsynaptic inhibition in spinal cord, brain stem and some higher brain regions. Several developmentally and regionally regulated GlyR isoforms exist, which result from a differential expression of the GlyR alpha (alpha1-alpha4) and beta subunit genes. Currently, very little is known about GlyRs containing the alpha4 subunit, whose existence was predicted from a partial genomic sequence. Here, we describe the isolation of complementary DNA (cDNA) sequences for the mouse and chick GlyR alpha4 subunits. We show that a mouse GlyR alpha4 subunit full-length cDNA directs the formation of functional homo-oligomeric strychnine-sensitive GlyRs in Xenopus laevis oocytes and mammalian cells, and that these resemble GlyRs composed of the alpha1 subunit in pharmacological profile and single-channel properties. In situ hybridization reveals high levels of GlyR alpha4 subunit transcripts in the embryonic (E13) chick spinal cord, lumbosacral sympathetic ganglia and dorsal root ganglia. The avian GlyR alpha4 subunit gene also shows male-specific expression in the developing genital ridge. The pharmacological profile of alpha4 subunit-containing receptors and deduced location of the avian GlyR alpha4 subunit are consistent with it being a component of the embryonic excitatory GlyRs previously identified in sympathetic neurons. Our data also suggest a novel role for GlyRs in the maturation of reproductive organs.

Amino Acid Sequence↗

PubMed2000

Interaction of the C-terminal tail region of the metabotropic glutamate receptor 7 with the protein kinase C substrate PICK1.

Group III metabotropic glutamate receptors (mGluRs) are highly enriched in the presynaptic terminals of glutamatergic synapses where they mediate feedback inhibition of neurotransmitter release. Here, we used the yeast two-hybrid system to identify a direct interaction of the C-terminal tail region of mGluR7 with the rat homologue of the protein kinase C substrate PICK1. This interaction is specifically mediated by the very C-terminal amino acids of the receptor and can be reconstituted in human embryonic kidney 293 cells by transfection of full-length mGluR7 and PICK1 cDNAs. Quantitative beta-galactosidase assays revealed that among the different group III mGluRs, mGluR7 is the major PICK1 binding partner although other subfamily members can also interact with PICK1. These data indicate that PDZ domain-containing proteins might contribute to the presynaptic localization of group III mGluRs.

Amino Acid Sequence↗

PubMed2000

Novel CNS glutamate receptor subunit genes of Drosophila melanogaster.

We report the identification and characterization of two genes from Drosophila melanogaster that encode novel ionotropic glutamate receptor proteins, named DGluR-IB and DNMDAR-II, and that are located on chromosome 3L, region 67AB, and the X chromosome, position 2B, respectively. The DGluR-IB full-length cDNA was isolated from Drosophila embryonic and head libraries. The encoded protein of 1,095 amino acids displays high sequence identity (73%) to DGluR-IA. The DNMDAR-II gene was identified by sequence-homology searches in databases. The deduced protein shows moderate sequence identity (29-31%) to the mouse NMDAR2A-D receptor subunits. Whole-mount in situ hybridization on embryos revealed DGluR-IB and DNMDAR-II transcripts in the CNS. Immunofluorescence analysis of the adult fly brain indicates that the DGluR-IB protein is expressed in neurons implicated in the regulation of the circadian clock.

Animals↗

PubMed2000

Crystallization and preliminary X-ray analysis of squid neuronal Sec1.

Sec1 protein family members are involved in the regulation of all intracellular SNARE-mediated (SNARE = soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) vesicle-fusion processes in a step preceding membrane fusion and have been shown to interact with t-SNAREs. To better understand the structural basis and the role of Sec1 in the regulation of the SNARE-complex formation, neuronal Sec1 from the squid Loligo pealei has been expressed and crystallized; this invertebrate protein shows a high sequence homology to the human neuronal Sec1, Munc18a. Here, the production of diffraction-quality native crystals, which belong to space group P3(1)21 and diffract to 3.3 A resolution, is described. In addition, selenomethionyl n-Sec1 crystals in space groups P3(1)21 and P2(1) have been generated. Preliminary analysis of the monoclinic space group indicates that these crystals diffract to a resolution higher than 2.5 A.

Animals↗