Biomedical subjects

C C Garner

Publications and source records attributed to C C Garner.

At least 37 records · Page 2Linked to original sources

PubMed1999

Identification of a cis-acting dendritic targeting element in MAP2 mRNAs.

In neurons, a limited number of mRNAs have been identified in dendritic processes, whereas other transcripts are restricted to the cell soma. Here we have investigated the molecular mechanisms underlying extrasomatic localization of mRNAs encoding microtubule-associated protein 2 (MAP2) in primary neuronal cultures. Vectors expressing recombinant mRNAs were introduced into hippocampal and sympathetic neurons using DNA transfection and microinjection protocols, respectively. Chimeric mRNAs containing the entire 3' untranslated region of MAP2 transcripts fused to a nondendritic reporter mRNA are detected in dendrites. In contrast, RNAs containing MAP2 coding and 5' untranslated regions or tubulin sequences are restricted to the cell soma. Moreover, 640 nucleotides from the MAP2 3' untranslated region (UTR) are both sufficient and essential for extrasomatic localization of chimeric mRNAs in hippocampal and sympathetic neurons. Thus, a cis-acting dendritic targeting element that is effective in two distinct neuronal cell types is contained in the 3' UTR of MAP2 transcripts. The observation of RNA granules in dendrites implies that extrasomatic transcripts seem to assemble into multimolecular complexes that may function as transport units.

3' Untranslated Regions↗

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↗

PubMed1999

Differential expression of the presynaptic cytomatrix protein bassoon among ribbon synapses in the mammalian retina.

Bassoon is a 420-kDa presynaptic protein which is highly concentrated at the active zones of nerve terminals of conventional synapses, both excitatory glutamatergic and inhibitory GABAergic, in rat brain. It is thought to be involved in the organization of the cytomatrix at the site of neurotransmitter release. In the retina, there are two structurally and functionally distinct types of synapses: ribbon and conventional synapses. Antibodies against bassoon were applied to sections of rat and rabbit retina. Strong punctate immunofluorescence was found in the outer and inner plexiform layers. Using pre- and post-embedding immunostaining and electron microscopy, bassoon was localized in the outer plexiform layer at ribbon synapses formed by rods and cones but was absent from basal synaptic contacts formed by cones. In the inner plexiform layer a different picture emerged. As in the brain, bassoon was found at conventional inhibitory GABAergic synapses, made by amacrine cells, but it was absent from the bipolar cell ribbon synapses. These data demonstrate differences in the molecular composition of the presynaptic apparatuses of outer and inner plexiform layer ribbon synapses. Thus, differential equipment with cytomatrix proteins may account for the functional differences observed between the two types of ribbon synapses in the retina.

Animals↗

PubMed1998

Caldendrin, a novel neuronal calcium-binding protein confined to the somato-dendritic compartment.

Using antibodies against synaptic protein preparations, we cloned the cDNA of a new Ca2+-binding protein. Its C-terminal portion displays significant similarity with calmodulin and contains two EF-hand motifs. The corresponding mRNA is highly expressed in rat brain, primarily in cerebral cortex, hippocampus, and cerebellum; its expression appears to be restricted to neurons. Transcript levels increase during postnatal development. A recombinant C-terminal protein fragment binds Ca2+ as indicated by a Ca2+-induced mobility shift in SDS-polyacrylamide gel electrophoresis. Antisera generated against the bacterial fusion protein recognize a brain-specific protein doublet with apparent molecular masses of 33 and 36 kDa. These data are confirmed by in vitro translation, which generates a single 36-kDa polypeptide, and by the heterologous expression in 293 cells, which yields a 33/36-kDa doublet comparable to that found in brain. On two-dimensional gels, the 33-kDa band separates into a chain of spots plausibly due to differential phosphorylation. This view is supported by in situ phosphorylation studies in hippocampal slices. Most of the immunoreactivity is detectable in cytoskeletal preparations with a further enrichment in the synapse-associated cytomatrix. These biochemical data, together with the ultra-structural localization in dendrites and the postsynaptic density, strongly suggest an association with the somato-dendritic cytoskeleton. Therefore, this novel Ca2+-binding protein was named caldendrin.

Amino Acid Sequence↗

PubMed1998

Immunocytochemical localization of the synapse-associated protein SAP102 in the rat retina.

An elaborate network of transmitter receptors, synapse associated proteins (SAPs), and cytoskeletal elements, generally known as the postsynaptic density, is involved with efficient synaptic signaling. The localization of the synapse associated protein SAP102 was studied in the rat retina by using immunocytochemical methods. Immunofluorescence for SAP102 was most prominent in the inner plexiform layer (IPL). It had a punctate appearance, suggesting a synaptic clustering of SAP102 in the IPL. Electron microscopy by use of pre-embedding immunocytochemistry showed that SAP102 is concentrated in the IPL in processes which are postsynaptic at bipolar cell ribbon synapses (dyads). As a rule, only one of the two postsynaptic members of the dyad was labeled for SAP102. Double-labeling experiments were performed in order to find out whether SAP102 is involved with the clustering the N-methyl-D-aspartate (NMDA) receptor 2A subunit (NR2A). Only a fraction (approximately 23%) of the SAP102 clusters expressed NR2A, suggesting SAP102 is also associated with other subunits or receptors. Distinct SAP102 labeling was also present in horizontal cell processes in the outer plexiform layer (OPL), which are inserted as lateral elements into photoreceptor ribbon synapses (triads). The optic nerve fibre layer was also diffusely immunoreactive for SAP102. The postsynaptic aggregation of SAP102 at bipolar cell dyads and at photoreceptor triads suggests SAP102 is associated with the clustering of transmitter receptors. However, the labeling of the optic nerve fibre layer indicates additional functions of SAP102 in the retina.

Animals↗

PubMed1998

SAP97 is associated with the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR1 subunit.

Rapid glutamatergic synaptic transmission is mediated by ionotropic glutamate receptors and depends on their precise localization at postsynaptic membranes opposing the presynaptic neurotransmitter release sites. Postsynaptic localization of N-methyl-D-aspartate-type glutamate receptors may be mediated by the synapse-associated proteins (SAPs) SAP90, SAP102, and chapsyn-110. SAPs contain three PDZ domains that can interact with the C termini of proteins such as N-methyl-D-aspartate receptor subunits that carry a serine or threonine at the -2 position and a valine, isoleucine, or leucine at the very C terminus (position 0). We now show that SAP97, a SAP whose function at the synapse has been unclear, is associated with alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors. AMPA receptors are probably tetramers and are formed by two or more of the four AMPA receptor subunits GluR1-4. GluR1 possesses a C-terminal consensus sequence for interactions with PDZ domains of SAPs. SAP97 was present in AMPA receptor complexes immunoprecipitated from detergent extracts of rat brain. After treatment of rat brain membrane fractions with the cross-linker dithiobis(succinimidylpropionate) and solubilization with sodium dodecylsulfate, SAP97 was associated with GluR1 but not GluR2 or GluR3. In vitro experiments with recombinant proteins indicate that SAP97 specifically associates with the C terminus of GluR1 but not other AMPA receptor subunits. Our findings suggest that SAP97 may be involved in localizing AMPA receptors at postsynaptic sites through its interaction with the GluR1 subunit.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Bassoon, a novel zinc-finger CAG/glutamine-repeat protein selectively localized at the active zone of presynaptic nerve terminals.

The molecular architecture of the cytomatrix of presynaptic nerve terminals is poorly understood. Here we show that Bassoon, a novel protein of >400,000 Mr, is a new component of the presynaptic cytoskeleton. The murine bassoon gene maps to chromosome 9F. A comparison with the corresponding rat cDNA identified 10 exons within its protein-coding region. The Bassoon protein is predicted to contain two double-zinc fingers, several coiled-coil domains, and a stretch of polyglutamines (24 and 11 residues in rat and mouse, respectively). In some human proteins, e.g., Huntingtin, abnormal amplification of such poly-glutamine regions causes late-onset neurodegeneration. Bassoon is highly enriched in synaptic protein preparations. In cultured hippocampal neurons, Bassoon colocalizes with the synaptic vesicle protein synaptophysin and Piccolo, a presynaptic cytomatrix component. At the ultrastructural level, Bassoon is detected in axon terminals of hippocampal neurons where it is highly concentrated in the vicinity of the active zone. Immunogold labeling of synaptosomes revealed that Bassoon is associated with material interspersed between clear synaptic vesicles, and biochemical studies suggest a tight association with cytoskeletal structures. These data indicate that Bassoon is a strong candidate to be involved in cytomatrix organization at the site of neurotransmitter release.

Amino Acid Sequence↗

PubMed1998

Functional analysis of the guanylate kinase-like domain in the synapse-associated protein SAP97.

SAP97 is a membrane cytoskeletal protein localized at the presynaptic nerve terminals of type 1 asymmetric synapses. It has been implicated in the assembly of synapses and in particular in the localization and clustering of ion channels. The C-terminal GK domain of SAP97 shares a high degree of sequence similarity with low-molecular-mass guanylate kinases. These enzymes are involved in the guanine nucleotide metabolic cycle and in the maintenance of GTP/GDP pools required for example in Ras-mediated cell signaling. It has therefore been hypothesized that SAP97 plays an essential role in cellular signaling by regulating the guanine nucleotide pools at synaptic junctions. Here, we test this hypothesis by assessing whether the GK domain in SAP97 encodes an authentic guanylate kinase. We show that the GK domain in and of itself does not encode an active guanylate kinase, that it cannot be activated by its binding partner GKAP and that flanking regions are not acting as inhibitory regulators for enzymatic activity. Thus, it would appear that the GK domain of SAP97 is not involved in the metabolism of guanine nucleotides required for signaling events.

Adaptor Proteins, Signal Transducing↗

PubMed1998

SAP90 binds and clusters kainate receptors causing incomplete desensitization.

The mechanism of kainate receptor targeting and clustering is still unresolved. Here, we demonstrate that members of the SAP90/PSD-95 family colocalize and associate with kainate receptors. SAP90 and SAP102 coimmunoprecipitate with both KA2 and GluR6, but only SAP97 coimmunoprecipitates with GluR6. Similar to NMDA receptors, GluR6 clustering is mediated by the interaction of its C-terminal amino acid sequence, ETMA, with the PDZ1 domain of SAP90. In contrast, the KA2 C-terminal region binds to, and is clustered by, the SH3 and GK domains of SAP90. Finally, we show that SAP90 coexpressed with GluR6 or GluR6/KA2 receptors alters receptor function by reducing desensitization. These studies suggest that the organization and electrophysiological properties of synaptic kainate receptors are modified by association with members of the SAP90/PSD-95 family.

Amino Acid Sequence↗

PubMed1998

Subcellular targeting and cytoskeletal attachment of SAP97 to the epithelial lateral membrane.

The synapse-associated protein SAP97 is a member of a novel family of cortical cytoskeletal proteins involved in the localization of ion channels at such membrane specializations as synaptic junctions. These multidomain proteins have binding sites for protein 4.1, GKAPs/SAPAPs, voltage- and ligand-gated ion channels and cell-adhesion molecules containing C-terminal T/SXV motifs. In this study, we evaluated the contribution of individual domains in SAP97 to its selective recruitment and attachment to the cortical cytoskeleton in epithelial cells. We find that the PDZ, SH3 and GK domains, as well as the I3 insert in SAP97, are not essential for subcellular targeting, though both PDZ1-2 domains and the I3 insert affect the efficiency of localization. Instead, we show that the first 65 amino acid residues in SAP97, which are absent from SAP90/PSD-95 and SAP102, direct the selective subcellular localization and can mediate at least one point of attachment of SAP97 to the cytoskeleton assembled at sites of cell-cell contact. Our data demonstrate that it is the sequences unique to SAP97 that direct its subcellular targeting to the epithelial lateral membrane.

Adaptor Proteins, Signal Transducing↗

PubMed1998

E-cadherin mediated cell adhesion recruits SAP97 into the cortical cytoskeleton.

Members of the SAP family of synapse-associated proteins have recently emerged as central players in the molecular organization of synapses. In this study, we have examined the mechanism that localizes one member, SAP97, to sites of cell-cell contact. Utilizing epithelial CACO-2 cells and fibroblast L-cells as model systems, we demonstrate that SAP97 is associated with the submembranous cortical cytoskeleton at cell-cell adhesion sites. Furthermore, we show that its localization into this structure is triggered by E-cadherin. Although SAP97 can be found in an E-cadherin/catenin adhesion complex, this interaction seems to be mediated by the attachment of SAP97 to the cortical cytoskeleton. Our results are consistent with a model in which SAP97 is recruited to sites of cell-cell contact via an E-cadherin induced assembly of the cortical cytoskeleton.

Actins↗

PubMed1997

Synaptic clustering of the cell adhesion molecule fasciclin II by discs-large and its role in the regulation of presynaptic structure.

The cell adhesion molecule Fasciclin II (FASII) is involved in synapse development and plasticity. Here we provide genetic and biochemical evidence that proper localization of FASII at type I glutamatergic synapses of the Drosophila neuromuscular junction is mediated by binding between the intracellular tSXV bearing C-terminal tail of FASII and the PDZ1-2 domains of Discs-Large (DLG). Moreover, mutations in fasII and/or dlg have similar effects on presynaptic ultrastructure, suggesting their functional involvement in a common developmental pathway. DLG can directly mediate a biochemical complex and a macroscopic cluster of FASII and Shaker K+ channels in heterologous cells. These results indicate a central role for DLG in the structural organization and downstream signaling mechanisms of cell adhesion molecules and ion channels at synapses.

Animals↗

PubMed1997

Functional expression of rat synapse-associated proteins SAP97 and SAP102 in Drosophila dlg-1 mutants: effects on tumor suppression and synaptic bouton structure.

The synapse-associated proteins SAP97 and SAP102 are mammalian proteins that are structurally related to the Drosophila tumor suppressor protein DlgA. Previous analyses revealed that DlgA is essential for the integrity of epithelia and neuromuscular synapses. Here we show that synaptic bouton structure is severely affected in mutant larvae carrying the dlg-1(XI-2) allele. We have tested SAP97 and SAP102 for functional homology to DlgA by heterologous expression in Drosophila. Both SAP97 and SAP102 can suppress tumor formation in dlg-1 mutant flies and mimic DlgA at larval neuromuscular junctions. Neuronal expression of SAP97 or SAP102 is required for morphological restoration of synaptic boutons, indicating that presynaptic DlgA function is essential for establishing structurally intact motor nerve terminals at larval neuromuscular junctions.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Interaction of the N-methyl-D-aspartate receptor complex with a novel synapse-associated protein, SAP102.

Ionotropic glutamate receptors are known to cluster at high concentration on the postsynaptic membrane of excitatory synapses, but the mechanism by which this occurs is poorly understood. Studies on the neuromuscular junction and central inhibitory synapses suggest that clustering of neurotransmitter receptors requires its interaction with a cytoplasmic protein. Recently, in vitro studies have shown that members of the N-methyl--aspartate (NMDA) class of glutamate receptors interact with a synapse-associated protein, SAP90 (PSD-95). However, evidence for the in vivo interaction of NMDA receptors with SAPs is still lacking. In the present study, we demonstrate the specific interaction between SAP102, a novel synapse-associated protein, and the NMDA receptor complex from the rat cortical synaptic plasma membranes using co-immunoprecipitation techniques. No association was observed between SAP102 and GluR1, a member of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate class of glutamate receptors. To identify the domain on the NMDA receptor responsible for this interaction, we constructed hexahistidine fusion proteins from different regions of the NR1a and NR2 subunits of the NMDA receptor. Immunoblot overlay experiments showed that while the C-terminal domain of the NR2 subunit displayed strong binding, the NR1a intracellular C-terminal tail did not interact with SAP102. The site of interaction was more precisely located to the last 20 amino acids of the NR2 subunit as indicated by the interaction of the synthetic peptide with SAP102. In summary, we demonstrate here for the first time an in vivo interaction between the native NMDA receptor complex and a synapse-associated protein. These results suggest that SAP102 may play an important role in NMDA receptor clustering and immobilization at excitatory synapses.

Animals↗

PubMed1996

Molecular characterization of dendritically localized transcripts encoding MAP2.

Transcripts encoding high molecular weight (Hwt) isoforms of microtubule-associated protein 2 (MAP2) have been localized in the dendritic compartment of neurons. In contrast, nearly all other neuronal messages, including transcripts encoding low molecular weight (Lwt) MAP2 isoforms, are restricted to cell somas. The mechanisms underlying the dendritic localization of Hwt-MAP2 transcripts are not known. In non-neuronal systems, mRNAs, are localized via signal sequences situated in their 3' untranslated regions (3' UTRs). In this study, we have localized the putative dendritic targeting element (DTE) in Hwt-MAP2 mRNAs by comparing the nucleotide sequences of the somatically localized 6 kb Lwt-MAP2 transcripts with the dendritcally localized 9 kb messages. Our analysis shows that both 6 kb and 9 kb transcripts have identical 3' - and 5'- UTRs, precluding the possibility that the DTE lies in these regions. Within the coding region a single segment that is unique to 9 kb Hwt MAP2 transcripts was identified. These findings suggest that the DTE lies within the 4 kb RNA segment that encodes the projection domain of Hwt-MAP2.

Animals↗