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At least 973 records · Page 54Linked to original sources
PubMed2000

Mechanisms of initiation and termination of signalling by neuropeptide receptors: a comparison with the proteinase-activated receptors.

Biological responses to neuropeptides are rapidly attenuated by overlapping mechanisms that include peptide degradation by cell-surface proteases, receptor uncoupling from heterotrimeric G-proteins and receptor endocytosis. We have investigated the mechanisms that terminate the proinflammatory effects of the neuropeptide substance P (SP), which are mediated by the neurokinin 1 receptor (NK1R). Neutral endopeptidase degrades SP in the extracellular fluid and is one of the first mechanisms to terminate signalling. G-protein receptor kinases and second-messenger kinases phosphorylate the NK1R to permit interaction with beta-arrestins, which uncouple the receptor from G-proteins to terminate the signal. SP-induces NK1R endocytosis by a beta-arrestin-dependent mechanism, which also involves the GTPases dynamin and Rab5a. Endocytosis contributes to desensitization by depleting receptors from the cell surface. Disruption of these mechanisms results in uncontrolled stimulation and disease. Thus the deletion of neutral endopeptidase in mice exacerbates inflammation of many tissues. There are similarities and distinct differences in the mechanisms that regulate signalling by neuropeptide receptors and other G-protein-coupled receptors, in particular those that are activated irreversibly by proteolysis.

Animals↗
PubMed2000

Constitutive endocytosis of GABAA receptors by an association with the adaptin AP2 complex modulates inhibitory synaptic currents in hippocampal neurons.

Type A GABA receptors (GABA(A)) mediate the majority of fast synaptic inhibition in the brain and are believed to be predominantly composed of alpha, beta, and gamma subunits. Although changes in cell surface GABA(A) receptor number have been postulated to be of importance in modulating inhibitory synaptic transmission, little is currently known on the mechanism used by neurons to modify surface receptor levels at inhibitory synapses. To address this issue, we have studied the cell surface expression and maintenance of GABA(A) receptors. Here we show that constitutive internalization of GABA(A) receptors in hippocampal neurons and recombinant receptors expressed in A293 cells is mediated by clathrin-dependent endocytosis. Furthermore, we identify an interaction between the GABA(A) receptor beta and gamma subunits with the adaptin complex AP2, which is critical for the recruitment of integral membrane proteins into clathrin-coated pits. GABA(A) receptors also colocalize with AP2 in cultured hippocampal neurons. Finally, blocking clathrin-dependant endocytosis with a peptide that disrupts the association between amphiphysin and dynamin causes a large sustained increase in the amplitude of miniature IPSCs in cultured hippocampal neurons. These results suggest that GABA(A) receptors cycle between the synaptic membrane and intracellular sites, and their association with AP2 followed by recruitment into clathrin-coated pits represents an important mechanism in the postsynaptic modulation of inhibitory synaptic transmission.

Adaptor Protein Complex 2↗
PubMed2000

The products of the Drosophila stoned locus interact with synaptic vesicles via synaptotagmin.

The stoned locus of Drosophila melanogaster encodes two novel proteins, stonedA (STNA) and stonedB (STNB), both of which are expressed in the nervous system. Flies with defects at the stoned locus have abnormal behavior and altered synaptic transmission. Genetic interactions, in particular with the shibire (dynamin) mutation, indicated a presynaptic function for stoned and suggested an involvement in vesicle cycling. Immunological studies revealed colocalization of the stoned proteins at the neuromuscular junction with the integral synaptic vesicle protein synaptotagmin (SYT). We show here that stoned interacts genetically with synaptotagmin to produce a lethal phenotype. The STNB protein is found by co-immunoprecipitation to be associated with synaptic vesicles, and glutathione S-transferase pull-downs demonstrate an in vitro interaction between the micro2-homology domain of STNB and the C2B domain of the SYTI isoform. The STNA protein is also found in association with vesicles, and it too exhibits an in vitro association with SYTI. However, we find that the bulk of STNA is in a nonmembranous fraction. By using the shibire mutant to block endocytosis, STNB is shown to be present on some synaptic vesicles before exocytosis. However, STNB is not associated with all synaptic vesicles. We hypothesize that STNB specifies a subset of synaptic vesicles with a role in the synaptic vesicle cycle that is yet to be determined.

Animals↗
PubMed2001

Interaction of stoned and synaptotagmin in synaptic vesicle endocytosis.

The Drosophila dicistronic stoned locus encodes two distinctive presynaptic proteins, Stoned A (STNA) and Stoned B (STNB); STNA is a novel protein without homology to known synaptic proteins, and STNB contains a domain with homology to the endocytotic protein AP50. Both Stoned proteins colocalize precisely with endocytotic proteins including the AP2 complex and Dynamin in the "lattice network" characteristic of endocytotic domains in Drosophila presynaptic terminals. FM1-43 dye uptake studies in stoned mutants demonstrate a striking decrease in the size of the endo-exo-cycling synaptic vesicle pool and loss of spatial regulation of the vesicular recycling intermediates. Mutant synapses display a significant delay in vesicular membrane retrieval after depolarization and neurotransmitter release. These studies suggest that the Stoned proteins play a role in mediating synaptic vesicle endocytosis. We have documented previously a highly specific synaptic mislocalization and degradation of Synaptotagmin I in stoned mutants. Here we show that transgenic overexpression of Synaptotagmin I rescues stoned embryonic lethality and restores endocytotic recycling to normal levels. Furthermore, overexpression of Synaptotagmin I in otherwise wild-type animals results in increased synaptic dye uptake, indicating that Synaptotagmin I directly regulates the endo-exo-cycling synaptic vesicle pool size. In parallel with recent biochemical studies, this genetic analysis strongly suggests that Stoned proteins regulate the AP2-Synaptotagmin I interaction during synaptic vesicle endocytosis. We conclude that Stoned proteins control synaptic transmission strength by mediating the retrieval of Synaptotagmin I from the plasma membrane.

Adaptor Protein Complex 2↗
PubMed2002

P2X receptor trafficking in neurons is subunit specific.

P2X receptors within the CNS mediate excitatory synaptic transmission and also act presynaptically to modulate neurotransmitter release. We have studied the targeting and trafficking of P2X4 and P2X2 receptors heterologously expressed in cultured olfactory bulb neurons. Homomeric P2X4 receptors had a punctate distribution, and many of the puncta colocalized with early endosomes. In contrast, P2X2 receptors were primarily localized at the plasma membrane. By antibody-labeling of surface receptors in living neurons, we showed that P2X4 receptors undergo rapid constitutive internalization and subsequent reinsertion into the plasma membrane, whereas P2X2 receptors were not regulated in such a way. The internalization of P2X4 receptors was dynamin-dependent, and the binding of ATP enhanced the basal rate of retrieval in a Ca2+-independent manner. The presence of the P2X4 subunit in a P2X4/6 heteromer governed the trafficking properties of the receptor. P2X receptors acted presynaptically to enhance the release of glutamate, suggesting that the regulated cycling of P2X4-containing receptors might provide a mechanism for modulation of synaptic transmission.

Adenosine Triphosphate↗
PubMed2002

Endophilin is critically required for synapse formation and function in Drosophila melanogaster.

Studies in cell-free systems and the lamprey giant synapse have implicated crucial roles for amphiphysin and endophilin in synaptic transmission. However, null mutants at the amphiphysin locus of Drosophila are viable and have no demonstrable synaptic vesicle-recycling defect. This has necessitated a re-examination of the role of Src homology 3 domain-containing proteins in synaptic vesicle recycling. In this report, we show that endophilin-deficient eye clones in Drosophila have an altered electroretinogram. A characteristic of this defect is its aggravation during heightened visual stimulation. It is shown that endophilin is primarily required in the nervous system. Decreased endophilin activity results in alterations in the neuromuscular junction structure and physiology. Immunofluorescence studies show colocalization of endophilin with dynamin consistent with a possible role in synaptic vesicle recycling.

5' Untranslated Regions↗
PubMed2003

Endophilin promotes a late step in endocytosis at glial invaginations in Drosophila photoreceptor terminals.

Retrieval of synaptic vesicles from the membrane of neurons is crucial to maintain normal rates of neurotransmitter release. Photoreceptor terminals of the fly's eye release neurotransmitter in a tonic manner. They therefore rely heavily on vesicle regeneration. Null mutations in endophilin (endo) block clathrin-mediated endocytosis at the Drosophila neuromuscular junction, where previous analysis of hypomorphic mutations has suggested a function for Endophilin (Endo) before vesicle fission, during membrane bending. Here, at fly photoreceptor synapses, we show that Endo is localized to synaptic vesicles at sites of endocytosis that are glial invaginations called capitate projections, and that when the photoreceptor synapses lack Endo they are impaired in their ability to release neurotransmitter. Detailed ultrastructural analysis of endo null mutant photoreceptor synapses fails to reveal a defect at early stages of vesicle reformation but, instead, reveals an accumulation of clusters of electron-dense, apparently nonfunctional, late endocytotic vesicles. Using dynamin;endo double-mutant photoreceptors, we provide further evidence that ultimately the function of Endophilin is required late in endocytosis, allowing vesicles to progress through the synaptic vesicle cycle.

Adaptor Proteins, Signal Transducing↗
PubMed1990

Mx proteins: antiviral proteins by chance or by necessity?

The interferon-inducible Mx1 protein is responsible for inborn resistance of mice to influenza. It is now recognized that this protein is a member of a family of interferon-inducible, putative GTP-binding proteins found in many organisms. Thus, these proteins, called the Mx proteins, are found in species that are naturally infected with influenza virus, and also in species that are not. Some Mx proteins display a broader antiviral profile than the one observed for Mx1 in mice. Others, however, may not be antiviral. Two recently discovered GTP-binding proteins, Vps1p in yeast and dynamin in rat, are also related to Mx1. These proteins are synthesized constitutively and serve basic cellular functions.

Animals↗
PubMed1991

[Cell division and the microtubular cytoskeleton].

Kinetochore microtubules result from an interaction between astral microtubules and the kinetochore of the chromosomes after breakdown of the nuclear envelope at the end of prophase. In this process, the end of a microtubule projecting from one of the polar regions contacts the primary constriction of a chromosome. The latter then undergoes rapid poleward movement. Concerning the mechanism of anaphase chromosome movement, the motive force for the chromosome-to-pole movement appears to be generated at the kinetochore or in the region very close to it. It has not been determined whether chromosomes propel themselves along stationary kinetochore microtubules by a motor at the kinetochore, or they are pulled poleward by a traction fiber consisting of kinetochore microtubules and associated motors. As chromosomes move poleward coordinate disassembly of kinetochore microtubules might occur from their kinetochore ends. In diatom and yeast spindles, elongation of the spindle in anaphase (anaphase B) may be explained by microtubule assembly at polar microtubule ends in the spindle mid-zone and sliding of the antiparallel microtubules from the opposite poles. The sliding force appears to be generated through an ATP-dependent microtubule motor. In isolated sea urchin spindles, the microtubule assembly at the equator alone might provide the force for spindle elongation, although, in addition, involvement of microtubule sliding by a GTP-requiring mechanochemical enzyme cannot be excluded. Discussions were made on possible participation in anaphase chromosome movement of such microtubule motors as dynein, kinesin, dynamin and the claret segregation protein.

Adenosine Triphosphate↗
PubMed1998

Receptor-specific allelic exclusion of TCRV alpha-chains during development.

Expression of a single Ag receptor on lymphocytes is maintained via allelic exclusion that generates cells with a clonal receptor repertoire. We show in normal mice and mice expressing functionally rearranged TCR alphabeta transgenes that allelic exclusion at the TCR alpha locus is not operational in immature thymocytes, whereas most mature T cells express a single TCRV alpha-chain. TCRV alpha allelic exclusion in mature thymocytes is regulated through a CD45 tyrosine phosphatase-mediated signal during positive selection. Using functional and genetic systems for selection of immature double TCRV alpha+ thymocytes, we show that peptide-specific ligand recognition provides the signal for allelic exclusion, i.e., mature T cells maintain expression of the ligand-specific TCRV alpha-chain, but lose the nonfunctional receptor. Whereas activation of TCRV beta-chains or CD3epsilon leads to receptor internalization, TCRV alpha ligation promotes retention of the TCR on the cell surface. Although both TCRV alpha- and TCRV beta-chains trigger phosphotyrosine signaling, only the TCRV beta-chain mediates membrane recruitment of the GTPase dynamin. These data indicate that TCRV alpha-directed signals for positive selection control allelic exclusion in T cells, and that developmental signals can select for single receptor usage.

Alleles↗
PubMed2000

Differences in protein level between neonatal and adult brain.

Age is a physiological condition that affects protein expression. We investigated differences in protein level between newborn and adult brains. Brain tissue extracts from male and female adult and neonatal rats were analyzed by two-dimensional gel electrophoresis. Gel comparison revealed the presence of many differences, of qualitative and quantitative nature, between a neonatal and adult brain. The most significant age-related difference concerned alpha-fetoprotein, which was detected in the brain of neonatal rats only. The levels of 22 proteins, including dihydropyrimidinase-related proteins 1,3, and 4 and 14-3-3 proteins, were higher in the neonatal brain, whereas the levels of 28 proteins, including dihydropyrimidinase-related protein 2, dynamin-1 and many enzymes were higher in the adult brain. We did not detect a consistent sex-related difference in the brain proteins. An inconsistency was observed in the location of the spot representing glial fibrillary acidic protein in the male brain.

Aging↗
PubMed1993

Mx proteins: GTPases involved in the interferon-induced antiviral state.

Mx proteins have molecular masses between 70 and 80 kDa and their synthesis is tightly regulated by interferons in mammalian and non-mammalian vertebrates. Some Mx proteins function as intracellular mediators of the interferon-induced antiviral state. When suitable cDNA constructs were constitutively expressed in mouse 3T3 cells the mouse nuclear Mx1 protein conferred selective resistance to influenza virus. The human cytoplasmic MxA protein conferred resistance to influenza virus and vesicular stomatitis virus but not to other viruses. Mx1 blocks influenza virus mRNA synthesis within the nucleus of infected cells. Mx1 presumably interacts with the influenza virus polymerase subunit PB2, because overexpression of PB2 titrates out the Mx1 block. MxA does not inhibit mRNA synthesis of influenza virus; it inhibits a subsequent cytoplasmic viral multiplication step. A possible target is the transport of newly synthesized influenza virus polymerase proteins back to the nucleus. Inhibition by MxA of vesicular stomatitis virus, which replicates in the cytoplasm, is at the transcriptional level. Parts of the N-terminal halves of all known Mx proteins are highly conserved. They contain the typical GTP-binding motif and show significant homology to other members of a new family of GTPases that includes rat dynamin, Drosophila Shibire and the yeast proteins Vps1/Spo15 and Mgm1. Purified Mx1 and MxA proteins possess GTPase activity. The GTP/GDP conversion rates are about 40 per min, and Km values about 700 microM. Mx1 and MxA variants with mutations in the GTP-binding sequences that violate the consensus are unable to confer virus resistance in vivo or to hydrolyse GTP in vitro, suggesting that GTPase activity is necessary for antiviral activity of Mx proteins. We hypothesize that the antivirally active Mx proteins (directly or indirectly) bind to polymerase proteins of susceptible viruses, thereby abolishing normal viral polymerase function. Interaction of Mx with viral targets is probably a GTP-dependent process.

Amino Acid Sequence↗
PubMed2004

Deficit of in vivo mitochondrial ATP production in OPA1-related dominant optic atrophy.

Dominant optic atrophy has been associated with mutations in the OPA1 gene, which encodes for a dynamin-related GTPase, a mitochondrial protein implicated in the formation and maintenance of mitochondrial network and morphology. We used phosphorus magnetic resonance spectroscopy to assess calf muscle oxidative metabolism in six patients from two unrelated families carrying the c.2708-2711delTTAG deletion in exon 27 of the OPA1 gene. The rate of postexercise phosphocreatine resynthesis, a measure of mitochondrial adenosine triphosphate production rate, was significantly delayed in the patients. Our in vivo results show for the first time to our knowledge a deficit of oxidative phosphorylation in OPA1-related DOA.

Adenosine Triphosphate↗
PubMed2005

OPA1 expression in the normal rat retina and optic nerve.

Autosomal dominant optic atrophy (DOA) is the most common form of hereditary optic neuropathy. DOA presents in the first decade of life and manifests as progressive vision loss. In DOA retinal ganglion cells and the optic nerve degenerate by an unknown mechanism. The gene mutated in DOA, Optic Atrophy Type 1 (OPA1), encodes a dynamin-related GTPase implicated in mitochondrial fusion and maintenance of the mitochondrial network and genome. Here, we determine which cell types in the normal retina and the optic nerve express OPA1. In the normal rat retina, OPA1 is expressed in the ganglion cell layer as well as in the outer plexiform layer, the inner nuclear layer, and the inner plexiform layer. In the ganglion cell layer, OPA1 is expressed predominantly in retinal ganglion cells. By contrast, OPA1 protein is low or undetectable in astrocytes and oligodendrocytes of the optic nerve. Additionally, OPA1 protein is present in axonal mitochondria. Last, OPA1 expression is present in mitochondria of processes and cell bodies of purified retinal ganglion cells and of the RGC-5 cell line. Thus, OPA1 is predominantly expressed in retinal ganglion cells of the normal rat retina and axons of the optic nerve. These findings may explain the selective vulnerability of retinal ganglion cells to OPA1 loss of function.

Animals↗
PubMed2006

Immunogold localization of phocein in dendritic spines.

Phocein, a widely expressed intracellular protein involved in clathrin- and dynamin-dependent membrane dynamics, has been shown to interact with members of the striatin family of proteins, striatin, SG2NA, and zinedin. Immunogold labeling was performed to assess subcellular localization of phocein in neurons of the rodent cerebellar cortex and hippocampal Ammon's horn. Most of the phocein-bound gold particles were located within dendritic thorns and spines of the cerebellar Purkinje cells and hippocampal pyramidal neurons, as observed previously for striatin in striatal neurons. The postsynaptic profiles containing phocein were engaged in asymmetric synapses with the main types of afferents in the cerebellum and in the hippocampus. In the cerebellum, phocein-bound immunogold particle numbers ranged from 1-20 in approximately 50% of the Purkinje cell spines. In these spines most of the immunogold particles were found in the neuroplasm ( approximately 70%) and on nonsynaptic plasma membrane domains and related structures such as endocytic-like profiles ( approximately 18%). As soon as the first postnatal week, phocein was detected in the Purkinje cell somatic and dendritic thorns making asymmetric synapses with climbing fibers. During the following weeks the protein was located in the dendritic spines, as observed in the adult molecular layer. Finally, double immunogold labeling revealed a distribution of phocein and SG2NA suggesting that the two proteins could interact in the Purkinje cell spines. The early postnatal expression of phocein, a protein involved in membrane dynamics, suggests that it may have functional relevance in dendritic remodeling during development and potentially in spine plasticity during adulthood.

Animals↗
PubMed2002

Oligodendrocytes express an alpha/beta-interferon-susceptible Mx gene: molecular characterization of the encoded protein.

Interferons (INFs) are cytokines that exert a plethora of biological activities by interacting with receptors and inducing the synthesis of effector proteins. One such effector is an antiviral protein called Mx, widely distributed among vertebrates. Mx proteins belong to the superfamily of large GTPases, which includes dynamins, products of the Drosophila shibire gene, the yeast Vps1p, and others. Oligodendrocytes (OLGs) synthesize, assemble, and maintain myelin in the central nervous system. In an effort to identify genes implicated in establishing a myelinogenic phenotype, we isolated a 2,494 bp cDNA that encodes a protein of 654 amino acids, which are 95% identical to ovine Mx (from endometrium of pregnant sheep) and 83% similar to human MxA. Cultured OLGs express the mRNA and protein. We present structural features of this protein and speculate on the origin of its induction and the functional significance of its presence in OLGs. The finding that OLGs express an IFN-susceptible protein raises interesting possibilities in light of the evidence that beta-IFN is therapeutically beneficial for the treatment of multiple sclerosis.

Amino Acid Sequence↗
PubMed1997

The expression of endosomal rab proteins correlates with endocytic rate in rat liver cells.

Hepatic endocytosis is characterized by a division of labor between the different cell types with respect to endocytosis, which is mediated by receptors expressed on their cell surface. We have investigated the expression of GTPases of the rab family in rat liver parenchymal and endothelial cells. Small GTPases of the rab protein family control distinct steps of intracellular transport both in the secretory and the endocytic pathway. As controls have been employed the normal rat kidney (NRK) cell line and brain tissue, neuronal cells are known to express high levels of components of the endocytic machinery (clathrin, adaptins, dynamin, uncoating adenosine triphosphatase, etc.). Endothelial cells were found to express four to seven times more rab4, rab5, and rab7 than parenchymal cells. A similar relationship was found between the endocytic rates in the two cell types; the rate of internalization from the plasma membrane of mannose receptors in rat liver endothelial cells was 2.3 pools/min, whereas the corresponding value for internalization of the galactose receptor in parenchymal liver cell was 0.27 pools/min (comparable with the rate of transferrin internalization in NRK cells). Both immunofluorescence and subcellular fractionation experiments showed that rab5 and rab7 were associated with compartments along the endocytic pathway. Brain tissue showed a similar high expression of endocytic components (rab4, rab5, and rab7) as liver endothelial cells, whereas lower values were found in NRK cells. We also analyzed the following proteins involved in endocytosis: clathrin, alpha-adaptin, beta-adaptin, and rabaptin-5. These proteins showed the same pattern of expression as the rab proteins. In conclusion, the results obtained with liver cells corroborate the data obtained in transfected cells and support the notion that rab proteins may be involved in controlling the endocytic rate in liver cells.

Animals↗
PubMed2003

Fourteen novel OPA1 mutations in autosomal dominant optic atrophy including two de novo mutations in sporadic optic atrophy.

The OPA1 gene, encoding a dynamin-related GTPase that plays a role in mitochondrial biogenesis, is implicated in most cases of autosomal dominant optic atrophy (ADOA). Sixty-nine pathogenic OPA1 mutations have been reported so far. Most of these are truncating mutations located in the GTPase domain coding region (exons 8-16) and at the 3'-end (exons 27-28). We screened 44 patients with typical ADOA using PCR-sequencing. We also tested 20 sporadic cases of bilateral optic atrophy compatible with ADOA. Of the 18 OPA1 mutations found, 14 have never been previously reported. The novel mutations include one nonsense mutation, 3 missense mutations, 6 deletions, one insertion and 3 exon-skipping mutations. Two of these are de novo mutations, which were found in 2 patients with sporadic optic atrophy. The recurrent c.2708_2711delTTAG mutation was found in 2 patients with a severe congenital presentation of the disease. These results suggest that screening for OPA1 gene mutations may be useful for patients with optic atrophy who have no affected relatives, or when the presentation of the disease is atypical as in the case of early onset optic atrophy.

Alternative Splicing↗