Src-mediated tyrosine phosphorylation of NR2 subunits of N-methyl-D-aspartate receptors protects from calpain-mediated truncation of their C-terminal domains.
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Src-mediated tyrosine phosphorylation of N-methyl-d-aspartate receptor subunits has been shown to modify the functional properties of N-methyl-d-aspartate receptors. Moreover, calpain-mediated truncation of N-methyl-d-aspartate receptor subunits has been found to alter the structure of the receptors. In the present study, we first used immunoprecipitation with a variety of antibodies against N-methyl-d-aspartate receptor subunits and anti-phosphotyrosine antibodies to show that tyrosine-phosphorylated subunits of N-methyl-d-aspartate receptor are protected against calpain-mediated truncation of their C-terminal domains. A GST fusion protein containing the C-terminal domain of NR2A was used to identify the calpain cutting sites in the C-terminal domain. One site was identified at residues 1278-1279, corresponding to one of the preferred calpain truncation sites. This site is adjacent to a consensus sequence for Src-mediated tyrosine phosphorylation, and Src-mediated tyrosine phosphorylation of the GST-NR2A C-terminal fusion protein also inhibited calpain-mediated truncation of the fusion protein. We propose that phosphorylation of NR2 subunits and the resulting inhibition of calpain-mediated truncation of their C-terminal domains provide for the stabilization of the N-methyl-d-aspartate receptors in postsynaptic structures. (+info)
SNAP-25 functional domains in SNARE core complex assembly and glutamate release of cerebellar granule cells.
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Synaptosomal associated protein of 25 kDa (SNAP-25) is a member of the SNARE protein complex that has been implicated in synaptic vesicle docking and fusion. In this report, we have generated SNAP-25 mutants and assayed their functions in SNARE complex formation and glutamate release from cultured rat cerebellar granule cells. In vitro binding studies show that a deletion mutant lacking the C-terminal 181-206 amino acid sequence inhibits the formation of the SNARE core complex. Additional deletion of an N-terminal 1-31 amino acid sequence abolished this inhibitory activity. Adenovirus-mediated gene transfer is used to overexpress wild-type and mutant SNAP-25 in cerebellar granule cells. Neurons overexpressing the wild-type protein show slight reductions in glutamate release, ranging from 10 to 15% in both the developing and mature granule cells. A 30-35% inhibition is obtained with the C-terminal deletion mutant, and the inhibitory effect is abolished in the N- and C-terminal double deletion mutant. These results demonstrate that the SNARE core complex exists in a dynamic and reversible state, and the formation of the core complex is necessary for neurotransmitter release in neurons. (+info)
Membrane-delimited coupling between sigma receptors and K+ channels in rat neurohypophysial terminals requires neither G-protein nor ATP.
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Receptor-mediated modulation of ion channels generally involves G-proteins, phosphorylation, or both in combination. The sigma receptor, which modulates voltage-gated K+ channels, is a novel protein with no homology to other receptors known to modulate ion channels. In the present study patch clamp and photolabelling techniques were used to investigate the mechanism by which sigma receptors modulate K+ channels in peptidergic nerve terminals. The sigma receptor photoprobe iodoazidococaine labelled a protein with the same molecular mass (26 kDa) as the sigma receptor protein identified by cloning. The sigma receptor ligands pentazocine and SKF10047 modulated K+ channels, despite intra-terminal perfusion with GTP-free solutions, a G-protein inhibitor (GDPbetaS), a G-protein activator (GTPgammaS) or a non-hydrolysable ATP analogue (AMPPcP). Channels in excised outside-out patches were modulated by ligand, indicating that soluble cytoplasmic factors are not required. In contrast, channels within cell-attached patches were not modulated by ligand outside a patch, indicating that receptors and channels must be in close proximity for functional interactions. Channels expressed in oocytes without receptors were unresponsive to sigma receptor agonists, ruling out inhibition through a direct drug interaction with channels. These experiments indicate that sigma receptor-mediated signal transduction is membrane delimited, and requires neither G-protein activation nor protein phosphorylation. This novel transduction mechanism is mediated by membrane proteins in close proximity, possibly through direct interactions between the receptor and channel. This would allow for more rapid signal transduction than other ion channel modulation mechanisms, which in the present case of neurohypophysial nerve terminals would lead to the enhancement of neuropeptide release. (+info)
The Ets transcription factor GABP is required for postsynaptic differentiation in vivo.
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At chemical synapses, neurotransmitter receptors are concentrated in the postsynaptic membrane. During the development of the neuromuscular junction, motor neurons induce aggregation of acetylcholine receptors (AChRs) underneath the nerve terminal by the redistribution of existing AChRs and preferential transcription of the AChR subunit genes in subsynaptic myonuclei. Neural agrin, when expressed in nonsynaptic regions of muscle fibers in vivo, activates both mechanisms resulting in the assembly of a fully functional postsynaptic apparatus. Several lines of evidence indicate that synaptic transcription of AChR genes is primarily dependent on a promoter element called N-box. The Ets-related transcription factor growth-associated binding protein (GABP) binds to this motif and has thus been suggested to regulate synaptic gene expression. Here, we assessed the role of GABP in synaptic gene expression and in the formation of postsynaptic specializations in vivo by perturbing its function during postsynaptic differentiation induced by neural agrin. We find that neural agrin-mediated activation of the AChR epsilon subunit promoter is abolished by the inhibition of GABP function. Importantly, the number of AChR aggregates formed in response to neural agrin was strongly reduced. Moreover, aggregates of acetylcholine esterase and utrophin, two additional components of the postsynaptic apparatus, were also reduced. Together, these results are the first direct in vivo evidence that GABP regulates synapse-specific gene expression at the neuromuscular junction and that GABP is required for the formation of a functional postsynaptic apparatus. (+info)
A novel SNAP25-caveolin complex correlates with the onset of persistent synaptic potentiation.
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We have identified synaptic protein complexes in intact rat hippocampal slices using the rapid chemical cross-linking reagent paraformaldehyde. Cellular proteins were rapidly cross-linked, solubilized, separated electrophoretically by SDS-PAGE, and then identified immunologically. Multiple complexes containing syntaxin, the synaptosomal-associated protein of 25 kDa (SNAP25), and vesicle-associated membrane protein (VAMP) were observed to coexist in a single hippocampal slice including a 100 kDa cross-linked protein complex that exhibited the same electrophoretic migration as a member of the previously identified SDS-resistant soluble N-ethylmaleimide-sensitive fusion attachment protein receptor "core" of the 20 S complex. A VAMP-synaptophysin complex, reported previously in vitro, was also observed in the hippocampal slices. This study links biochemical and physiological studies involving presynaptic proteins implicated in secretion and confirms that these proteins that have been studied extensively previously in the presence of detergent do form "bona fide" cellular complexes. Importantly, we have also detected additional novel protein complexes that do not correspond to complexes identified previously in vitro. After the induction of persistent synaptic potentiation, an abundant 40 kDa SNAP25-caveolin1 complex was observed. The SNAP25-caveolin1 complex was not abundant in control slices and, therefore, represents the first demonstration of a reorganization of protein complexes in intact hippocampal slices during the induction of synaptic potentiation. The interaction between caveolin1 and SNAP25 was confirmed biochemically by demonstration of the association of caveolin with recombinant-immobilized SNAP25 and by the coimmunoprecipitation of SNAP25 using caveolin-specific antisera. Caveolin1, like SNAP25, was observed to be abundant in isolated hippocampal nerve terminals (synaptosomes). Immunofluorescent studies demonstrated that both SNAP25 and caveolin1 are present in neurons and colocalize in axonal varicosities. These results suggest that a short-lasting SNAP25-caveolin interaction may be involved in the early phase of synaptic potentiation. (+info)
F-actin is concentrated in nonrelease domains at frog neuromuscular junctions.
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To gain insight into the role of F-actin in the organization of synaptic vesicles at release sites, we examined the synaptic distribution of F-actin by using a unique synaptic preparation of frog target-deprived nerve terminals. In this preparation, imaging of the synaptic site was unobstructed by the muscle fiber cytoskeleton, allowing for the examination of hundreds of synaptic sites in their entirety in whole mounts. At target-deprived synaptic sites F-actin was distributed in a ladder-like pattern and was colocalized with beta-fodrin. Surprisingly, F-actin stain, which we localized to the nerve terminal itself, did not overlap a synaptic vesicle marker, suggesting that it was concentrated in nonrelease domains of nerve terminals between clusters of synaptic vesicles. These findings suggest that the majority of the presynaptic F-actin is not involved in tethering synaptic vesicles. Instead, the strategic presynaptic positioning of this cytoskeletal meshwork in nonrelease domains of the nerve terminal suggests alternate functions such as restricting synaptic vesicles to release domains, recycling synaptic vesicles, or stabilizing the nerve terminal. (+info)
Nerve terminals form but fail to mature when postsynaptic differentiation is blocked: in vivo analysis using mammalian nerve-muscle chimeras.
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To better understand the role of the postsynaptic cell in the differentiation of presynaptic terminals, we transplanted muscles that lacked postsynaptic differentiation from mutant mice into normal adult immunocompatible hosts and attached the host nerve to the grafts. Host motor axons innervated wild-type grafted muscle fibers and established normal appearing chimeric neuromuscular junctions. By repeated in vivo imaging, we found that these synapses were stably maintained. Results were different when nerves entered transplanted muscles derived from mice lacking muscle-specific receptor tyrosine kinase (MuSK) or rapsyn, muscle-specific components required for postsynaptic differentiation. Initial steps in presynaptic differentiation (e.g., formation of rudimentary arbors and vesicle clustering at terminals) occurred when wild-type neurites contacted MuSK- or rapsyn deficient muscle fibers, either in vivo or in vitro. However, wild-type terminals contacting MuSK or rapsyn mutant muscle fibers were unable to mature, even when the chimeras were maintained for up to 7 months. Moreover, in contrast to the stability of wild-type synapses, wild-type nerve terminals in mutant muscles underwent continuous remodeling. These results suggest that postsynaptic cells supply two types of signals to motor axons: ones that initiate presynaptic differentiation and others that stabilize the immature contacts so that they can mature. Normal postsynaptic differentiation appears to be dispensable for initial stages of presynaptic differentiation but required for presynaptic maturation. (+info)
Mechanisms for ovariectomy-induced hyperalgesia and its relief by calcitonin: participation of 5-HT1A-like receptor on C-afferent terminals in substantia gelatinosa of the rat spinal cord.
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Chronic treatment with calcitonin in osteoporotic patients alleviates the pain associated with this condition by an unknown mechanism. In ovariectomized rats that develop osteoporosis and hyperalgesia, we examined whether a functional change in serotonergic systems in the spinal dorsal horn was involved, using whole-cell recordings from substantia gelatinosa neurons in spinal cord slices and [(3)H]8-hydroxy-2(di-n-propylamino)tetralin ([(3)H]8-OH-DPAT) binding. Hyperalgesia could be attributed to the elimination of presynaptic inhibition by 5-HT of glutamatergic primary C-afferent terminals and an associated decrease in the density of [(3)H]8-OH-DPAT binding sites whose receptors are neither 5-HT(1A)- nor 5-HT(7)-subtype. These changes in serotonergic systems were restored after chronic treatment with calcitonin. Reversal of 5-HT receptor changes by calcitonin treatment may provide an explanation for its analgesic actions in patients. (+info)