[show abstract][hide abstract] ABSTRACT: Neurotransmitter release proceeds by Ca(2+)-triggered, SNARE-complex-dependent synaptic vesicle fusion. After fusion, the ATPase NSF and its cofactors α- and βSNAP disassemble SNARE complexes, thereby recycling individual SNAREs for subsequent fusion reactions. We examined the effects of genetic perturbation of α- and βSNAP expression on synaptic vesicle exocytosis, employing a new Ca(2+) uncaging protocol to study synaptic vesicle trafficking, priming, and fusion in small glutamatergic synapses of hippocampal neurons. By characterizing this protocol, we show that synchronous and asynchronous transmitter release involve different Ca(2+) sensors and are not caused by distinct releasable vesicle pools, and that tonic transmitter release is due to ongoing priming and fusion of new synaptic vesicles during high synaptic activity. Our analysis of α- and βSNAP deletion mutant neurons shows that the two NSF cofactors support synaptic vesicle priming by determining the availability of free SNARE components, particularly during phases of high synaptic activity.
[show abstract][hide abstract] ABSTRACT: In the mammalian CNS, each neuron typically receives thousands of synaptic inputs from diverse classes of neurons. Synaptic transmission to the postsynaptic neuron relies on localized and transmitter-specific differentiation of the plasma membrane with postsynaptic receptor, scaffolding, and adhesion proteins accumulating in precise apposition to presynaptic sites of transmitter release. We identified protein interactions of the synaptic adhesion molecule neuroligin 2 that drive postsynaptic differentiation at inhibitory synapses. Neuroligin 2 binds the scaffolding protein gephyrin through a conserved cytoplasmic motif and functions as a specific activator of collybistin, thus guiding membrane tethering of the inhibitory postsynaptic scaffold. Complexes of neuroligin 2, gephyrin and collybistin are sufficient for cell-autonomous clustering of inhibitory neurotransmitter receptors. Deletion of neuroligin 2 in mice perturbs GABAergic and glycinergic synaptic transmission and leads to a loss of postsynaptic specializations specifically at perisomatic inhibitory synapses.
[show abstract][hide abstract] ABSTRACT: Synaptic cell adhesion and synaptogenesis are thought to involve the interaction of neuroligin, a postsynaptic transmembrane protein, with its presynaptic ligand neurexin. Neuroligin also interacts with SAP90/PSD95, a multidomain scaffolding protein thought to recruit proteins to postsynaptic sites. Using expression of GFP-tagged versions of neuroligin in cultured hippocampal neurons, we find that neuroligin is targeted to synapses via intracellular sequences distinct from its SAP90/PSD95 binding site. A neuroligin mutant lacking the intracellular domain fails to target to synapses. These data indicate that postsynaptic targeting of neuroligin does not rely on the scaffolding action of SAP90/PSD95 and is not induced by binding to presynaptic neurexin. Neuroligin is rather targeted to synapses via a postsynaptic mechanism, which may precede and be necessary for subsequent recruitment of neurexin and other neuroligin interactors such as SAP90/PSD95, suggesting a pivotal position for neuroligin in a putative hierarchy of interactions assembling or stabilizing synapses.
Molecular and Cellular Neuroscience 12/2004; 27(3):227-35. · 3.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: The postsynaptic specialisation at glutamatergic synapses is composed of a network of proteins located within the membrane and the underlying postsynaptic density. The strong interconnectivity between the protein components is mediated by a limited number of interaction modes. Particularly abundant are PDZ domain-mediated interactions. An obstacle in understanding the fidelity of postsynaptic processes involving PDZ domains is the high degree of overlap with respect to their binding specificities. Focussing on transsynaptic adhesion molecules, we used the yeast two-hybrid system to obtain an overview of the binding specificities of selected C-terminal PDZ binding motifs. Neuroligin, a postsynaptic cell surface protein that spans the synaptic cleft and interacts with beta-neurexin, served as a starting point. Neuroligin binds to the PDZ domain-containing proteins PSD95, SAP102, Chapsyn110, S-SCAM, Magi1 and 3, Shank1 and 3, Pick1, GOPC, SPAR, Semcap3 and PDZ-RGS3. Next, we examined the relationship between neuroligin and synaptic cell adhesion molecules or glutamate receptor subunits with respect to PDZ-mediated interactions. We found a limited overlap in the PDZ-domain binding specificities of neuroligin with those of Sidekick2 and Ephrin-B2. In contrast, Syndecan2 and IgSF4 show no overlap with the PDZ-domain specificity of neuroligin, instead, they bind to GRIP and syntenin. The AMPA receptor subunit GluR2 interacts with Semcap3 and PDZ-RGS3, whereas the kainate receptor subunits GluR5 and GluR6 show weak interactions with PSD95. In summary, we can sketch a complex pattern of overlap in the binding specificities of synaptic cell surface proteins towards PDZ-domain proteins.
[show abstract][hide abstract] ABSTRACT: Small glutamine-rich tetratricopeptide repeat-containing protein (SGT) is a ubiquitously expressed cochaperone of heat shock cognate protein of 70 kDa (Hsc70). SGT binds to the C terminus of Hsc70, a site used by several tetratricopeptide repeat-containing binding partners to recruit Hsc70 into complexes of diverse function. We describe here an isoform of SGT with 60% amino acid sequence identity that we name betaSGT. In contrast to the previously published alphaSGT, betaSGT is almost exclusively expressed in brain. Both isoforms of SGT possess similar binding properties toward Hsc70 and cysteine string protein, a synaptic vesicle-associated J-domain-containing protein. In addition, SGTs oligomerize without preferences among isoforms. The distribution of protein binding motifs on SGTs reveals a modular structure. The N-terminal domains mediate oligomerization. Binding to Hsc70 is impaired by mutations of basic residues within the central tetratricopeptide repeat domain of betaSGT, indicating a two-carboxylate clamp as the binding mode. The tetratricopeptide repeats are also necessary for binding to the cysteine string protein. However, this binding mode is distinct from the two-carboxylate clamp that is involved in Hsc70 binding. The C-terminal regions of SGTs might constitute independent protein interaction domains. We conclude that betaSGT is likely to cooperate with alphaSGT as co-chaperone of Hsc70 in the brain. The modular structure of SGTs allows them to recruit client proteins to Hsc70 and to direct the resulting complex toward downstream proteins that take over the respective client proteins.
Journal of Biological Chemistry 11/2003; 278(40):38376-83. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Synaptic plasticity underlies many essential processes in the brain, including learning and memory. In their perspective, Meyer and Brose discuss new work (Pak and Sheng) that elucidates one possible mechanism of use-dependent synaptic plasticity in neurons. This mechanism involves the Snk kinase-induced degradation of the SPAR protein leading to alterations in the morphology of postsynaptic dendritic spines
[show abstract][hide abstract] ABSTRACT: The tubulin-binding protein gephyrin copurifies with the inhibitory glycine receptor (GlyR) and is essential for its postsynaptic localization. Here we have analyzed the interaction between the GlyR and recombinant gephyrin and identified a gephyrin binding site in the cytoplasmic loop between the third and fourth transmembrane segments of the beta subunit. GlyR alpha subunits and GABAA receptor proteins failed to bind recombinant gephyrin. However, insertion of an 18 residue segment of the GlyR beta subunit into the GABAA receptor beta 1 subunit conferred gephyrin binding both in an overlay assay and in transfected mammalian cells. These results indicate that beta subunit expression is essential for the formation of a postsynaptic GlyR matrix.
[show abstract][hide abstract] ABSTRACT: The tubulin-binding protein gephyrin copurifies with the inhibitory glycine receptor (GIyR) and is essential for its postsynaptic localization. Here we have analyzed the interaction between the GIyR and recombinant gephyrin and identified a gephyrin binding site in the cytoplasmic loop between the third and fourth transmembrane segments of the β subunit. GIyR α subunits and GABAA receptor proteins failed to bind recombinant gephyrin. However, insertion of an 18 residue segment of the GIyR β subunit into the GABAA receptor β1 subunit conferred gephyrin binding both in an overlay assay and in transfected mammalian cells. These results indicate that β subunit expression is essential for the formation of a postsynaptic GIyR matrix.
[show abstract][hide abstract] ABSTRACT: The inhibitory glycine receptor (GlyR) mediates postsynaptic inhibition in spinal cord, brain stem and other regions of the vertebrate central nervous system. Biochemical and molecular approaches have identified different developmentally and regionally regulated GlyR isoforms that result from the differential expression of at least four genes coding for different variants of the ligand-binding alpha subunit. Molecular studies have allowed identification of GlyR subunit domains implicated in ligand binding, channel formation and receptor assembly. At the postsynaptic membrane, the GlyR colocalizes with a 93-kDa tubulin-binding peripheral membrane protein, gephyrin. Antisense inhibition of gephyrin expression prevents GlyR accumulation at postsynaptic membrane specialization. Thus, gephyrin is essential for postsynaptic receptor topology.
Journal of Physiology-Paris 02/1994; 88(4):243-8. · 0.82 Impact Factor