Calmodulin dependence of presynaptic metabotropic glutamate receptor signaling.

Glutamatergic neurotransmission is controlled by presynaptic metabotropic glutamate receptors (mGluRs). A subdomain in the intracellular carboxyl-terminal tail of group III mGluRs binds calmodulin and heterotrimeric guanosine triphosphate-binding protein (G protein) betagamma subunits in a mutually exclusive manner. Mutations interfering with calmodulin binding and calmodulin antagonists inhibit G protein-mediated modulation of ionic currents by mGluR 7. Calmodulin antagonists also prevent inhibition of excitatory neurotransmission via presynaptic mGluRs. These results reveal a novel mechanism of presynaptic modulation in which Ca(2+)-calmodulin is required to release G protein betagamma subunits from the C-tail of group III mGluRs in order to mediate glutamatergic autoinhibition.

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.

PKC phosphorylation of a conserved serine residue in the C-terminus of group III metabotropic glutamate receptors inhibits calmodulin binding.

Group III metabotropic glutamate receptors (mGluRs) serve as presynaptic receptors that mediate feedback inhibition of glutamate release via a Ca(2+)/calmodulin (CaM)-dependent mechanism. In vitro phosphorylation of mGluR7A by protein kinase C (PKC) prevents its interaction with Ca(2+)/CaM. In addition, activation of PKC leads to an inhibition of mGluR signaling. Here, we demonstrate that disrupting CaM binding to mGluR7A by PKC in vitro is due to phosphorylation of a highly conserved serine residue, S862. We propose charge neutralization of the CaM binding consensus sequence resulting from phosphorylation to constitute a general mechanism for the regulation of presynaptic mGluR signaling.

Interaction of a synaptobrevin (VAMP)-syntaxin complex with presynaptic calcium channels.

Nerve terminal protein complexes implicated in exocytosis were examined by immuno-isolation from rat brain synaptosomes. Immunoprecipitation with anti-syntaxin or anti-VAMP antibodies revealed a syntaxin-SNAP25-VAMP-synaptotagmin complex. Anti-VAMP antibodies also trapped a distinct VAMP-synaptophysin complex. A similar fraction (about 70%) of N-type calcium channels ([125I]omega conotoxin GVIA receptors), was immunoprecipitated by either anti-syntaxin or anti-VAMP antibodies, but not by anti-synaptophysin antibodies (< 4%). The majority of N- but not L-type calcium channels ([3H]PN200-110 receptors), appear to be associated with a synaptic vesicle prefusion complex.

Expression of synaptotagmin and syntaxin associated with N-type calcium channels in small cell lung cancer.

The presence of synaptic proteins involved in excitation/secretion coupling was examined in ten small cell lung cancer lines. N-Type calcium channels (omega-conotoxin receptors), synaptotagmin (p65) and syntaxin (HPC-1) were detected in eight. Co-immunoprecipitation experiments indicated that syntaxin can form a complex with synaptotagmin and calcium channels. The expression of synaptotagmin in small cell lung cancer may elicit an autoimmune response that reduces transmitter release at the nerve terminal.

Interaction of synaptotagmin with voltage gated calcium channels: a role in Lambert-Eaton myasthenic syndrome?

Plasma from patients with Lambert-Eaton myasthenic syndrome (LEMS), an autoimmune disease of neuromuscular transmission, contains antibodies that bind to the synaptic vesicle protein synaptotagmin. Synaptotagmin associates with calcium channels and appears to regulate synaptic vesicle docking at the plasma membrane prior to rapid neurotransmitter release. Autoantibodies directed against a synaptotagmin-calcium channel complex may be involved in the etiology of LEMS. In the majority of patients LEMS is associated with small cell lung cancer (SCLC). We have detected the expression of proteins of the secretory pathway, including synaptotagmin, syntaxin and N-type calcium channels, in a panel of SCLC tumor lines. These observations are compatible with the hypothesis that the initial autoimmune response in LEMS is triggered by the tumor.

Protein interactions implicated in neurotransmitter release.

Biochemical evidence indicates that the exocytotic release of neurotransmitters involves both evolutionary conserved membrane proteins, the SNAREs, as well as ubiquitous cytosolic fusion proteins, NSF and SNAPs. We have analyzed the biochemical properties and the physiological effects of these proteins. Our data suggest models how NSF, SNAPs and SNAREs may function in neurotransmitter exocytosis.

Synaptotagmin: a Lambert-Eaton myasthenic syndrome antigen that associates with presynaptic calcium channels.

Plasma from patients with Lambert-Eaton myasthenic syndrome (LEMS), an autoimmune disease of neuromuscular transmission, contains antibodies that immunoprecipitate 125I-omega-conotoxin GVIA labeled-calcium channels solubilized from rat brain. These antibodies label a 58-kDa protein in Western blots of partially purified 125I-omega-conotoxin receptor preparations. Monoclonal antibody 1D12, produced by immunizing mice with synaptic membranes, has similar properties as these LEMS IgG. 1D12 antigen was purified by immunoaffinity chromatography and shown to bind LEMS IgG. The antigen was identified by immunoscreening a rat brain cDNA library with mAb 1D12 and found to have strong homology to the synaptic vesicle protein synaptotagmin. These antibodies immunoprecipitate calcium channels by binding to synpatotagmin, an associated protein. We suggest that the interaction between synaptotagmin and omega-conotoxin sensitive calcium channels plays a role in docking synaptic vesicles at the plasma membrane prior to rapid neurotransmitter release. Autoantibody binding to a synaptotagmin-calcium channel complex may be involved in the etiology of LEMS.

Role of NSF in neurotransmitter release: a peptide microinjection study at the crayfish neuromuscular junction.

Peptides that inhibit the SNAP-stimulated ATPase activity of N-ethylmaleimide-sensitive fusion protein (NSF-2, NSF-3) were injected intra-axonally to study the role of this protein in the release of glutamate at the crayfish neuromuscular junction. Macropatch recording was used to establish the quantal content and to construct synaptic delay histograms. NSF-2 or NSF-3 injection reduced the quantal content, evoked by either direct depolarization of a single release bouton or by axonal action potentials, on average by 66 +/- 12% (mean +/- SD; n = 32), but had no effect on the time course of release. NSF-2 had no effect on the amplitude or shape of the presynaptic action potential nor on the excitatory nerve terminal current. Neither NSF-2 nor NSF-3 affected the shape or amplitude of single quantal currents. Injection of a peptide with the same composition as NSF-2, but with a scrambled amino acid sequence, failed to alter the quantal content. We conclude that, at the crayfish neuromuscular junction, NSF-dependent reactions regulate quantal content without contributing to the presynaptic mechanisms that control the time course of release.

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.

Calcium extrusion from mammalian photoreceptor terminals.

Ribbon synapses of vertebrate photoreceptors constantly release glutamate in darkness. Transmitter release is maintained by a steady influx of calcium through voltage-dependent calcium channels, implying the presence of a mechanism that is able to extrude calcium at an equal rate. The two predominant mechanisms of intracellular calcium extrusion are the plasma membrane calcium ATPase (PMCA) and the Na+/Ca2+-exchanger. Immunohistochemical staining of retina sections revealed strong immunoreactivity for the PMCA in rod and cone terminals, whereas staining for the Na+/Ca2+-exchanger was very weak. The PMCA was localized to the plasma membrane along the sides of the photoreceptor terminals and was excluded from the base of the terminals where the active zones are located. The amplitude of a calcium-activated chloride current was used to monitor the intracellular calcium concentration. An upper limit for the time required to remove intracellular free calcium is obtained from two time constants measured for the calcium-activated chloride current tail currents: one of 50 msec and a second of 190 msec. Calcium extrusion was inhibited in the absence of intracellular ATP or in the presence of the PMCA inhibitor orthovanadate, but was unaffected by replacement of external Na+ with Li+. The data indicate that the PMCA, rather than the Na+/Ca2+-exchanger, is the predominant mechanism for calcium extrusion from photoreceptor synaptic terminals.

Purification of the N-type calcium channel associated with syntaxin and synaptotagmin. A complex implicated in synaptic vesicle exocytosis.

omega-Conotoxin-sensitive N-type calcium channels control neurotransmitter release at the nerve terminal and interact with proteins implicated in secretion. Solubilized omega-conotoxin receptors from rat brain synaptic membrane were immunoprecipitated by antibodies against calcium channel alpha 1 subunits, syntaxin, and a 105-kDa plasma membrane protein. A multimeric complex, composed of calcium channel subunits, and synaptic proteins that showed varying degrees of association, was purified by a procedure involving anti-syntaxin immunoaffinity chromatography. A 250-kDa N-type alpha 1 subunit, containing cAMP-dependent phosphorylation site(s), was identified by photoaffinity labeling with 125I-azidonitrobenzoyl omega-conotoxin and immunoblotting with sequence-directed antibodies. An immunologically related 210-kDa form of the alpha 1 subunit was detected that displayed different pharmacological and regulatory properties. Protein bands of 140, 70, 58, and 35 kDa comigrated with purified alpha 1 subunits upon sucrose gradient centrifugation, whereas the 105-kDa protein was removed. The 58- and 35-kDa bands contained, respectively, the synaptic vesicle protein synaptotagmin and syntaxin, a plasma membrane protein that binds synaptic vesicle proteins. Purified omega-contoxin receptors were quantitatively immunoprecipitated by anti-syntaxin antibodies. These proteins may constitute an isolated exocytotic complex in which the N-type calcium channel tightly interacts with a synaptic vesicle docking site.

PICK1 is required for the control of synaptic transmission by the metabotropic glutamate receptor 7.

Both postsynaptic density and presynaptic active zone are structural matrix containing scaffolding proteins that are involved in the organization of the synapse. Little is known about the functional role of these proteins in the signaling of presynaptic receptors. Here we show that the interaction of the presynaptic metabotropic glutamate (mGlu) receptor subtype, mGlu7a, with the postsynaptic density-95 disc-large zona occludens 1 (PDZ) domain-containing protein, PICK1, is required for specific inhibition of P/Q-type Ca(2+) channels, in cultured cerebellar granule neurons. Furthermore, we show that activation of the presynaptic mGlu7a receptor inhibits synaptic transmission and this effect also requires the presence of PICK1. These results indicate that the scaffolding protein, PICK1, plays an essential role in the control of synaptic transmission by the mGlu7a receptor complex.

The synaptic vesicle protein synaptotagmin associates with calcium channels and is a putative Lambert-Eaton myasthenic syndrome antigen.

Immunoglobulin G fractions from patients with Lambert-Eaton myasthenic syndrome (LEMS), an autoimmune disease of neuromuscular transmission, immunoprecipitate 125I-labeled omega-conotoxin GVIA-labeled calcium channels solubilized from rat brain. A 58-kDa antigen was detected by probing Western blots of partially purified calcium channels with LEMS plasma and IgG and was shown to be the relevant antigen in omega-conotoxin receptor immunoprecipitation. Monoclonal antibody 1D12, produced by immunizing mice with synaptic membranes, has properties similar to these autoimmune IgGs in both immunoprecipitation and Western blotting assays. 1D12 antigen was purified by immunoaffinity chromatography and shown to bind LEMS IgG. The antigen was identified by screening a rat brain cDNA library with 1D12 and was found to have strong homology to the synaptic vesicle membrane protein synaptotagmin. Our results indicate therefore that these antibodies immunoprecipitate omega-conotoxin receptors by binding to synaptotagmin that is associated with calcium channels. We suggest that the interaction between synaptotagmin and the voltage-gated calcium channel plays a role in docking synaptic vesicles at the plasma membrane prior to rapid neurotransmitter release and that autoantibody binding to a synaptotagmin-calcium-channel complex may be involved in the etiology of LEMS.

Antigens associated with N- and L-type calcium channels in Lambert-Eaton myasthenic syndrome.

In Lambert-Eaton myasthenic syndrome neurotransmitter release is reduced by an autoimmune response directed against the calcium channel complex of the nerve terminal. Autoantibodies were detected by immunoprecipitation assays using solubilized receptors labeled with ligands selective for N-type (125I-omega conotoxin GVIA) and L-type ([3H]PN200-110) calcium channels. Sera with a high antibody titer (> 3 nM) against rat brain N-type channels contained autoantibodies that immunoprecipitated neuronal and muscle L-type channels. These IgG fractions stained a 55-kDa protein in immunoblots of purified skeletal muscle dihydropyridine receptor, suggesting that they contain autoantibodies against the beta subunit of the calcium channel. A distinct antibody population in the same fractions reacted with a nerve terminal 65-kDa protein that is unrelated to the beta subunit and displays properties similar to those of synaptotagmin.

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.

Mapping of calmodulin and Gbetagamma binding domains within the C-terminal region of the metabotropic glutamate receptor 7A.

Ca(2+)/calmodulin (Ca(2+)/CaM) and the betagamma subunits of heterotrimeric G-proteins (Gbetagamma) have recently been shown to interact in a mutually exclusive fashion with the intracellular C terminus of the presynaptic metabotropic glutamate receptor 7 (mGluR 7). Here, we further characterized the core CaM and Gbetagamma binding sequences. In contrast to a previous report, we find that the CaM binding motif localized in the N-terminal region of the cytoplasmic tail domain of mGluR 7 is conserved in the related group III mGluRs 4A and 8 and allows these receptors to also bind Ca(2+)/CaM. Mutational analysis of the Ca(2+)/CaM binding motif is consistent with group III receptors containing a conventional CaM binding site formed by an amphipathic alpha-helix. Substitutions adjacent to the core CaM target sequence selectively prevent Gbetagamma binding, suggesting that the CaM-dependent regulation of signal transduction involves determinants that overlap with but are different from those mediating Gbetagamma recruitment. In addition, we present evidence that Gbetagamma uses distinct nonoverlapping interfaces for interaction with the mGluR 7 C-terminal tail and the effector enzyme adenylyl cyclase II, respectively. Although Gbetagamma-mediated signaling is abolished in receptors lacking the core CaM binding sequence, alpha subunit activation, as assayed by agonist-dependent GTPgammaS binding, was not affected. This suggests that Ca(2+)/CaM may alter the mode of group III mGluR signaling from mono- (alpha) to bidirectional (alpha and betagamma) activation of downstream effector cascades.