A novel calmodulin-binding protein, belonging to the WD-repeat family, is localized in dendrites of a subset of CNS neurons.

A rat brain synaptosomal protein of 110,000 M(r) present in a fraction highly enriched in adenylyl cyclase activity was microsequenced (Castets, F., G. Baillat, S. Mirzoeva, K. Mabrouk, J. Garin, J. d'Alayer, and A. Monneron. 1994. Biochemistry. 33:5063-5069). Peptide sequences were used to clone a cDNA encoding a novel, 780-amino acid protein named striatin. Striatin is a member of the WD-repeat family (Neer, E.J., C.J. Schmidt, R. Nambudripad, and T.F. Smith. 1994. Nature (Lond.). 371:297-300), the first one known to bind calmodulin (CaM) in the presence of Ca++. Subcellular fractionation shows that striatin is a membrane-associated, Lubrol-soluble protein. As analyzed by Northern blots, in situ hybridization, and immunocytochemistry, striatin is localized in the central nervous system, where it is confined to a subset of neurons, many of which are associated with the motor system. In particular, striatin is conspicuous in the dorsal part of the striatum, as well as in motoneurons. Furthermore, striatin is essentially found in dendrites, but not in axons, and is most abundant in dendritic spines. We propose that striatin interacts, through its WD-repeat domain and in a CaM/Ca(++)-dependent manner, with one or several members of a surrounding cluster of molecules engaged in a Ca(++)-signaling pathway specific to excitatory synapses.

Na(v)1.7 and Na(v)1.3 are the only tetrodotoxin-sensitive sodium channels expressed by the adult guinea pig enteric nervous system.

The types of sodium channels that are expressed by neurons shape the rising phase of action potentials and influence patterns of action potential discharge. With regard to the enteric nervous system (ENS), there is uncertainty about which channels are expressed, and in particular it is unknown whether Na(v)1.7 is present. We designed specific probes for the guinea pig Na(v)1.7 alpha subunit as well as for the other tetrodotoxin (TTX)-sensitive alpha subunits (Na(v)1.1, Na(v)1.2, Na(v)1.3, and Na(v)1.6) in order to perform in situ hybridization (ISH) histochemistry on guinea pig myenteric ganglia. We established that only Na(v)1.7 mRNA and Na(v)1.3 mRNA are expressed in these ganglia. The ISH signal for Na(v)1.7 transcripts was found in seemingly all the myenteric neurons. The expression of the Na(v)1.3 alpha subunit was confirmed by immunohistochemistry in a large proportion (62%) of the myenteric neuron population. This population included enteric sensory neurons. Na(v)1.6 immunoreactivity, absent from myenteric neurons, was detected in glial cells only when a high anti-Na(v)1.6 antibody concentration was used. This suggests that the Na(v)1.6 alpha subunit and mRNA are present only at low levels, which is consistent with the fact that no Na(v)1.6 mRNA could be detected in the ENS by ISH. The fact that adult myenteric neurons are endowed with only two TTX-sensitive alpha subunits, namely, Na(v)1.3 and Na(v)1.7, emphasizes the singularity of the ENS. Both these subunits, known to have slow-inactivation kinetics, are well adapted for generating action potentials from slow excitatory postsynaptic potentials, a mode of synaptic transmission that applies to all ENS neuron types.

Molecular cloning and characterization of phocein, a protein found from the Golgi complex to dendritic spines.

Phocein is a widely expressed, highly conserved intracellular protein of 225 amino acids, the sequence of which has limited homology to the sigma subunits from clathrin adaptor complexes and contains an additional stretch bearing a putative SH3-binding domain. This sequence is evolutionarily very conserved (80% identity between Drosophila melanogaster and human). Phocein was discovered by a yeast two-hybrid screen using striatin as a bait. Striatin, SG2NA, and zinedin, the three mammalian members of the striatin family, are multimodular, WD-repeat, and calmodulin-binding proteins. The interaction of phocein with striatin, SG2NA, and zinedin was validated in vitro by coimmunoprecipitation and pull-down experiments. Fractionation of brain and HeLa cells showed that phocein is associated with membranes, as well as present in the cytosol where it behaves as a protein complex. The molecular interaction between SG2NA and phocein was confirmed by their in vivo colocalization, as observed in HeLa cells where antibodies directed against either phocein or SG2NA immunostained the Golgi complex. A 2-min brefeldin A treatment of HeLa cells induced the redistribution of both proteins. Immunocytochemical studies of adult rat brain sections showed that phocein reactivity, present in many types of neurons, is strictly somato-dendritic and extends down to spines, just as do striatin and SG2NA.

Striatin, a calmodulin-dependent scaffolding protein, directly binds caveolin-1.

Caveolins are scaffolding proteins able to collect on caveolae a large number of signalling proteins bearing a caveolin-binding motif. The proteins of the striatin family, striatin, SG2NA, and zinedin, are composed of several conserved, collinearly aligned, protein-protein association domains, among which a putative caveolin-binding domain [Castets et al. (2000) J. Biol. Chem. 275, 19970-19977]. They are associated in part with membranes. These proteins are mainly expressed within neurons and thought to act both as scaffolds and as Ca(2+)-dependent signalling proteins [Bartoli et al. (1999) J. Neurobiol. 40, 234-243]. Here, we show that (1) rat brain striatin, SG2NA and zinedin co-immunoprecipitate with caveolin-1; (2) all are pulled down by glutathione-S-transferase (GST)-caveolin-1; (3) a fragment of recombinant striatin containing the putative caveolin-binding domain binds GST-caveolin-1. Hence, it is likely that the proteins of the striatin family are addressed to membrane microdomains by their binding to caveolin, in accordance with their putative role in membrane trafficking [Baillat et al. (2001) Mol. Biol. Cell 12, 663-673].

Distribution of striatin, a newly identified calmodulin-binding protein in the rat brain: an in situ hybridization and immunocytochemical study.

Striatin, a 110-kDa protein, is the first member of the tryptophane-aspartate repeat protein family known to bind calmodulin in the presence of Ca2+. We examined the distribution of striatin and its mRNA in the rat central nervous system (CNS) by using immunocytochemistry and in situ hybridization, respectively. Striatin immunostaining and mRNA labeling patterns are generally concordant. Regions showing the most intense staining are the dorsal striatum, nucleus accumbens (anterior and shell parts), olfactory tubercle, red nucleus, subthalamic nucleus, cranial nerve motor nuclei, and layer IX of the spinal cord (motoneurons). Low levels of both striatin and its mRNA are detected in the cerebral cortex, thalamus, septum, amygdala, hippocampus, midbrain and cerebellum. Striatin-immunoreactive neuronal processes are found predominantly in the structures containing striatin-positive neurons, suggesting that these labeled processes represent dendritic arborization rather than axonal processes. Except for the medial forebrain bundle, all axonal fiber tracts examined are devoid of striatin immunolabeling. These data show that the somatodendritic localization of striatin, previously described in the striatum, may be a main feature of the subcellular distribution of this protein throughout the CNS. Although widely distributed in neurons throughout the rat CNS, striatin is expressed prominently in the structures belonging to the motor system, suggesting that this protein may play a preponderant role in motor control.

Relationships between striatin-containing neurons and cortical or thalamic afferent fibres in the rat striatum. An ultrastructural study by dual labelling.

Striatin, a recently isolated rat brain calmodulin-binding protein belonging to the WD-repeat protein family, is thought to be part of a calcium signal transduction pathway presumably specific to excitatory synapses, at least in the striatum. This study was aimed to specify the cellular and subcellular localization of striatin, and to determine the possible synaptic relationships between the two main excitatory afferent pathways, arising from the cerebral cortex and the thalamus, and the striatin-containing elements, in the rat striatum. Anterograde tract-tracing by means of biotinylated dextran amine injection in the frontoparietal cerebral cortex or the parafascicular nucleus of the thalamus was combined with immunogold detection of striatin. Striatin-immunoreactivity was confined to the neuronal somatodendritic compartment, including spines. Whereas 90-95% of the striatal neurons were striatin-positive, only about 50% of the sections of dendritic spines engaged in asymmetrical synaptic contacts exhibited striatin labelling. Among the sections of striatin-immunopositive dendritic spines, the number of immunogold particles ranged from one to more than seven, indicating an heterogeneity of the spine labelling. Moreover, within each class of spines presenting at least two silver-gold particles, the distribution of the particles varied from a clear-cut alignment under the postsynaptic densities (24-33% of spines) to a location distant from the synaptic area. In the cell bodies and dendrites, striatin labelling was usually not associated with the cytoplasmic membrane nor with the postsynaptic densities. In the striatum ipsilateral to the tracer injections, only 34.8% of the synaptic contacts formed by corticostriatal afferents involved striatin-positive elements (slightly labelled dendritic spines), whereas 56.7% of the synaptic contacts formed by thalamostriatal boutons were made on striatin-positive targets (mostly dendrites). In both cases, striatin labelling was usually not associated with the postsynaptic density. Most of the immunoreactive dendritic spines were in contact with unidentified afferents. These data reveal that striatin is expressed in the vast majority of the cell bodies of striatal spiny neurons, but is heterogeneously distributed among the dendritic spines of those neurons. Data also indicate a preferential relationship between striatin-containing structures and afferents from the parafascicular thalamic nucleus with respect to the frontoparietal cerebral cortex. But, at the dendritic spine level, striatin may be involved in signal transduction mechanisms involving as yet unidentified excitatory afferents to striatal neurons.

Transcriptional regulation of the human S100 beta gene.

S100 beta is a calcium-binding protein produced and secreted by glial cells in the central and peripheral nervous systems. S100 beta promotes neuronal differentiation and survival but may be detrimental to cells if overexpressed. The selective overproduction of S100 beta has been implicated in the progression of the neuropathological changes in Alzheimer's disease. In addition, at high concentrations, S100 beta stimulates toxic intracellular pathways in cultured cells. To begin to define the regulation of S100 beta expression, we characterized the human S100 beta promoter and mapped its upstream regulatory elements by using a luciferase reporter system. The functional S100 beta promoter was localized to a region -168/ +697 containing 168 bp upstream of the transcription initiation site of the gene. This minimal promoter was active in a variety of cell types, including those of glial, neuronal, and non-neural origin. The human S100 beta promoter activity is regulated by both positive and negative regulatory elements located upstream in the 5' flanking DNA regions. The regions -788/ -391 and -1012/ -788 contain strong positive, cell type-specific regulatory elements. Negative regulatory elements were mapped to the more distal -4437/ -1012 and -1012/ -788 regions of the gene. The -4437/ -1012 negative element suppressed promoter activity in all cell types examined, except C6 glioma cells. These data demonstrate that the expression of the human S100 beta gene is under complex transcriptional regulation that allows for precise control of the S100 beta level in the nervous system.

CK2 accumulation at the axon initial segment depends on sodium channel Nav1.

Accumulation of voltage-gated sodium channel Nav1 at the axon initial segment (AIS), results from a direct interaction with ankyrin G. This interaction is regulated in vitro by the protein kinase CK2, which is also highly enriched at the AIS. Here, using phosphospecific antibodies and inhibition/depletion approaches, we showed that Nav1 channels are phosphorylated in vivo in their ankyrin-binding motif. Moreover, we observed that CK2 accumulation at the AIS depends on expression of Nav1 channels, with which CK2 forms tight complexes. Thus, the CK2-Nav1 interaction is likely to initiate an important regulatory mechanism to finely control Nav1 phosphorylation and, consequently, neuronal excitability.

Zinedin, SG2NA, and striatin are calmodulin-binding, WD repeat proteins principally expressed in the brain.

Striatin is an intracellular protein characterized by four protein-protein interaction domains, a caveolin-binding motif, a coiled-coil structure, a calmodulin-binding domain, and a WD repeat domain, suggesting that it is a signaling or a scaffold protein. Down-regulation of striatin, which is expressed in a few subsets of neurons, impairs the growth of dendrites as well as rat locomotor activity (Bartoli, M., Ternaux, J. P., Forni, C., Portalier, P., Salin, P., Amalric, M., and Monneron, A. (1999) J. Neurobiol. 40, 234-243). Zinedin, a "novel" protein described here, and SG2NA share with striatin identical protein-protein interaction domains and the same overall domain structure. A phylogenetic analysis supports the hypothesis that they constitute a multigenic family deriving from an ancestral gene. DNA probes and antibodies raised against specific domains of each protein showed that zinedin is mainly expressed in the central nervous system, whereas SG2NA, of more widespread occurrence, is mainly expressed in the brain and muscle. All three proteins are both cytosolic and membrane-bound. All three bind calmodulin in the presence of Ca(2+). In rat brain, SG2NA and striatin are generally not found in the same neurons. Both localize to the soma and dendrites, suggesting that they share a similar type of addressing and closely related functions.

S100 beta stimulates inducible nitric oxide synthase activity and mRNA levels in rat cortical astrocytes.

The glia-derived, neurotrophic protein S100 beta has been implicated in development and maintenance of the nervous system. However, S100 beta has also been postulated to play a role in mechanisms of neuropathology, because of its specific localization and selective overexpression in Alzheimer's disease. To begin to address the question of whether S100 beta can induce potentially toxic signaling pathways, we examined the effects of the protein on nitric oxide synthase (NOS) activity in cultures of rat cortical astrocytes. S100 beta treatment of astrocytes induced a time- and dose-dependent increase in accumulation of the NO metabolite, nitrite, in the conditioned medium. The S100 beta- stimulated nitrite production was blocked by cycloheximide and by the NOS inhibitor N-nitro-L-arginine methylester, but not by the inactive D-isomer of the inhibitor. Direct measurement of NOS enzymatic activity in cell extracts and analysis of NOS mRNA levels showed that the NOS activated by S100 beta addition is the calcium-independent, inducible isoform. Furthermore, the specificity of the effects of S100 beta on activation of NOS was demonstrated by the inability of S100 alpha and calmodulin to induce an increase in nitrite levels. Our data indicate that S100 beta can induce a potent activation of inducible NOS in astrocytes, an observation that might have relevance to the role of S100 beta in neuropathology.

A monoclonal antibody directed against the catalytic site of Bacillus anthracis adenylyl cyclase identifies a novel mammalian brain catalytic subunit.

A brain adenylyl cyclase was shown to contain an epitope closely related to that specified by a conserved sequence containing a nucleotide-binding consensus sequence GXXXXGKS and located in the catalytic sites of bacterial, calmodulin-dependent adenylyl cyclases [Goyard, S., Orlando, C., Sabatier, J.-M., Labruyere, E., d'Alayer, J., Fontan, G., van Rietschoten, J., Mock, M., Danchin, A., Ullmann, A., & Monneron, A. (1989) Biochemistry 28, 1964-1967]. A monoclonal antibody, mab 164, produced against a peptide corresponding to this conserved sequence specifically inhibited the Bordetella pertussis adenylyl cyclase. It also specifically inhibited rat and rabbit brain synaptosomal adenylyl cyclases. The extent of inhibition depended upon the type of enzyme purification, reaching 90% for the calmodulin-sensitive species of enzyme and 20-35% for the forskolin-agarose-retained species. The extent of inhibition in a given fraction also depended upon the effector present. mab 164 reacted on Western blots of forskolin-agarose-retained fractions with a 175-kDa component and did not recognize the Gs alpha stimulatory subunit. Consequently, the 175-kDa protein was considered as a good candidate for an adenylyl cyclase catalyst. The adenylyl cyclase activity contained in forskolin-agarose-retained fractions was further purified on calmodulin-Sepharose. On Western blots of such fractions, mab 164 reacted with a 140-kDa protein, a component that appeared to derive from the 175-kDa protein enriched in the previous step. The kcat of this 140-kDa presumptive adenylyl cyclase was estimated to be of the order of 600 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

A brain synaptosomal adenylyl cyclase of high specific activity is photolabeled with azido-ATP.

Partially purified adenylyl cyclase preparations of high specific activity (60 +/- 10 mumol cAMP/(mg.min)) were obtained from rat brain synaptosomes (Orlando, C., d'Alayer, J., Baillat, G., Castets, F., Jeannequin, O., Mazié, J. C., & Monneron, A. (1992) Biochemistry 31, 3215-3222). Adenylyl cyclase activity was stimulated 4-fold by Ca2+/calmodulin and 2-fold by forskolin or by Mn2+. These preparations contained two major proteins of 140 and 110 kDa. The 140-kDa protein was identified as the neural cell adhesion molecule. The 110-kDa protein was specifically recognized by affinity-purified antibodies directed against a peptide corresponding to sequence 976-1013 of adenylyl cyclase type I. It was photolabeled by [alpha-32P]8- and 2-N3ATP in a light-dependent manner and was by far the most heavily labeled component of FC fractions. Saturation was obtained with 30 microM [32P]8-N3ATP. Photoinsertion of N3ATP into the protein was largely prevented by ATP or adenylyl imidodiphosphate but not by ADP, AMP, or adenosine. A modest incorporation of N3cAMP and photoinsertion of [alpha-32P]N3GTP into the 110-kDa protein were observed. Although some of the properties of the synaptosomal 110-kDa protein described here would match those expected from adenylyl cyclase type I, it appears that its specific activity is on the order of 1 mmol cAMP/(mg.min), about 200-fold that measured for brain adenylyl cyclases type I.

Cloning of human striatin cDNA (STRN), gene mapping to 2p22-p21, and preferential expression in brain.

Rat striatin, a recently discovered calmodulin-binding protein belonging to the WD repeat family, is expressed in neurons, mostly in the striatum and motor and olfactory systems. Striatin is localized in the somato-dendritic compartment of neurons, mainly in the spines. It may play a role in dendritic Ca2+ signaling. Here we report the cloning and sequencing of human striatin cDNA (HGMW-approved symbol STRN), the localization of the gene to chromosome 2p22-p21, and its preferential expression in brain. The human cDNA sequence is predicted to encode a 780-amino-acid protein possessing eight WD repeats. Striatin is highly conserved between rat and human with 96% identity and 98% similarity at the amino acid level. Since the Caenorabditis elegans genome also contains a closely related striatin coding sequence, the function of striatin is likely to be well conserved.