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PubMed1994

Molecular cloning and characterization of retinal photoreceptor guanylyl cyclase-activating protein.

Guanylyl cyclase-activating protein (GCAP) is thought to mediate Ca(2+)-sensitive regulation of guanylyl cyclase (GC), a key event in recovery of the dark state of rod photoreceptors following light exposure. Here, we characterize GCAP from several vertebrate species by molecular cloning and provide evidence that GCAP contains a heterogeneously acylated N-terminal region that interacts with GC. Vertebrate GCAPs consist of 201-205 amino acids, and sequence analysis indicates the presence fo three EF hand Ca(2+)-binding motifs. These results establish that GCAP is a novel photoreceptor-specific member of a large family of Ca(2+)-binding proteins and suggest that it participates in the Ca(2+)-binding proteins and suggest that it participates in the Ca(2+)-sensitive activation of GC.

Amino Acid Oxidoreductases↗
PubMed2004

MGluRs regulate the expression of neuronal calcium sensor proteins NCS-1 and VILIP-1 and the immediate early gene arg3.1/arc in the hippocampus in vivo.

The metabotropic glutamate receptor (mGluR) agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) is involved in several forms of hippocampal synaptic plasticity. DHPG application can induce slow-onset potentiation, a form of long-term potentiation (LTP), in the dentate gyrus and in the CA1 region in vivo. The induction of LTP correlates with increased expression levels of neuronal calcium sensor (NCS), considered as key elements for plasticity. In this study we investigated mGluR- and time-dependent changes in the expression of two different NCS proteins. Following DHPG application in vivo NCS-1 and VILIP-1 expression increased, with significant levels reached after 8 and 24h. The effect was attenuated by treatment with the group I mGluR specific antagonist S-4-carboxyphenylglycine. The immediate early gene (IEG) arg3.1/arc showed highest expression levels 2h after DHPG-treatment. Therefore, mGluRs at concentrations which induce synaptic plasticity regulate the expression of IEGs and NCS proteins in different time frames and thus contribute to late phases of synaptic plasticity.

Animals↗
PubMed2004

Cellular and subcellular localization of the inhibitory glycine receptor in hippocampal neurons.

Inhibitory glycine receptors are most abundant in spinal cord and brainstem, and glycinergic synapses have a well-established role in the regulation of locomotor behavior. Little is known about the function of glycine receptors in cortex and hippocampus, where GABA plays a dominant role in synaptic inhibition. Therefore, we have investigated tissue and cellular expression of glycine receptor alpha-subunits. Western blot and immunohistochemical analyses reveal the presence of glycine receptors in hippocampal tissue. Immunocytochemical experiments in hippocampal cultures show prominent cellular expression of glycine receptors in pyramidal neurons and GAD-positive interneurons similar to the calcium-binding protein VILIP-1 with widespread hippocampal distribution. On the subcellular level we found co-staining of GlyR and the presynaptic marker synapsin I. Furthermore, co-staining with GAD at synaptic terminals indicated partial co-localization of GABA- and glycine receptors.

Animals↗
PubMed2002

Functional analysis of calcium-binding EF-hand motifs of visinin-like protein-1.

Visinin-like protein-1 (VILIP-1), a myristoylated calcium sensor protein with three EF-hand motifs, modulates adenylyl cyclase activity. It translocates to membranes when a postulated "calcium-myristoyl switch" is triggered by calcium-binding to expose its sequestered myristoyl moiety. We investigated the contributions of the EF-hand motifs to the translocation of VILIP-1 to membranes and to the modulation of adenylyl cyclase activity. Mutation of residues crucial for binding calcium within each one of the EF-hand motifs indicated that they all contributed to binding calcium. Simultaneous mutations of all of the three EF-hand motifs completely abolished VILIP-1's ability to bind calcium, attenuated but did not eliminate its modulation of adenylyl cyclase activity, and abolished its calcium-dependence for association with cellular membranes. These results show that the calcium-binding EF-hand motifs of VILIP-1 do not have an essential role in modulating adenylyl cyclase activity but instead have a structural role in activating the "calcium-myristoyl switch" of VILIP-1.

Adenylyl Cyclases↗
PubMed2003

Group I mGlu receptors regulate the expression of the neuronal calcium sensor protein VILIP-1 in vitro and in vivo: implications for mGlu receptor-dependent hippocampal plasticity?

Metabotropic glutamate (mGlu) receptors are involved in several forms of synaptic plasticity in the rat hippocampus. Agonists which activate group I mGlu receptors induce slow-onset potentiation without prior tetanization in the hippocampal area CA1. Activation of group I mGlu receptors induces protein synthesis which may contribute to mGlu receptor-dependent forms of long-term plasticity. Calcium-binding proteins are widely considered to comprise key elements for synaptic plasticity. Therefore, we investigated whether the calcium sensor protein VILIP-1 is associated with group I mGlu receptor-mediated plasticity in the dentate gyrus (DG) in vivo.Application of either the group I and II mGlu agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (ACPD) or the selective group I agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) resulted in slow-onset potentiation in the DG of adult rats. In hippocampal cell cultures both agonists elicited an enhanced expression of VILIP-1. In situ hybridization revealed strong hippocampal expression of VILIP-1 and intracerebral application of DHPG to adult rats significantly enhanced hippocampal VILIP-1 expression. The DHPG effects in both, hippocampal cultures and in vivo, were prevented by the group I mGlu receptor antagonist 4-Carboxyphenylglycine (4CPG). Calcium sensor proteins thus appear to be regulated by mGlu receptors in an activity-dependent manner. A specific role for group I mGlu receptors is evident. Furthermore, the sensor proteins may function as molecular switches for the long-term regulation of synaptic plasticity.

Animals↗
PubMed2001

Immunochemical assessment of neural visinin-like calcium-binding protein 3 expression in rat brain.

Expression of neural visinin-like calcium-binding protein 3 (NVP3) was assessed by immunoblot and immunohistochemical analyses in rat brain. NVP3 was markedly expressed in the cerebellum, at a concentration of 9.5microM. On SDS-PAGE, native NVP3 migrated at 23kDa, identical to the recombinant myristoyl-form, but somewhat faster than the non-myristoyl-form. Both forms bound 3 moles of calcium. The myristoyl-form exhibited a cooperativity in binding calcium and calcium-dependent membrane-binding, but the non-myristoyl-form did not. At 3 months, NVP3 was primarily localized in the Purkinje cells, with intense staining in the cell bodies, dendrites and axons. The cerebellar granule cells and basal nuclear neurons were faintly stained. During development of the cerebellum, NVP3-positive Purkinje cells first appeared on post-natal day 14 (P14). The staining intensity then increased and plateaued on P28. Labeling showed a tendency to accumulate in the dendrites and nerve terminals in a fine granular pattern. During aging process, NVP3 levels decreased by 43% at 12 months and 68% at 24 months, while the levels of NVP1, synaptophysin and drebrin were preferentially preserved. These results suggest that NVP3 is involved in dendritic arborization and postsynaptic function in cerebellar Purkinje cells and that presynaptic nerve terminals are another functional site of the protein.

Aging↗
PubMed2003

Brain region-specific changes in the expression of calcium sensor proteins after repeated applications of ketamine to rats.

We investigated the cellular distribution of three calcium sensor proteins, visinin-like protein-1 (VILIP-1), VILIP-3, and hippocalcin, in different rat brain areas after repeated administration of the non-competitive N-methyl-D-aspartate receptor antagonist ketamine. In comparison to controls we observed an increase in the density of VILIP-1 immunoreactive (IR) hippocampal interneurons and presubicular nerve cells in ketamine treated rats, whereas the density of VILIP-1 expressing cells was decreased in the Nuc. accumbens of these rats. No alterations were seen in the distribution patterns of VILIP-3. The density of hippocalcin-expressing neurons was increased in the cingulate cortex of drug-treated rats. Our experiments show that repeated injections of subanesthetic doses of ketamine induce subtle changes in the cellular distribution of calcium sensor proteins which in part resemble those recently described in postmortem brains of human schizophrenics [Bernstein, H.-G., Braunewell, K.-H., Spilker, C., Danos, P., Baumann, B., Funke, S., Diekmann, S., Gundelfinger, E.D. and Bogerts, B., NeuroReport, 13 (2002) 393-396].

Animals↗
PubMed1996

Distribution of visinin-like protein (VILIP) immunoreactivity in the hippocampus of the Mongolian gerbil (Meriones unguiculatus).

Visinin-like protein (VILIP) is a neuronal EF-hand Ca(2+)-binding protein. In the chick brain, it is widely expressed, e.g. in neurons of the visual pathway and the cerebellum. In the cerebellum, a presynaptic localization of VILIP in glutamatergic parallel- and climbing-fiber terminals has been observed. Here, we describe the distribution of immunoreactivity (IR) detected by antibodies against chick VILIP in the gerbil hippocampus at the light and electron microscopic level. VILIP antibodies stain neurons in the whole hippocampal formation including pyramidal cells in the CA1 and CA3 region of the Ammon's horn and granule cells of the dentate gyrus. In CA3 neurons, VILIP-IR is localized in dendrites and dendritic spines.

Animals↗
PubMed1997

Calcium- and myristoyl-dependent subcellular localization of the neuronal calcium-binding protein VILIP in transfected PC12 cells.

Wild-type neuronal calcium-binding protein VILIP (visinin-like protein), and a myristoylation mutant of VILIP which lacks the consensus sequence for N-terminal myristoylation, have been stably transfected in PC12 cells. Immunocytochemical studies of VILIP-transfected PC12 cells have revealed the wild-type VILIP is strongly concentrated at the cell membrane, particularly at cell-cell contact sites, but is also distributed throughout the cytosol at moderate levels. In contrast, myristoylation-mutant VILIP shows a more even distribution, with significantly less association at cell-cell contact sites. Western blot analysis of subcellular fractions has shown that wild-type VILIP associates in a calcium-dependent manner with membrane fractions, whereas the myristoylation mutant only weakly associates with this fraction. Therefore, a calcium-myristoyl switch seems to be a major, but not sole determinant for the association of VILIP with membranes in living cells.

Animals↗
PubMed1997

Low level expression of calcium-sensor protein VILIP induces cAMP-dependent differentiation in rat C6 glioma cells.

Wild-type visinin-like-protein (VILIP) and a myristoylation-deficient VILIP mutant, when stably expressed at low levels in C6 cells, enhances or reduces the basal cAMP-level, respectively. The morphology of wild-type VILIP-transfected cells resembles that of differentiated astrocytes, whereas the myristoylation mutant shows a phenotype similar to parental cells, but with reduced cell growth. In both parental and myristoylation mutant cells a differentiated phenotype similar to that produced by wild-type VILIP-transfected cells is inducible with 8-bromo-cAMP. The changed morphology parallels an increase in the expression of the astrocytic differentiation marker glial fibrillary acidic protein (GFAP) in wild-type VILIP-transfected and cAMP-differentiated cells, but a decrease of GFAP in myristoylation mutant cells. These results suggest that depending on myristoylation, low level ectopic expression of VILIP affects basal cAMP homeostasis differentially, thereby influencing differentiation of C6 model cells.

1-Methyl-3-isobutylxanthine↗
PubMed2000

The neuronal EF-hand calcium-binding protein visinin-like protein-3 is expressed in cerebellar Purkinje cells and shows a calcium-dependent membrane association.

Visinin-like protein-3 is a member of the family of intracellular neuronal calcium sensors belonging to the superfamily of EF-hand proteins. Members of this family are involved in the calcium-dependent regulation of signal transduction cascades. To gain insights into the characteristics of visinin-like protein-3, we have generated specific antibodies against visinin-like protein-3 and determined the developmental and tissue distribution of the protein and its exact cellular and subcellular localization. Expression of visinin-like protein-3 protein appeared late in development mainly in the cerebellum. It is strongly expressed in cerebellar Purkinje cells. The protein expression results were further confirmed by in situ hybridization and compared with hippocalcin messenger RNA localization. Native cerebellar visinin-like protein-3 was shown to bind calcium and to associate in a calcium-dependent manner with membrane fractions during subcellular fractionation. Recombinant wild-type visinin-like protein-3 was shown to be N-terminally myristoylated in transfected cells. The membrane association was strongly reduced for the non-myristoylated mutant of visinin-like protein-3 in transfected cells. These results suggest that visinin-like protein-3, which is mainly expressed in Purkinje cells in vivo, shows a calcium-dependent association with cell membranes which is mediated by a calcium-myristoyl switch.

Aging↗
PubMed2003

Calcium-myristoyl switch, subcellular localization, and calcium-dependent translocation of the neuronal calcium sensor protein VILIP-3, and comparison with VILIP-1 in hippocampal neurons.

Neuronal calcium sensor (NCS) proteins including the subfamily of visinin-like-proteins (VILIPs) are involved in regulation of various signaling cascades. One molecular regulation mechanism is the calcium-myristoyl switch. VILIPs show a calcium-dependent membrane association in brain homogenates; however, differences in calcium-induced conformation changes and degree of membrane association are reported. Little is known about differences in the calcium-myristoyl switch in living cells leading to localization of VILIPs to distinct subcellular compartments. Therefore, we studied the calcium-dependent localization of green fluorescent protein (GFP)-tagged VILIP-3 in living cell lines and hippocampal neurons and compared it with that of GFP-VILIP-1. Interestingly, the observed fast and reversible calcium-myristoyl switch of VILIP-3-GFP and VILIP-1-GFP differed, e.g., in calcium-dependent translocation to Golgi membranes. Similarily, the calcium-dependent localization of endogenously expressed VILIP-3 and -1 in dendrites differed. Thus, VILIPs co-expressed in the same neuron show clear differences in calcium-dependent localization which may allow neurons a highly selective response to various calcium stimuli.

Animals↗
PubMed2002

Evidence for different functional properties of the neuronal calcium sensor proteins VILIP-1 and VILIP-3: from subcellular localization to cellular function.

The visinin-like-proteins VILIP-1 and -3 are EF-hand calcium-binding proteins and belong to the family of neuronal calcium sensor (NCS) proteins. Members of this family are involved in the calcium-dependent regulation of signal transduction cascades mainly in the nervous system. VILIP-1 and VILIP-3 are expressed in different populations of neuronal cells. To gain insights into the different functional characteristics of VILIP-1 and -3, we have compared the localization of the proteins in intact cells and the calcium-dependent membrane association in subcellular fractions. Furthermore, we have investigated the different functional properties of the two proteins in activating cGMP signal pathways and have defined different sets of protein interaction partners. Our data indicate that VILIP-3, which is mainly expressed in Purkinje cells, and VILIP-1, which is expressed in granule cells in the cerebellum, show a different calcium-dependent subcellular localization, may activate different cellular signaling pathways, and thus have signaling functions which seem to be cell-type specific.

Animals↗
PubMed1999

Regional and cellular distribution of neural visinin-like protein immunoreactivities (VILIP-1 and VILIP-3) in human brain.

Neural visinin-like proteins (VILIPs) are members of the neuronal subfamily of intracellular EF-hand calcium sensor proteins termed the NCS family, which are thought to play important roles in cellular signal transduction. While numerous studies suggest a wide but uneven distribution of these proteins in rat and chicken brain, their location in, and possible significance for, the human brain, remains to be established. We used specific polyclonal antisera to map the human brain for VILIP-1 and VILIP-3 immunoreactivities. VILIP-1 was detected in cortical pyramidal cells and interneurons, septal, subthalamic and hippocampal neurons (subfields CA1 and CA4 pyramidal cells and especially hilar interneurons) as well as in cerebellar Golgi, basket, granule, stellate and dentate nucleus neurons. Purkinje cells were free of immunoreaction. VILIP-3 was more restricted in its distribution. It was identified in cerebellar Purkinje cells and a subpopulation of granule neurons. Further, neurons belonging to different nuclei of the brain stem and multiple subcortical nerve cells stained for visinin-like protein 3. A weak immunoreaction appeared in cortical and hippocampal neurons. Intracellularly the immunoreactivity appeared in the perikarya, dendrites and some axons. Sometimes, immunostaining was found in the neuropil. Glia did not express visinin-like proteins. Our findings support, from a neuroanatomical viewpoint, the idea that these calcium sensor proteins may be of relevance for neuronal signalling in the human CNS.

Animals↗
PubMed2001

Increased number of nitric oxide synthase immunoreactive Purkinje cells and dentate nucleus neurons in schizophrenia.

There is growing interest in the cerebellum as a site of neuropathological changes in schizophrenia. Reports showing that schizophrenics have higher nitric oxide synthase (NOS) activity and MAPKinase levels in the vermis, point to possible aberrations in the cerebellar signal transduction of schizophrenics. It has been speculated that Ca(2+)-dependent extracellular to intracellular signal transduction may be disrupted in the cerebellum of schizophrenics. We decided to test this hypothesis by studying the nitrergic system and markers of the Ca(2+)-triggered signal cascade in the cerebellum of schizophrenics, depressives and controls. The cellular distribution of two calcium sensor proteins (VILIP-1 and VILIP-3) and of neuronal NOS immunoreactivity was studied morphometrically in the flocculonodulus, the inferior vermis and the dentate nucleus of 9 schizophrenics, 7 depressive patients and 9 matched controls. In comparison to controls and depressed patients there were fewer Nissl-stained neurons in the dentate nucleus of schizophrenics. The number of NOS-expressing Purkinje neurons was however strongly increased. In the flocculonodulus and the vermis no differences between the groups were found with regard to the density of Nissl-stained Purkinje cells. The number of NOS-expressing Purkinje neurons was increased in schizophrenics, however. No differences between schizophrenics, depressives and controls were found in the number of VILIP-1 immunoreactive dentate nucleus neurons and VILIP-3 immunoreactive vermal and flocculonodular Purkinje cells. Our data provide further histochemical evidence in favor of structural abnormalities in discrete cerebellar regions of schizophrenics. They confirm and extend earlier reports of increased cerebellar NOS immunoreactivity in schizophrenia and point to possible neurodevelopmental disturbances. Our failure to show an altered expression of two calcium sensor proteins possibly points to a less important role of calcium signaling in cerebellar pathology of the disease.

Adult↗
PubMed1999

Age-related changes in expression of hippocalcin and NVP2 in rat brain.

Expression of hippocalcin and neural visinin-like calcium-binding protein 2 (NVP2) in aging rat brain was investigated by immunoblot and immunohistochemical analyses. In 3-month old rats, hippocalcin and NVP2 were present at high concentrations in hippocampal and cerebral pyramidal cells and dentate granule cells, with hippocalcin protein levels being five to ten times higher than NVP2 levels. Hippocalcin levels in hippocampus and cerebral cortex decreased by approximately 20% at 24 months. While the number of hippocalcin-positive cells in CA3, dentate gyrus and cerebral cortex were preserved, staining intensity decreased. In contrast, the number and staining intensity of hippocalcin-positive cells in CA1 were maintained. NVP2 levels in hippocampus and cerebral cortex decreased by approximately 30% at 24 months. In cerebral cortex, the number and intensity of NVP2-positive cells decreased. In CA1 through CA3 and in dentate gyrus, NVP2-positive cell numbers were preserved, but staining intensity decreased. In summary, the loss of hippocalcin and NVP2 in aging rat brain may be associated with age-related impairment of postsynaptic functions.

Aging↗
PubMed2005

Visinin-like protein-1 is a potent inhibitor of cell adhesion and migration in squamous carcinoma cells.

Tumor cell invasion is a highly integrated and complex process comprising several biologically distinct functions such as cell adhesion, motility, proteolysis, etc. Visinin-like protein-1 (VILIP-1), a member of the neuronal EF-hand calcium-sensor protein family, plays a role in regulating tumor cell invasiveness of mouse squamous cell carcinoma (SCC). VILIP-1 enhances cyclic adenosine monophosphate levels through PKA induction. However, the mechanism by which VILIP-1 reduces cell invasiveness is not well understood. In this study, we show that VILIP-1 decreased cell adhesion and migration/invasiveness of highly invasive mouse SCC cells. Forced expression of VILIP-1 reduced cell adhesion to fibronectin in parallel to downregulating alphav and alpha5 integrin subunit levels. VILIP-1 overexpression also led to decreased migration ability. Conversely, short hairpin RNA-mediated VILIP-1 knock-down of SCC cells' characterized by little or no invasiveness, correlated with increased adhesion to fibronectin and enhanced expression of alphav and alpha5 integrin subunits together with increased cell migration. Function-blocking assays with inhibitory anti-alpha5 and anti-alphav integrin antibodies showed that both subunits contributed to cell adhesion, migration, and invasiveness of highly invasive SCC cell lines. These results point to a critical role of VILIP-1 in regulating cell adhesion and migration by downregulation of fibronectin receptor expression. Decreased or absent VILIP-1 expression in SCC cell subpopulations may lead to a more advanced malignant phenotype characterized by changes in adhesive ability and increased cell motility, suggestive of a tumor suppressor function.

Animals↗
PubMed1997

The neuronal calcium-sensor protein VILIP modulates cyclic AMP accumulation in stably transfected C6 glioma cells: amino-terminal myristoylation determines functional activity.

VILIP (visinin-like protein) is a member of the neuronal subfamily of EF-hand calcium sensor proteins. Members of this family are involved in the calcium-dependent regulation of the desensitization of signal cascades in retinal photoreceptors. To gain insight into the function of VILIP in cell signaling, we have transfected wild-type VILIP and mutant VILIP lacking the myristoylation consensus sequence into C6 glioma cells. Expression of wild-type VILIP did not significantly influence the desensitization of beta-adrenergic receptors, which are coupled to adenylyl cyclase in C6 cells. However, VILIP expression increased the beta-adrenergic receptor-stimulated cyclic AMP (cAMP) level in these cells severalfold. The stimulatory effect was also observed after direct stimulation of the adenylyl cyclase with forskolin, indicating that VILIP acts downstream of receptor and G protein in the beta-adrenergic signaling pathway in C6 cells. In contrast, the nonmyristoylated mutant of VILIP reduced cellular cAMP levels in C6 cells. Myristoylated wild-type VILIP was associated in a calcium-dependent manner with membrane fractions during subcellular fractionation, presumably owing to a calcium-myristoyl switch. In contrast, association of nonmyristoylated mutant VILIP with membranes was strongly reduced. Thus, myristoylation and most likely the calcium-dependent membrane association of VILIP are important prerequisites for the activating effect of wild-type VILIP on cAMP accumulation in C6 cells. These results suggest that VILIP acts as a calcium sensor molecule that modulates cell signaling cascades, possibly by direct or indirect regulation of adenylyl cyclase activity.

Animals↗