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PubMed1995

Ca(2+)-dependent interaction of recoverin with rhodopsin kinase.

Recoverin (Rv) is a myristoylated Ca(2+)-binding protein present primarily in bovine photoreceptors. It represents a newly identified family of neuronal specific Ca(2+)-binding proteins that includes neurocalcin, hippocalcin, and guanylyl cyclase-activating protein. To investigate the function of Rv in photoreceptors, we identified proteins that bind immobilized Rv in a Ca(2+)-dependent manner. Rhodopsin kinase (RK), interphotoreceptor retinoid-binding protein, and tubulin interact with Rv in the presence of Ca2+. The importance of the Rv/RK interaction was further characterized. RK, purified using immobilized Rv as an affinity matrix, catalyzed the light-dependent and Ca(2+)-independent incorporation of phosphates into rhodopsin when reconstituted with urea-stripped rod outer segment membranes. When only a small fraction (0.04%) of rhodopsin was photolyzed, as many as 700 phosphates were incorporated per photolyzed rhodopsin, a phenomenon known as "high gain" phosphorylation. When recoverin was added, the activity of RK became sensitive to free Ca2+, with EC50 = 3 microM. The N-terminal myristoyl residue of Rv enhances the inhibitory effect of Rv and introduces cooperativity to the Ca(2+)-dependent inhibition of rhodopsin phosphorylation. Rv neither interacts with other members of the G-protein-coupled receptor kinase family such as beta-adrenergic receptor kinase 1 nor inhibits beta-adrenergic receptor kinase 1 activity. The specific and Ca(2+)-dependent Rv/RK interaction is necessary for the inhibitory effect of Rv on rhodopsin phosphorylation and may play an important role in photoreceptor light adaptation.

Amino Acid Sequence↗
PubMed2000

Immunocytochemical localization and crystal structure of human frequenin (neuronal calcium sensor 1).

Frequenin, a member of a large family of myristoyl-switch calcium-binding proteins, functions as a calcium-ion sensor to modulate synaptic activity and secretion. We show that human frequenin colocalizes with ARF1 GTPase in COS-7 cells and occurs in similar cellular compartments as the phosphatidylinositol-4-OH kinase PI4Kbeta, the mammalian homolog of the yeast kinase PIK1. In addition, the crystal structure of unmyristoylated, calcium-bound human frequenin has been determined and refined to 1.9 A resolution. The overall fold of frequenin resembles those of neurocalcin and the photoreceptor, recoverin, of the same family, with two pairs of calcium-binding EF hands and three bound calcium ions. Despite the similarities, however, frequenin displays significant structural differences. A large conformational shift of the C-terminal region creates a wide hydrophobic crevice at the surface of frequenin. This crevice, which is unique to frequenin and distinct from the myristoyl-binding box of recoverin, may accommodate a yet unknown protein ligand.

Amino Acid Sequence↗
PubMed2001

Instead of binding calcium, one of the EF-hand structures in guanylyl cyclase activating protein-2 is required for targeting photoreceptor guanylyl cyclase.

Guanylyl cyclase activator proteins (GCAPs) are calcium-binding proteins closely related to recoverin, neurocalcin, and many other neuronal Ca(2+)-sensor proteins of the EF-hand superfamily. GCAP-1 and GCAP-2 interact with the intracellular portion of photoreceptor membrane guanylyl cyclase and stimulate its activity by promoting tight dimerization of the cyclase subunits. At low free Ca(2+) concentrations, the activator form of GCAP-2 associates into a dimer, which dissociates when GCAP-2 binds Ca(2+) and becomes inhibitor of the cyclase. GCAP-2 is known to have three active EF-hands and one additional EF-hand-like structure, EF-1, that deviates form the EF-hand consensus sequence. We have found that various point mutations within the EF-1 domain can specifically affect the ability of GCAP-2 to interact with the target cyclase but do not hamper the ability of GCAP-2 to undergo reversible Ca(2+)-sensitive dimerization. Point mutations within the EF-1 region can interfere with both the activation of the cyclase by the Ca(2+)-free form of GCAP-2 and the inhibition of retGC basal activity by the Ca(2+)-loaded GCAP-2. Our results strongly indicate that evolutionary conserved and GCAP-specific amino acid residues within the EF-1 can create a contact surface for binding GCAP-2 to the cyclase. Apparently, in the course of evolution GCAP-2 exchanged the ability of its first EF-hand motif to bind Ca(2+) for the ability to interact with the target enzyme.

Amino Acid Sequence↗
PubMed2004

Fission yeast homolog of neuronal calcium sensor-1 (Ncs1p) regulates sporulation and confers calcium tolerance.

The neuronal calcium sensor (NCS) proteins (e.g. recoverin, neurocalcins, and frequenin) are expressed at highest levels in excitable cells, and some of them regulate desensitization of G protein-coupled receptors. Here we present NMR analysis and genetic functional studies of an NCS homolog in fission yeast (Ncs1p). Ncs1p binds three Ca2+ ions at saturation with an apparent affinity of 2 microm and Hill coefficient of 1.9. Analysis of NMR and fluorescence spectra of Ncs1p revealed significant Ca2+-induced protein conformational changes indicative of a Ca2+-myristoyl switch. The amino-terminal myristoyl group is sequestered inside a hydrophobic cavity of the Ca2+-free protein and becomes solvent-exposed in the Ca2+-bound protein. Subcellular fractionation experiments showed that myristoylation and Ca2+ binding by Ncs1p are essential for its translocation from cytoplasm to membranes. The ncs1 deletion mutant (ncs1Delta) showed two distinct phenotypes: nutrition-insensitive sexual development and a growth defect at high levels of extracellular Ca2+ (0.1 m CaCl(2)). Analysis of Ncs1p mutants lacking myristoylation (Ncs1p(G2A)) or deficient in Ca2+ binding (Ncs1p(E84Q/E120Q/E168Q)) revealed that Ca2+ binding was essential for both phenotypes, while myristoylation was less critical. Exogenous cAMP, a key regulator for sexual development, suppressed conjugation and sporulation of ncs1Delta, suggesting involvement of Ncs1p in the adenylate cyclase pathway turned on by the glucose-sensing G protein-coupled receptor Git3p. Starvation-independent sexual development of ncs1Delta was also complemented by retinal recoverin, which controls Ca2+-regulated desensitization of rhodopsin. In contrast, the Ca2+-intolerance of ncs1Delta was not affected by cAMP or recoverin, suggesting that the two ncs1Delta phenotypes are mechanistically independent. We propose that Schizosaccharomyces pombe Ncs1p negatively regulates sporulation perhaps by controlling Ca2+-dependent desensitization of Git3p.

Alleles↗
PubMed2002

Calcium-modulated membrane guanylate cyclase in synaptic transmission?

Rod outer segment guanylate cyclase 1 (ROS-GC1) is a pivotal enzyme for vertebrate phototransduction and the systematically growing evidence point to its connection with processes other than phototransduction within and outside the retina. ROS-GC1 activity is regulated by Ca2+ in two opposite modes. This regulation is indirect and occurs through Ca+-binding proteins. At nanomolar Ca2+ concentrations, ROS-GC1 is activated by GCAPs and at micromolar Ca2+-concentrations, by S100beta and neurocalcin. The former mode operates in phototransduction and the latter was proposed to play a role in synaptic activity. The last possibility was supported by findings of ROS-GC1 expression not only in various retinal layers other than photoreceptor outer segments but also outside the retina, in pineal gland and olfactory bulb. If ROS-GC1 indeed is to play a role in neurotransmission its expression must be colocalized with its Ca2+-dependent regulators and with possible targets of an increased cyclic GMP concentration, cyclic nucleotide-gated channels or cyclic GMP-dependent protein kinase, in synaptic regions. In this review these aspects of ROS-GC1 expression in retina, pineal gland and olfactory bulb are discussed.

Animals↗
PubMed1997

Handling context-sensitivity in protein structures using graph theory: bona fide prediction.

We constructed five comparative models in a blind manner for the second meeting on the Critical Assessment of protein Structure Prediction methods (CASP2). The method used is based on a novel graph-theoretic clique-finding approach, and attempts to address the problem of interconnected structural changes in the comparative modeling of protein structures. We discuss briefly how the method is used for protein structure prediction, and detail how it performs in the blind tests. We find that compared to CASP1, significant improvements in building insertions and deletions and sidechain conformations have been achieved.

Amino Acid Sequence↗
PubMed2006

Correlation of visinin-like-protein-1 expression with clinicopathological features in squamous cell carcinoma of the esophagus.

EF-hand Ca(2+)-sensor proteins are key molecules for transducing Ca(2+) signals into physiological answers and changes in cytosolic Ca(2+) concentration control a variety of cellular responses, including proliferation, migration, and differentiation, which are relevant for tumor progression. The Ca(2+)-sensor visinin-like protein-1 (VILIP-1) has recently attracted major interest due to its putative tumor suppressor function. Whereas VILIP-1 is expressed in normal skin, it is downregulated in skin tumors in a murine tumor model. The aim of this study was to investigate the expression of the Ca(2+)-sensor VILIP-1 in squamous cell carcinoma of the esophagus and to correlate expression levels with clinicopathological features of the tumor. We examined VILIP-1 expression in 54 specimens of esophageal squamous cell carcinomas and 24 normal esophagus tissues, with immunohistochemical staining and immunofluorescence co-staining techniques. VILIP-1 expression was completely lost or significantly reduced in esophageal tumor tissue compared with normal squamous epithelium. Correlation with clinicopathological features indicated that there was significantly less VILIP-1 expression in lymph node positive (N = 1) versus lymph node negative (N = 0) tumors (P = 0.002). Although there was no significant difference between highly (G(1)), moderately (G(2)) and poorly differentiated (G(3)) tumors (P = 0.177), VILIP-1 expression in tumors is significantly correlated with the depth of tumor invasion (P = 0.028 between T1, T2, T3, and T4). In contrast, co-staining with the proliferation marker Ki-67 indicated no significant correlation with proliferation rates in tumors (Ki-67 index of the tumor). In summary, the expression of the Ca(2+)-sensor VILIP-1 was found to be lost during development of squamous cell carcinoma of the esophagus. The protein expression level significantly correlates with invasive features, such as depth of tumor invasion and local lymph node metastasis, but not with proliferation rate of tumor cells.

Aged↗
PubMed1996

The neuronal EF-hand Ca(2+)-binding protein VILIP: interaction with cell membrane and actin-based cytoskeleton.

VILIP is a member of the visinin/recoverin family of neuronal EF-hand Ca(2+)-binding proteins. Cell fractionation revealed cytoplasmic, membrane- and cytoskeleton-associated pools of VILIP. The association with the cytoskeletal protein fraction is Ca(2+)-dependent and may be mediated by direct interaction with actin. This is concluded from the observations that (i) Ca(2+)-loaded recombinant VILIP binds actin in an overlay assay; (ii) in the presence of Ca(2+), beta-actin co-immunoprecipitates with native VILIP from brain extracts, and (iii) actin and VILIP are co-localized in PC12 cells stably transfected with VILIP cDNA. The interaction of VILIP with the cortical cytoskeleton through actin may facilitate a Ca(2+)-dependent recruitment of VILIP to the cell membrane.

Actins↗
PubMed1995

Sequence characterization and genetic mapping of the human VSNL1 gene, a homologue of the rat visinin-like peptide RNVP1.

In the course of isolation and sequence analysis of microsatellite repeat containing human cDNAs, we have isolated the human homologue of the rat visinin-like peptide gene. The human gene shows a high degree of conservation at both the amino acid and the DNA sequence level. The (CA)n microsatellite repeat embedded in the 3' untranslated region of the gene is conserved between rat and human, along with the flanking DNA sequences. We have mapped the VSNL1 gene to the short arm of chromosome 2.

Amino Acid Sequence↗
PubMed2001

The neuronal calcium sensor protein VILIP-1 is associated with amyloid plaques and extracellular tangles in Alzheimer's disease and promotes cell death and tau phosphorylation in vitro: a link between calcium sensors and Alzheimer's disease?

To investigate whether the observed association of intracellular neuronal calcium sensor (NCS) proteins with amyloid plaques and neurofibrillar tangles in Alzheimer brains is linked to a possible neuroprotective or neurotoxic activity of the protein, we performed cytotoxicity tests in PC12 cells transfected with the calcium sensor protein VILIP-1 (visinin-like protein) and the calcium buffer protein calbindin-D28K. Whereas VILIP-1 expression enhanced the neurotoxic effect of ionomycin already at low ionophore concentrations, calbindin-D28K protected against ionomycin-induced cytotoxicity only at high ionomycin and therefore calcium concentrations. However, in double-transfected cells calbindin-D28K rescued VILIP-1-mediated cytotoxicity at low ionomycin concentrations. Since VILIP-1 was found to be associated with fibrillar tangles in Alzheimer brains, we tested whether VILIP-1 has an influence on tau hyperphosphorylation. VILIP-1 expression enhanced hyperphosphorylation of tau protein compared to nontransfected or calbindin-D28K-transfected cells. These results raise the possibility that the observed reduction in VILIP-1-expressing cells may indicate a selective vulnerability of these neurons and that the calcium sensor protein is involved in the pathophysiology of Alzheimer's disease. The calcium sensor protein may influence tau phosphorylation and have a role in calcium-mediated neurotoxicity opposed to the previously discovered protective effect of calcium buffer proteins.

Alzheimer Disease↗
PubMed1995

Delayed changes in neural visinin-like calcium-binding protein gene expression caused by acute phencyclidine administration.

Phencyclidine (PCP) induces a psychotomimetic state that closely resembles schizophrenia, and PCP-treated animals can serve as a model for schizophrenia. The effects of PCP on the gene expression of NVP-1, a novel Ca(2+)-binding protein, were studied in rats. After 24 hours, the NVP-1 mRNA level in the nucleus accumbens showed a significant decrease of 42%. This result suggests that alterations in Ca(2+)-binding protein may be involved in the pathology of PCP-induced psychosis and, presumably, schizophrenia.

Actins↗
PubMed1998

Haptenylation of antibodies during affinity purification: a novel and convenient procedure to obtain labeled antibodies for quantification and double labeling.

Haptenylation of primary antibodies is a useful technique for multiple purposes. It is a technically straightforward procedure, as many haptens are available as N-hydroxysuccinimide esters or isothiocyanates. Unfortunately, the hapten group may become covalently attached to or close to the combining site of antibodies, lectins, or other ligand-binding proteins during the process of haptenylation. Thus, the interaction of the corresponding protein with its ligand may become severely hampered. To overcome this restriction, we developed a novel procedure for the haptenylation of polyclonal antibodies that combines purification and haptenylation. Haptenylation during adsorption to the affinity matrix combines two advantages: the antigen binding site is protected and the labeling procedure becomes most convenient, as overlabeled proteins and unreacted haptens are easily removed by simple washing. Haptenylation during adsorption to the affinity matrix is a two-phase reaction, which requires different conditions to the conventional procedure. To obtain such optimal conditions, stabilities and reactivities of N-hydroxysuccinimide esters and isothiocyanate groups were investigated with a newly developed assay. Based on this information, antibodies against two recently described calcium-binding proteins, NCS-1 and NVP-3, were biotinylated or digoxigenylated. The haptenylated antibodies were successfully applied for biochemical determination and simultaneous immunoenzymatic double labeling of the two proteins.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1996

Localization of the neural calcium-binding protein VILIP (visinin-like protein) in neurons of the chick visual system and cerebellum.

The visinin-like protein (VILIP) is a member of a recently discovered family of calcium sensors specifically expressed in neurons. Family members contain four potential calcium-binding domains commonly referred to as "EF-hand motifs". VILIP interacts in a calcium-dependent manner with the actin-based neuronal cytoskeleton and modulates the phosphorylation of G-protein-coupled receptors, i.e., rhodopsin, in vitro. Here, we have used antisera against VILIP to study its distribution in the chick brain. Immunostaining of subsets of neurons is observed throughout the brain. Generally, the distribution of VILIP coincides well with the distribution of VILIP transcripts as detected previously by in situ hybridization. The most intense expression is detected in the visual system and the cerebellum. In the visual system, neurons of the nuclei of the ascending tecto-fugal pathway are stained, as are the pretectal, isthmic, and oculomotor nuclei. VILIP immunoreactivity is found in cell bodies, dendrites, and synaptic structures. Thus, VILIP appears to be an excellent marker for the characterization of neurons of the visual pathway. In the cerebellum, VILIP immunoreactivity is detected in deep cerebellar nuclei and in a subset of granule cells, Golgi type II cells, basket cells, and stellate cells, whereas it is completely absent from Purkinje cells. Intense punctate staining in the molecular layer suggests that VILIP is transported from deep cerebellar nuclei and from granule cells to the glutamatergic climbing-fiber and parallel-fiber synapses, respectively, both of which terminate on Purkinje-cell dendrites. The localization of VILIP in these presynaptic terminals has been confirmed at the electron-microscopic level.

Animals↗
PubMed2006

Functional contribution of Ca2+ and Mg2+ to the intermolecular interaction of visinin-like proteins.

The interaction of human visinin-like protein 1 (VILIP1) and visinin-like protein 3 (VILIP3) with divalent cations (Mg2+, Ca2+, Sr2+ and Ba2+) was explored using circular dichroism and fluorescence measurement. These results showed that the four cations each induced a different subtle change in the conformation of VILIPs. Moreover, VILIP1 and VILIP3 bound with Ca2+ or Mg2+ in a cooperative manner. Studies on the truncated mutants showed that the intact EF-3 and EF-4 were essential for the binding of VILIP1 with Ca2+ and Mg2+. Pull-down assay revealed that Ca2+ and Mg2+ enhanced the intermolecular interaction of VILIPs, and led to the formation of homo- and hetero-oligomer of VILIPs. Together with previous findings that Ca2+-dependent localization of VILIPs may be involved in the regulation of distinct cascades and deprivation of Ca2+-binding capacity of VILIPs did not completely eliminate their activity, it is likely to reflect that Mg2+-bound VILIPs may play a role in regulating the biological function of VILIPs in response to a concentration fluctuation of Ca2+ in cells.

Amino Acid Sequence↗
PubMed1995

The development of neural visinin-like Ca(2+)-binding protein 2 immunoreactivity in the rat neocortex and hippocampus.

Neural visinin-like Ca(2+)-binding protein 2 (NVP2) immunoreactivity in the rat neocortex and hippocampus was barely detectable by immunoblot analysis on postnatal day 1 (P1), but increased during postnatal weeks 2-3, reaching a plateau on P28. Immunohistochemical analysis revealed moderate immunoreactivity firstly on P7 in some subsets of the hippocampal interneurons and in the hippocampal pyramidal cells and dentate granule cells. Immunoreactivity of the interneurons decreased during postnatal weeks 2-3 and disappeared by P28. In contrast, immunoreactivity of the cortical and hippocampal pyramidal cells and dentate granule cells abruptly increased during postnatal week 2. The distinctly immunoreactive cells were distributed throughout the neocortex, especially in the cortical plate, and the stratum pyramidale of Ammon's horn and granular layer of the dentate gyrus on P14. Immunoreactivity was homogeneously concentrated in the cell bodies and proximal dendrites at this stage, whereas thereafter immunoreactivity in the neuropil gradually increased, and underwent a relative decrease in the cell bodies. By P28, the higher and granular immunoreactivity in the neuropil covered whole layers of the neocortex, Ammon's horn and the dentate gyrus, the same as in adults. Differential expression of NVP2 in different neuron populations may reflect the differential functional consequences for neuronal development.

Animals↗
PubMed1992

VILIP, a cognate protein of the retinal calcium binding proteins visinin and recoverin, is expressed in the developing chicken brain.

Using a selective cloning approach we previously isolated a number of cDNAs of transcripts that are newly expressed during terminal differentiation of the chicken optic tectum. Here, we have characterized one of these cDNAs (OZ1) by Northern analysis and in situ hybridization. The OZ1 cDNA hybridizes to two transcripts of 1.6 kb and 2.9 kb which are widely expressed in the brain but not detectable in liver, heart or skeletal muscle. Cloning of overlapping cDNAs revealed that both transcripts encode the same open reading frame for a polypeptide of 191 amino acids. The deduced protein contains 4 EF-hand consensus motifs characteristic of calmodulin-like Ca(2+)-binding proteins. It displays 40% and 46% sequence identity with the retinal photoreceptor-specific Ca(2+)-binding proteins visinin and recoverin, respectively, and was termed VILIP (visinin-like protein). VILIP transcripts are also expressed in the retina. However, the expression pattern does not overlap with that of visinin or recoverin. The possible functional implications of the similarity to recoverin, which regulates guanylate cyclase activity of retinal rod cells in a Ca(2+)-dependent manner, are discussed.

Animals↗
PubMed1994

Immunohistochemical localization of neural visinin-like Ca(2+)-binding protein 2 in adult rat brain.

The distribution of neural visinin-like Ca(2+)-binding protein 2 (NVP2) in adult rat brain was analyzed by immunoblot and immunohistochemical methods. NVP2 immunoreactivity was expressed intensely in the pyramidal cells of the CA1 and 2 regions of Ammon's horn, the granule cells of the dentate gyrus and the pyramidal cells of the cerebral cortex layers II to VI, moderately in the pyramidal-shaped cells of the anterior olfactory nucleus and large spindle-shaped cells of the globus pallidus, and weakly in neurons in the nucleus accumbens and the anterior and dorsomedial thalamus. In most cell types, NVP2 immunoreactivity was located in the cytoplasm and plasma membrane of the cell bodies and dendrites.

Animals↗