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At least 19 recordsLinked to original sources
PubMed1993

Myristoylation of hippocalcin is linked to its calcium-dependent membrane association properties.

Hippocalcin, a recently identified Ca(2+)-binding protein of the recoverin family exclusively expressed in the hippocampus, has a primary structure containing three putative Ca(2+)-binding sites (EF-hands) and a possible NH2-terminal myristoylation site. 45Ca blots demonstrated that every three EF-hand domains, expressed as fusion proteins in Escherichia coli, bind Ca2+, indicating that hippocalcin binds 3 mol of Ca2+/mol of protein. To determine whether hippocalcin is myristoylated, hippocalcin mRNA was translated in vitro in the presence of [3H]myristic acid. 3H label was resistant to hydroxylamine treatment, and replacement of NH2-terminal glycine with alanine prevented 3H label incorporation, indicating that in vitro translated hippocalcin covalently bound [3H]myristic acid at the NH2-terminal glycine. In vitro translated hippocalcin is quantitatively myristoylated, as evidenced by an electrophoretic mobility shift of [35S]methionine-labeled protein on two-dimensional gels. Native hippocalcin comigrated precisely with the in vitro translated hippocalcin on two-dimensional gels, suggesting that native hippocalcin is myristoylated. Native and in vitro translated hippocalcins, but not non-myristoylated mutagenic (Gly1-Ala1) hippocalcin, displayed Ca(2+)-dependent membrane association, indicating that myristoylation participates in its Ca(2+)-dependent membrane association properties. In vitro translated hippocalcin bound to phospholipid vesicles somewhat, however, phospholipid association was insufficient for its membrane association properties, suggesting that the NH2-terminal myristoyl moiety on hippocalcin interacts with lipid bilayers and facilitates interaction with other membrane proteins.

Alanine↗
PubMed2000

Role of hippocalcin in Ca2+ -induced activation of phospholipase D.

The role of hippocalcin as a novel mediator in the PKC-independent Ca2+ -induced phospholipase D (PLD) activation pathway was investigated. Hippocalcin was expressed in the Sf9 insect cell expression system because the myristoylation of this protein is essential for its function. PLD and Cdc42 proteins were prepared from a rat brain cell membrane and cytosol, respectively. The recombinant hippocalcin was expressed in the Sf9 cell using expression vector pVL1393. The hippocalcin expressed was purified as a single band on PAGE following the hydrophobic phenyl HPLC and TSKgel G3000SW gel filtration HPLC. The molecular size of the rat brain hippocalcin expressed in this system was estimated to be 22 kDa. Myristoylated hippocalcin migrated faster than the non-myristoylated form on SDS-PAGE. Less than 10% of the total hippocalcin expressed was myristoylated in this baculovirus expression system. PLD was extracted from rat brain membranes and chromatographically enriched 70-fold. From the rat brain cytosol, Cdc42 was purified to near homogeneity. While hippocalcin alone did not activate PLD, it increased PLD activity activated with Cdc42 1.8-fold in the presence of calcium (300 nM free calcium). In the absence of calcium in the reaction mixture, the effect of hippocalcin to facilitate Cdc42-activated PLD activity was abolished. This result suggests that hippocalcin might be one of the regulatory proteins in the PKC-independent Ca2+ -mediated PLD activation pathway in conjunction with the Cdc42 protein.

Animals↗
PubMed1994

Expression of hippocalcin in the developing rat brain.

Expression of hippocalcin in the developing rat brain was investigated by a combination of Northern blot, in situ hybridization, immunoblot and immunohistochemical methods. In the hippocampus, hippocalcin mRNA and immunoreactivity first appeared in the CA3 pyramidal cells on embryonic day 19 (E19) and postnatal day 1 (P1), respectively, and extended throughout Ammon's horn. After P14, the hippocampal pyramidal cells, especially in the CA1 region, maintained the highest expression level among the brain regions. The dentate granule cells expressed a small amount of hippocalcin mRNA and immunoreactivity from P7 and maintained a low level through the developmental stages. In the cerebral cortex, hippocalcin mRNA and immunoreactivity appeared in the pyramidal cells of the piriform cortex from P1 and P4, respectively. Their expression extended throughout the cerebral cortex and reached the maximum level on P14, and then declined gradually with age to half of the maximum level by adults. In the cerebellum, a few Purkinje cells expressed a small amount of hippocalcin mRNA and immunoreactivity on P7. Their expression became evident in most of the Purkinje cells on P14 and increased gradually by P28. Then, their expression declined with age; however, the immunoreactivity was concentrated in the cell bodies and proximal segments of the dendrites in adults. These results suggest that the expression of hippocalcin mRNA and protein is strictly controlled by both the cell type and the developmental process and that hippocalcin plays a role in neuronal differentiation in the early stages of development and may relate to other neuronal function in the adult brain.

Animals↗
PubMed1998

Genomic structure and chromosomal mapping of the human and mouse hippocalcin genes.

In an attempt to elucidate the possible relationship of hippocalcin to neurological disorders, we isolated and analyzed the human and mouse hippocalcin genes. The human and mouse hippocalcin genes contain three exons and two introns, and span approximately 7 and 8kb, respectively. The exon/intron splice junctions of the human and mouse genes are all situated in exactly the same position and are not consistently placed with respect to the coding regions of the tandemly repeated EF-hand motifs. The amino acid sequences of human and mouse hippocalcins deduced from the genes are 100% identical. Within the 2-kb 3'-flanking sequences of the human and mouse genes, one conserved polyadenylation signal was identified at positions 762 and 823bp downstream from TAG, respectively. Within the 2.6-kb 5'-flanking sequences of the human and mouse genes, neither a canonical 'TATA' box nor a 'CAAT' box was found. Southern blot analysis of the human and mouse genomic DNAs demonstrated that the positive bands coincide exactly with those expected from the sequence of the cloned genes, indicating that the human and mouse hippocalcin genes are present as a single-copy gene. Fluorescence in-situ hybridization revealed that the human hippocalcin gene is located at chromosome 1 p34.2-35 and the mouse hippocalcin gene at chromosome 4 D2-D3.

Animals↗
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↗
PubMed1994

Hippocalcin expression in the brain of the Snell dwarf mutant mouse.

To determine factors contributing to the expression of the brain-derived protein, hippocalcin, we mapped its distribution in the brain of Snell pituitary dwarf mutant mice (dw) by immunohistochemical and immunoblot methods. Our findings are as follows. (1) In the hippocampus, hippocalcin immunoreactivity was found in the cell body and dendrites of pyramidal neurons of the normal controls and dw mice, although the intensity of immunoreactivity in the dw mice was lower. (2) In the cerebellum, hippocalcin immunoreactivity was strongly expressed in the Purkinje cell body of both the control and dw mice. However, the Purkinje cell dendrites were found to be more intensely stained in the dw mice than in the normal controls. (3) In the dw cerebral cortex, the pyramidal neurons of layers II to VI strongly expressed hippocalcin, whereas its expression in the controls was weak. (4) The amount of hippocalcin in the dw hippocampus was less than in the normal controls, whereas the amount in the dw cerebral cortex and cerebellum was greater. These results indicate that the developmental expression of hippocalcin in the dw brain is affected by the retarded maturation of the neuronal network due to the deficient hormonal state (the lack of growth and thyroid hormones).

Animals↗
PubMed1996

Characterization of the rat hippocalcin gene: the 5' flanking region directs expression to the hippocampus.

Hippocalcin is an EF-hand [Persechini A. et al. (1989) Trends Neurosci. 12, 462-467] Ca2+ binding protein encoded by a neuron-specific gene. A detailed atlas of hippocalcin messenger RNA expression in the adult rat brain was complied using in situ hybridization. Highest levels of messenger RNA are found in the hippocampus, where messenger RNA is localized in proximal dendrites of CA pyramidal cells. Expression is also seen in other brain regions, including the neocortex, caudate-putamen, taenia tecti, claustrum, olfactory tubercle, anterior olfactory nucleus, and granule cell and glomerular layers of the olfactory bulb. The rat hippocalcin gene spans approximately 9 kb and consists of three exons, separated by introns of 6.7 and 0.25 kb. Sequence analysis of the putative proximal promoter region identified two clusters of multiple E-box sites which may regulate the cell-specific expression. Two lacZ fusion constructs carrying 0.9 and 3.4 kb of rat hippocalcin gene upstream region were used to create transgenic mice. With the 3.4 kb construct, transgene expression varied between founder mice, but was always found in the dentate gyrus and CA1-CA4 regions of the hippocampus, thus partly mimicking the expression of the endogenous gene. For the 0.9 kb construct, the levels of lacZ expression were weaker and more variable. Neither construct showed expression in any peripheral tissues examined. To establish an in vitro model of transcriptional regulation, the 3.4 and 0.9 kb 5' upstream regions were fused to a promoterless reporter gene encoding chloramphenicol acetyltransferase and transiently transfected into the hippocalcin-positive NG-108 cells. The 3.4 kb construct was strongly expressed, whilst the 0.9 kb construct was not expressed. In this paper, we describe the detailed expression pattern of the rat hippocalcin gene, the gene structure and its neuron-specific promoter.

Animals↗
PubMed1995

Hippocalcin in rat retina. Comparison with calbindin-D28k, calretinin and neurocalcin.

The post-natal developmental expression in rat retina of four calcium-binding proteins belonging to the calmodulin-troponin-C family was investigated by immunohistochemistry using anti-calbindin-D28k, anti-calretinin, anti-hippocalcin and anti-neurocalcin polyclonal antibodies on paraffin sections from Wistar rat retinae aged from post-natal days 1 (P1), 5 (P5), 10 (P10), 20 (P20) to adulthood (8 weeks). Immunoblot using anti-hippocalcin and homogenates proteins from retina, cerebellar cortex, hippocampus and cerebellum was also performed. Hippocalcin immunoreactivity in adult rat retina was demonstrated by both immunohistochemistry and Western blot. During post-natal development, calbindin-D28k, calretinin and neurocalcin immunoreactivity were detected at P1 in ganglion cells, whereas hippocalcin immunoreactivity was seen later at P5 in this cell layer. In the amacrine cell layer, neurocalcin immunoreactivity was detected at P5 and hippocalcin at P10. Calbindin-D28k was labelling the immature horizontal cell, calretinin was detected in nearly all ganglion cells and in some amacrine cells since P1. These three calcium-binding proteins do not seem to play a role in synaptogenesis which takes place later. We confirmed that calbindin-D28k appeared to be a good marker for horizontal cells. The presence of hippocalcin, a myristoylated calcium-binding protein belonging to the recovering subfamily and previously localized in few brain areas has been detected for the first time in retina.

Animals↗
PubMed2000

NAIP interacts with hippocalcin and protects neurons against calcium-induced cell death through caspase-3-dependent and -independent pathways.

Inhibitor-of-apoptosis proteins (IAPs), including neuronal apoptosis inhibitory protein (NAIP), inhibit cell death. Other IAPs inhibit key caspase proteases which effect cell death, but the mechanism by which NAIP acts is unknown. Here we report that NAIP, through its third baculovirus inhibitory repeat domain (BIR3), binds the neuron-restricted calcium-binding protein, hippocalcin, in an interaction promoted by calcium. In neuronal cell lines NSC-34 and Neuro-2a, over-expression of the BIR domains of NAIP (NAIP-BIR1-3) counteracted the calcium-induced cell death induced by ionomycin and thapsigargin. This protective capacity was significantly enhanced when NAIP-BIR1-3 was co-expressed with hippocalcin. Over-expression of the BIR3 domain or hippocalcin alone did not substantially enhance cell survival, but co-expression greatly increased their protective effects. These data suggest synergy between NAIP and hippocalcin in facilitating neuronal survival against calcium-induced death stimuli mediated through the BIR3 domain. Analysis of caspase activity after thapsigargin treatment revealed that caspase-3 is activated in NSC-34, but not Neuro-2a, cells. Thus NAIP, in conjunction with hippocalcin, can protect neurons against calcium-induced cell death in caspase-3-activated and non-activated pathways.

Animals↗
PubMed1994

Molecular cloning of human hippocalcin cDNA and chromosomal mapping of its gene.

We have isolated a cDNA clone encoding human hippocalcin from a human hippocampus cDNA library. This clone (hHLP1) consists of 840 nucleotides, including the entire open reading frame of 582 nucleotides, 10 nucleotides of the 5' leader and 248 nucleotides of the 3' noncoding regions. Comparison of the human hippocalcin sequence with the corresponding rat sequence revealed an amino acid identity of 100% and nucleotide identity of 92%. Northern blot analysis showed that a single transcript at a position corresponding to 2.0 kb was detected only in the brain. The human hippocalcin gene was mapped to chromosome 1 by amplification of human hippocalcin-specific DNA fragment on DNA from human-rodent somatic cell hybrids by using the polymerase chain reaction.

Amino Acid Sequence↗
PubMed1993

Hippocalcin: a calcium-binding protein of the EF-hand superfamily dominantly expressed in the hippocampus.

Hippocalcin is a recently identified Ca(2+)-binding protein with three EF-hand structures, dominantly expressed in the hippocampal pyramidal layer. The complete amino acid sequence of hippocalcin deduced from the cDNA is composed of 195 residues, has a calculated molecular mass of 22,574 daltons, and has a striking sequence homology to those of visinin, recoverin, S-modulin, neurocalcins and neural visinin-like proteins. Hippocalcin binds 3 mol of Ca2+ per mol of protein at submicromolar Ca2+ levels, and associates the plasma membrane in a Ca(2+)-dependent manner. Hippocalcin is myristoylated at its NH2-terminal glycine residue, and this modification is a key event in terms of its membrane-association property.

Amino Acid Sequence↗
PubMed1993

Distribution of hippocalcin mRNA and immunoreactivity in rat brain.

Distribution of hippocalcin in rat brain was analysed by in situ hybridization and immunohistochemical methods. Hippocalcin mRNA and immunoreactivity were expressed more intensely in the pyramidal cells of the hippocampus, intensely in the Purkinje cells of the cerebellum, moderately in the dentate granule cells and pyramidal cells of cerebral cortex layers II-VI and weakly in the large neuronal cells of the caudate-putamen. Some discrepancies in the localization of hippocalcin mRNA and immunoreactivity were noted in the mamillary nuclei, anterior part of the thalamus and the septal nuclei. In most cell types, hippocalcin immunoreactivity was localized in the cytoplasm and plasma membrane of cell bodies and dendrites.

Animals↗
PubMed1998

Recoverin and hippocalcin distribution in the lamprey (Lampreta fluviatilis) retina.

Recoverin is a calcium-sensing protein which is involved in the transduction of light in vertebrate photoreceptors. It is also detected in other retina cell types in which its function is not yet elucidated, and is an autoantigen in a cancer-associated degenerative disease of the retina. Recently, hippocalcin, an homologous protein of recoverin, belonging to the same family of fatty acylated EF-hand calcium binding proteins was described in mammals. The immunohistochemical studies presented in this paper demonstrate, that, in the retina of the lamprey, an Agnathan considered the living ancestor of actual jawed vertebrates, recoverin was present in all photoreceptors and, to a lesser extent in subpopulations of amacrine and ganglion cells whereas hippocalcin was detected in numerous amacrine and ganglion cells and in the inner segments of long photoreceptors. The existence of these calcium-binding proteins shows that they have a high degree of conservation during evolution. Their presence in the same cells that in jawed vertebrates (photoreceptors and ganglion cells for recoverin; amacrine and ganglion cells for hippocalcin) suggests that some retinal functions are well conserved but because they were also found in different cell types than in other species (amacrine for recoverin; photoreceptors for hippocalcin), they may have functions more specific to the lamprey retina.

Animals↗
PubMed1997

DNA regions supporting hippocalcin gene expression in cell lines.

The rat hippocalcin gene -3.2 to +0.6 kb region activates reporter gene expression in the NG108-15 and PC12 neuronal cell lines, but not in NIH3T3 or HEK-293 cells. Three fragments (-3.2 to -2.6, -2.6 to -2.3 and -2.3 to -1.8 kb) weakly activate transcription, and "-1.8 to -1.5" kb is a strong activator. Thus cell type-specific expression of the rat hippocalcin gene is regulated by distributed elements in the -3.2 to -1.5 kb region.

3T3 Cells↗
PubMed1992

Molecular cloning of hippocalcin, a novel calcium-binding protein of the recoverin family exclusively expressed in hippocampus.

We have isolated a cDNA clone encoding a novel calcium-binding protein of the recoverin family from rat brain cDNA library. This clone (PCB11) has 588 nucleotides in the open reading frame including the termination codon, 174 nucleotides of the 5' leader and 800 nucleotides of the 3' noncoding region. The complete amino acid sequence deduced from the cDNA is composed of 195 residues, has a calculated molecular mass of 22,574 Daltons, and contains three putative calcium-binding domains of the EF-hand structure. The deduced amino acid sequence has a striking sequence homology to those of the retinal recoverin family (recoverin, visinin, P26, 23kD protein, S-modulin) and the brain-derived recoverin family (P23k, 21-kDa CaBP and neurocalcin). Northern blot, in situ hybridization, immunoblot and immunohistochemical analyses revealed that the protein is exclusively expressed in pyramidal layer of the hippocampus. The protein was therefore designated hippocalcin.

Amino Acid Sequence↗
PubMed1994

Molecular cloning of a novel calcium-binding protein structurally related to hippocalcin from human brain and chromosomal mapping of its gene.

A cDNA clone (hHLP2) encoding a novel calcium-binding protein structurally related to hippocalcin has been isolated from the human hippocampus cDNA library. The primary structure consists of 193 amino acids, and contains three EF-hand structures and a possible NH2-terminal myristoylation site. A single transcript at a position corresponding to 1.7 kilobases was detected only in the brain. The hHLP2 gene was mapped to human chromosome 2.

Amino Acid Sequence↗
PubMed2002

Differential use of myristoyl groups on neuronal calcium sensor proteins as a determinant of spatio-temporal aspects of Ca2+ signal transduction.

The localizations of three members of the neuronal calcium sensor (NCS) family were studied in HeLa cells. Using hippocalcin-EYFP and NCS-1-ECFP, it was found that their localization differed dramatically in resting cells. NCS-1 had a distinct predominantly perinuclear localization (similar to trans-Golgi markers), whereas hippocalcin was present diffusely throughout the cell. Upon the elevation of intracellular Ca(2+), hippocalcin rapidly translocated to the same perinuclear compartment as NCS-1. Another member of the family, neurocalcin delta, also translocated to this region after a rise in Ca(2+) concentration. Permeabilization of transfected cells using digitonin caused loss of hippocalcin and neurocalcin delta in the absence of calcium, but in the presence of 10 microm Ca(2+), both proteins translocated to and were retained in the perinuclear region. NCS-1 localization was unchanged in permeabilized cells regardless of calcium concentration. The localization of NCS-1 was unaffected by mutations in all functional EF hands, indicating that its localization was independent of Ca(2+). A minimal myristoylation motif (hippocalcin-(1-14)) fused to EGFP resulted in similar perinuclear targeting, showing that localization of these proteins is because of the exposure of the myristoyl group. This was confirmed by mutation of the myristoyl motif of NCS-1 and hippocalcin that resulted in both proteins remaining cytosolic, even at elevated Ca(2+) concentration. Dual imaging of hippocalcin-EYFP and cytosolic Ca(2+) concentration in Fura Red-loaded cells demonstrated the kinetics of the Ca(2+)/myristoyl switch in living cells and showed that hippocalcin rapidly translocated with a half-time of approximately 12 s after a short lag period when Ca(2+) was elevated. These results demonstrate that closely related Ca(2+) sensor proteins use their myristoyl groups in distinct ways in vivo in a manner that will determine the time course of Ca(2+) signal transduction.

Adaptor Protein Complex gamma Subunits↗