From the AG Molecular and Cellular Neurobiology,
Institute for Medical Psychology, Otto-von-Guericke-University, 39120 Magdeburg, Germany, the § Department of Neurochemistry and
Molecular Biology, Leibniz Institute for Neurobiology, 39118 Magdeburg,
Germany, the ¶ AG Molecular Neuroendocrinology, Institute for
Anatomy, Westfälische Wilhelms-University, 48149 Münster,
Germany, the
Institute for Pharmacology and Toxicology,
Otto-von-Guericke-University, 39120 Magdeburg, Germany, and the
** Department of Neurobiology, University of Alabama at Birmingham,
Birmingham, Alabama 35294-0027
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ABSTRACT |
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Using antibodies against synaptic protein preparations, we cloned the cDNA of a new Ca2+-binding protein. Its C-terminal portion displays significant similarity with calmodulin and contains two EF-hand motifs. The corresponding mRNA is highly expressed in rat brain, primarily in cerebral cortex, hippocampus, and cerebellum; its expression appears to be restricted to neurons. Transcript levels increase during postnatal development. A recombinant C-terminal protein fragment binds Ca2+ as indicated by a Ca2+-induced mobility shift in SDS-polyacrylamide gel electrophoresis. Antisera generated against the bacterial fusion protein recognize a brain-specific protein doublet with apparent molecular masses of 33 and 36 kDa. These data are confirmed by in vitro translation, which generates a single 36-kDa polypeptide, and by the heterologous expression in 293 cells, which yields a 33/36-kDa doublet comparable to that found in brain. On two-dimensional gels, the 33-kDa band separates into a chain of spots plausibly due to differential phosphorylation. This view is supported by in situ phosphorylation studies in hippocampal slices. Most of the immunoreactivity is detectable in cytoskeletal preparations with a further enrichment in the synapse-associated cytomatrix. These biochemical data, together with the ultra-structural localization in dendrites and the postsynaptic density, strongly suggest an association with the somato-dendritic cytoskeleton. Therefore, this novel Ca2+-binding protein was named caldendrin.
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INTRODUCTION |
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Neurons are structurally and functionally highly polarized cells consisting of an axonal sending and a somato-dendritc receptive compartment. Communication between neurons occurs at synapses, which are asymmetric cell-cell contact sites that typically consist of specialized membrane structures and the underlying cytoskeleton. These cellular specializations derive from the axon of the presynaptic neuron and a dendrite of the postsynaptic neuron. Normal synaptic transmission as well as synaptic plasticity, i.e. use-dependent modulation of synaptic strength, critically depend on intracellular signaling processes on either side of the synapse. Although many of these signaling pathways are mediated by few common messengers, including Ca2+ or cAMP, local specificity is achieved by clustering protein components involved in these pathways at distinct subcellular sites. One such site is the postsynaptic density (PSD),1 an electron-dense proteinaceous structure of the postsynaptic cytoskeleton at excitatory synapses, which is thought to cluster together neurotransmitter receptors with components of signaling pathways (1-4).
Signaling by a great variety of external stimuli, including neurotransmitters growth factors and hormones converges at the level of the Ca2+ ion and is further mediated by diverse intracellular Ca2+-binding proteins (CaBPs) that either can modulate enzymes or structural proteins of the cytoskeleton or are enzymes or cytoskeletal proteins themselves (5). Some of these CaBPs, like calmodulin, are ubiquitously expressed in all cells and mediate a plethora of intracellular responses to Ca2+ signals. Others, e.g. members of the intracellular neuronal calcium sensor (NCS) family (6, 7) are restricted to the central nervous system and even to specific neural cell types.
Several neuronal CaBPs have been localized to the postsynaptic
compartment. These include -actinin-2 (8) and fodrin (9) as
structural components, as well as the regulatory molecule calmodulin (10), the protein phosphatase calcineurin (11), and the NCS protein
VILIP (12). Recent studies revealed an important role for
-actinin
and Ca2+/calmodulin in the
Ca2+-dependent attachment of NMDA receptors to
the synaptic cytomatrix (8, 13, 14). Moreover, calmodulin in its
Ca2+-bound state is able to bind and activate calmodulin
kinase II, a multifunctional kinase highly enriched in the PSD, which
is engaged in several processes of neuronal plasticity including the
induction of long term potentiation (15, 16). Calcineurin has been
shown to dephosphorylate the NMDA receptor in a
Ca2+-dependent manner, thereby shortening the
channel openings (17). Calcineurin overexpression affects the
transition of short term to long term memory and reveals a novel
intermediate phase of long term potentiation (18, 19). VILIP appears to
be involved in the cross-talk of cyclic nucleotide- and
Ca2+-regulated signaling pathways (20).
Using a combined biochemical and molecular approach to identify new synapse-associated proteins (21, 22), we cloned a cDNA encoding a novel neuron-specific polypeptide that was named caldendrin because it binds Ca2+ and is primarily localized in dendrites and neuronal somata. The protein was formerly called calp (calmodulin-like protein), indicating its similarity to calmodulin (22). The present study describes the cloning of the full-length caldendrin cDNA, the tissue distribution of the corresponding transcript, and the biochemical characterization and subcellular localization of the encoded protein.
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MATERIALS AND METHODS |
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Transcript Analysis-- Northern analysis was performed as described previously (23). For in situ hybridization, the following oligodeoxynucleotides (36-43-mers) were derived from the caldendrin cDNA: 95/60, 5'-CAA ATC TTC GGA AGT CCA GTC GTC CAT CTC CAT TGA GGT C-3' (nucleotides 962-923 of caldendrin cDNA); 95/58, 5'-TGG TTC TGA GAT CAA AGG GCT AGG CGA GCT GGG ACG GGC A-3' (nucleotides 1385-1346 of caldendrin cDNA); 95/59, 5'-GAC CTC AAT GGA GAT GGA CGA CTG GAC TTC CGA AGA TTT G-3' (nucleotides 923-962 of caldendrin cDNA) sense control. Labeling and hybridization were performed exactly as described recently (24).
In Vitro Transcription/Translation-- A coupled cell-free transcription/translation system (TNT T3, Promega) was used to generate the primary caldendrin translation product in vitro. Translation products were analyzed by SDS-PAGE and Western blotting.
Isolation of Subcellular Protein Fractions and Western Blotting-- Tissue from adult rats (total brain, neocortex, heart, or liver) was homogenized in 20 mM Tris buffer, pH 7.4, containing either 2 mM CaCl2 or 1 mM EDTA and protease inhibitor mixture (Boehringer, Mannheim, Germany). Soluble proteins were obtained as the supernatant after 100.000 × g centrifugation. After detergent extraction of the remaining pellet with 1% Triton X-100, the detergent-insoluble pellet was extracted with 1% SDS to obtain a fraction of cytoskeletal proteins. Subcellular membrane fractions were prepared essentially as described in Ref. 25 with some modifications from Ref. 26. For isolation of the synaptic junction proteins (PSD fraction), the synaptosomal fraction of the first gradient was diluted with 320 mM sucrose (60 ml/10 g of wet tissue) and an equal volume of 1% Triton X-100, 320 mM sucrose, and 12 mM Tris-HCl, pH 8.1. The suspension was kept on ice for 15 min and centrifuged for 30 min at 32,800 × g. The pellet was resuspended in 320 mM sucrose, 1 mM NaHCO3 (2.5 ml/10 g pf wet tissue) and fractionated in a second sucrose step gradient as described (25). Synaptic junction proteins (PSD fraction) were diluted with 320 mM sucrose, 1 mM NaHCO3 and an equal volume of 1% Triton X-100, 150 mM KCl solution, and pelleted for 20 min at 201,800 × g. All steps were carried out at 4 °C.
Extraction experiments of P2 pellets with various agents were performed as follows. P2 pellets were resuspended in homogenization buffer, aliquoted into six samples (200 mg of protein each) and centrifuged at 15,000 × g for 20 min. Each pellet was then resuspended in 0.5 ml of one of the extraction buffers, incubated for 15 min at 4 °C with gentle shaking, and centrifuged again for 15 min at 100,000 × g. The resulting pellets were washed in homogenization buffer and dissolved in 80 µl of gel loading buffer (27). The supernatants were precipitated with trichloroacetic acid, and the resulting pellets were dissolved in 80 µl of loading buffer. Separation of proteins by SDS-polyacrylamide gel electrophoresis on 5-20% gels under fully reducing conditions and transfer onto nitrocellulose were performed as described previously (21). For two-dimensional separation, the small gel two-dimensional technique described in Ref. 28 was applied using the Hoefer SE 250 two-dimensional system. Western blots were immunodeveloped by overnight incubation with primary antiserum (29) and immunoreactivity visualized using the ECL detection system (Amersham Buchler, Braunschweig, Germany).Transient Expression of Caldendrin in HEK293 Cells-- A caldendrin cDNA fragment that comprises nucleotides 1-1025 containing the entire open reading frame (ORF, see Fig. 1) was subcloned into the pRC/CMV vector (Invitrogen). Human embryonic kidney 293 (HEK293) cells were transiently transfected using the DAC-30 transfection reagent (Eurogentech) according to the manufacturer's instructions.
Immunocytochemistry-- Immunocytochemical staining was carried out using 7-µm microtome sections from rat brains, which were fixed by immersion in Bouin's fluid for 48 h, dehydrated, and embedded in paraplast.
Caldendrin was detected with a purified Ig fraction of the rabbit anti caldendrin polyclonal antibody diluted 1:3,000 using the peroxidase-antiperoxidase method. Antibody binding was visualized by incubating sections first with porcine anti-rabbit IgG (DAKO, Hamburg, Germany) and subsequently with rabbit peroxidase-antiperoxidase complex (DAKO). Subsequently, the detection reagent, 3,3-diaminobenzidine (DAB, Sigma, Munich, Germany), was applied. Some sections were counterstained with hematoxylin for morphological orientation. Controls were performed as follows: (i) preabsorption of the antibody with the antigen and (ii) omitting the first or secondary antibody. Electron microscopy was carried out using vibratome sections (50 µm) from rat brains fixed by perfusion with 3% acrolein, 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.35. Floating sections were stained with the Vectastain ABC staining kit (Vector, Burlingame, CA) according to the manufacturer's instructions. After color reaction with diaminobenzidine, the sections were extensively washed in 0.05 M Tris/HCl buffer (pH,7.4) (twice) and 0.1 M cacodylate buffer (pH 7.4) (twice) before being fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 h (4 °C). Subsequently sections were further washed in 0.1 M cacodylate buffer (pH 7.4) (twice) and doubly distilled water (ddH2O) (four times). Silver enhancement of the DAB product was performed as follows: Solution A consisted of 3% hexamethylenetetramine in ddH2O; solution B consisted of 5% silver nitrate (AgNO3) in ddH2O; solution C consisted 2.5% disodium tetraborate in ddH2O; solution D consisted of 0.05% tetrachloroauric(III) acid in ddH2O; solution E consisted of 2.5% sodium thiosulfate in ddH2O. First, sections were incubated for 10 min at 60 °C in a premade mixed solution (5 ml (A) + 250 µl (B) + 500 µl (C)) and afterward washed in distilled water (dH2O) (3 × 3 min). After these washing steps, sections were incubated in solution D at room temperature for 3 min, washed with dH2O (3 × 3 min), incubated in solution E for 3 min, and washed again for 3 × 3 min in dH2O. Subsequently, the sections were postfixed in 1% OsO4, dehydrated in ethanol, and embedded in epon. Parallel semithin sections were stained with toluidine blue for morphological orientation; ultrathin sections were contrasted with uranyl acetate/lead citrate prior to analysis with a Philips electron microscope.Preparation of Hippocampal Slices, in Situ Phosphorylation, and Immunoprecipitation-- For in situ phosphorylation studies, hippocampal slices were prepared and incubated in 100 µCi of 33P (final concentration in the incubation medium 50 µCi/ml) as described previously (30). After 90 min of labeling time, slices were rinsed with homogenization buffer A (10 mM Tris/HCl, 0.5 M NaCl, 1% Triton X-100, pH 7.4), homogenized, and spun at 100,000 × g for 1 h. The supernatant was immunoprecipitated with 10 µl of polyclonal rabbit antiserum preabsorbed to GammaBind Plus Sepharose (Pharmacia Biotech, Uppsala, Sweden). The remaining pellet was extracted with 1% SDS. For immunoprecipitation of caldendrin, the resulting supernatant was diluted with 10 mM Tris/HCl to give a final SDS concentration of 0.2%. Immunoprecipitation was performed as described previously (23). Non-binding proteins and immunoprecipitates were separated by SDS-PAGE and transferred onto nitrocellulose (0.45 µm pore size). Western blots were dried, and 33P incorporation was visualized using a Fujix BAS1000 Bioimager. Detection of the immunoprecipitate on Western blots was performed with a polyclonal mouse caldendrin antiserum.
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RESULTS |
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Identification and Cloning of Caldendrin cDNAs--
From a
collection of cDNA clones isolated by expression screening with
antisera against a rat brain synaptic protein preparation (22), one
cDNA clone, termed sap8i, encoded a protein fragment that displayed
distinct sequence similarity to rat calmodulin. The clone was used to
isolate a set of seven overlapping cDNAs ranging in size from 1 to
1.4 kilobases. They harbor an open reading frame of 298 amino acids
(Fig. 1A). The translation
initiation site was assigned to nucleotides 86-88 encoding the first
in-frame methionine. This region fits well with the consensus pattern
for eukaryotic translation initiation ((A/G)CC AUG (G/A); Ref. 31), including a 5' GC-rich region with only one mismatch at the 1 position (G instead of C). In vitro translation and
heterologous expression studies confirmed the use of this initiation
site (see below). A putative polyadenylation signal is formed by
nucleotides 1385-1390 (bold in Fig. 1A). The
deduced protein caldendrin has a calculated Mr
of 33,071 and a theoretical isoelectric point of 7.42. Whereas primary
structure analysis revealed no identifiable structural features in the
N-terminal half of the protein, the C-terminal part displays a high
degree of similarity to the entire reading frame of rat calmodulin. As
shown in an alignment of both proteins (Fig. 1B), two of the
four Ca2+-binding EF-hand structures found in calmodulin
are conserved in the novel protein. However, the remaining two miss
several essential residues in the consensus pattern. Within the aligned region sequence, identity and similarity between caldendrin and calmodulin are 42% and 57%, respectively. The N-terminal half of
caldendrin contains seven putative protein kinase C phosphorylation sites (arrows in Fig. 1A) and an unusually high
proline content (13%).
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Spatial and Temporal Expression Pattern of the Caldendrin Transcript-- Northern blot analysis revealed a single hybridizing band of 1.8 kilobases that is detectable in various brain regions with strongest expression in the cerebral cortex. No hybridization is detected in heart, muscle, liver, and C6 glioma cells (Fig. 2A). During brain development, hybridization signals were first observed in the second postnatal week, i.e. during period of synapse formation and terminal differentiation of the brain. After the initial period of increase caldendrin, mRNA levels remain essentially unchanged throughout later ontogenetic stages (Fig. 2B).
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Calcium Binding Activity of Recombinant Caldendrin Fragments-- A caldendrin cDNA fragment comprising nucleotides 500-979 (see Fig. 1) was generated via polymerase chain reaction and subcloned into the pQE30 vector to generate a bacterially expressed fusion protein including amino acids 139-298 and six histidine residues at the N terminus. This fusion protein has a calculated Mr of 19.386 kDa. In the presence of 1 mM Ca2+, a clear mobility shift in the PAGE gel can be observed, indicating the ability of the caldendrin C terminus to bind Ca2+ (Fig. 3). This shift is most plausibly based on conformational changes that occur in caldendrin upon Ca2+ binding as has been shown for other CaBP, e.g. calmodulin (32, 33) or VILIP (34).
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Characterization of Caldendrin-- The fusion protein was used to raise polyclonal caldendrin antisera in mice and rabbits. Three independent antisera specifically detect a protein doublet of 33/36 kDa in brain protein preparations (Fig. 4). This doublet is not recognized by either of the pre-immune sera (data not shown). To get a first clue to the biochemical nature of the two polypeptides, we tested their solubility in low salt buffer and detergent. The 33-kDa protein isoform is partly found in soluble protein fractions (SP in Fig. 4). The Triton X-100 extract of the remaining pellet does not contain significant amounts of either of the immunoreactive bands. The 36-kDa caldendrin polypeptide is almost exclusively found in the aqueous and detergent-insoluble cytoskeletal pellet (CP in Fig. 4). During postnatal brain development, the amounts of caldendrin immunoreactivity increase from the second to the eighth postnatal week both in soluble and cytoskeletal protein fractions. Heart and kidney protein preparations obtained from 8-week-old rats are devoid of caldendrin immunoreactivity (H and K in Fig. 4).
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Subcellular Localization of Caldendrin-- Subcellular fractionation by differential centrifugation of brain proteins demonstrated that caldendrin immunoreactivity is associated with nearly all particulate fractions including light and heavy membrane fractions and several synaptic fractions, like synaptosomes, synaptic membranes and the PSD fraction (Fig. 6A). The immunoreactivity in particular of the 36-kDa band is clearly enriched in the PSD fraction after the second washing step with Triton X-100 (T2 in Fig. 6A), indicating a tight association with the synaptic cytomatrix. However, caldendrin appears not to be an exclusively synaptic protein.
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Caldendrin as a Putative Substrate for Protein Kinases-- The primary structure of caldendrin contains seven putative protein kinase C phosphorylation sites (see Fig. 1). Therefore, we tested whether caldendrin is a possible substrate for protein kinases. Two-dimensional gel electrophoresis of immunoprecipitated caldendrin demonstrates that both isoforms differ in their migration behavior along the pH gradient (Fig. 8). Whereas the 36-kDa isoform appears as a single spot at a pI of 6.8-6.9 (calculated pI of 7.42), the 33-kDa band separates into a chain of at least three spots within the range of pH 5.9-6.8. This could be a consequence of phosphorylation, because a phosphate group added to a protein shifts the pI for 0.1 to 0.3 units (35).
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DISCUSSION |
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In this study, we describe the cloning and first characterization of rat caldendrin, a novel EF-hand Ca2+-binding protein selectively localized to the somato-dendritic compartment of neurons and highly enriched at postsynaptic structures. Sequence alignment shows that ~150 amino acids at the C terminus of caldendrin exhibit high similarity with the ubiquitous calcium mediator protein calmodulin. Two of the EF-hand domains of caldendrin, i.e. the second and fourth ones, deviate from the consensus pattern defined for functional EF-hands and therefore may have lost their Ca2+-binding activity.
The predicted Mr of 33,071 for caldendrin is in reasonable agreement with the apparent molecular mass of 33/36 kDa obtained from Western blots of rat brain proteins and confirms the assumed translation start site. The in vitro translation product generated from the full-length caldendrin cDNA comigrates with the upper band at 36 kDa. Therefore, we assume that this band represents the primary translation product. The biochemical modification or nature of the second isoform which migrates as a 33-kDa protein band is still unknown. Cross-reactivity with a different protein as well as origin from a differentially spliced transcript may be excluded, because HEK293 cells transfected with the caldendrin cDNA express both isoforms. This clearly indicates that the modification of the primary translation product is post-translational and occurs in heterologous expression systems as in neurons.
The modification is accompanied by an increased solubility of the protein. Both the mechanism of the cytoskeletal association of caldendrin and the partial release into the cytosol are currently unknown. A Ca2+-dependent translocation, as shown for the neuronal EF-hand Ca2+-sensor protein VILIP (34), can be excluded because the subcellular distribution of caldendrin is not altered by changes in the Ca2+ concentration during sample preparation.2
The post-translational modification of the primary polypeptide not only leads to an increased electrophoretic mobility in SDS-PAGE, it also modifies migration in pH gradients as observed on two-dimensional gels. The appearance of a chain of spots at 33 kDa may indicate the occurrence of different states of phosphorylation; however, it could also result from an artificial carbamylation during the sample preparation (35). Analysis of the caldendrin primary structure revealed several potential phosphorylation sites in the N-terminal portion, and incorporation of radioactively labeled phosphorus into caldendrin was shown under in situ conditions in hippocampal slice preparations. Interestingly, only the 33-kDa isoform incorporated 33P. This could point to an important role for phosphorylation in modulating biochemical properties of caldendrin. It is possible that an initial phosphorylation step of the 36-kDa isoform induces conformational alterations, which result in the observed change in migration behavior and solubility of the protein.
The potential of caldendrin to bind Ca2+ was shown in vitro with a bacterially expressed fusion protein. The electrophoretic mobility shift observed with this 18-kDa caldendrin fragment upon Ca2+ binding is not detectable with the full-length brain protein. A possible explanation may be that the conformational changes are compensated in the much larger full-length protein which exhibits a clear bipartite structure. The two parts are predicted to differ significantly in their physico-chemical features. Whereas the N-terminal half is highly basic (pI = 11.9; 13 positively charged amino acid residues), the Ca2+-binding C-terminal half is acidic (calculated pI = 4.5; 13 negatively charged residues). Therefore, intramolecular interactions are likely, which may be subject to regulation by Ca2+ binding and phosphorylation.
On the basis of their postulated function, the EF-hand CaBPs have usually been classified as "trigger" or "buffer" proteins. Whereas trigger proteins like calmodulin (for review, see Ref. 36) or the members of the NCS family change their conformation upon Ca2+ binding and then modulate a variety of channels or enzymes, buffer proteins like parvalbumin (reviewed in Ref. 37) are thought to play a more passive scavenger role to limit the rise in intracellular Ca2+ concentration. The modular organization of caldendrin supports a postulated trigger function. Analysis of the caldendrin N terminus, which appears not to be structurally related to any known protein, may help to understand the functional significance of this bipartite molecular organization.
Caldendrin seems to be preferentially expressed in brain regions with a laminar organization (i.e. cortex, hippocampus, and cerebellum) and in neuronal cells with a broad dendritic tree. So far, we have found no evidence for expression of the protein in neuroglia. Its distribution within the neuron appears to be restricted to the somato-dendritic compartment, a feature that it shares for example with MAP2, a cytoskeletal protein associated with microtubules (38). Both the biochemical solubilization characteristics and the subcellular fractionation studies lead us to conclude that caldendrin is associated with the cortical cytoskeleton. Caldendrin seems to be localized to the subplasmalemmal cortex of cell soma and dendrites, and both biochemical fractionation and electron microscopic investigation clearly demonstrate a synaptic localization of caldendrin. Although caldendrin is not exclusively localized to synapses we found that, in contrast to many other proteins of the dendritic cytoskeleton, immunoreactivity is enriched in the postsynaptic density fraction.
Several other synaptic proteins display similar intramolecular
dichotomies and therefore are good candidates to confer
Ca2+-dependent regulation to subcellular
structures or for the coupling to different signaling pathways.
Examples include the cytoskeletal proteins -spectrin, non-muscle
-actinins (39), and myosin. In the latter case, the calmodulin-like
domain represents the regulatory light chain (40). The
Ca2+-dependent Cys endopeptidase calpain also
contains EF-hand domains at the C terminus of each subunit that confers
Ca2+-regulation to the proteolytic action of the holoenzyme
(41). Another example is calcineurin, a
Ca2+-calmodulin-dependent protein phosphatase
that is composed of a catalytic subunit and a calmodulin-like
regulatory subunit (42). Based on gene structure analyses, it has been
suggested that these modular molecules, which are composed of one or
more Ca2+-binding EF-hand domains combined with other
functional domains, have evolved from a single genetically mobile
ancestral EF-hand motif (43).
The relatively late onset of transcription of the caldendrin gene during development is shared with several other neuronal CaBPs (44). This suggests that caldendrin expression is not crucial for synaptogenesis. However, in contrast to some other CaBPs, like hippocalcin (45) or calbindin-D28K (46), no decrease in protein levels in mature neurons was observed. The ontogenetic expression profile, therefore, suggests a functional role for caldendrin in the dendritic tree of terminally differentiated neurons.
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ACKNOWLEDGEMENTS |
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We thank C. Otto, K. Hartung, M. Marunde, A. Lewedag, and B. Kracht for expert technical assistance and Prof. P. W. Beesley for critical comments on the manuscript.
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FOOTNOTES |
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* This work was supported by Bundesministerium für Bildung und Forschung Grant TPA2 (Neurotrauma Magdeburg-Berlin) and Land Sachsen-Anhalt (FKZ 1883A/0025); by Deutsche Forschungsgemeinschaft Grants SFB 426/A1, KR-1255/4-1, and Wi 588/5-1; and by the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y17048.
To whom correspondence should be addressed: Dept. of
Neurochemistry and Molecular Biology, Leibniz Institute for
Neurobiology, P. O. Box 1860, D-39008 Magdeburg, Germany. Tel.:
49-391-6263223; Fax: 49-391-6263229; E-mail:
kreutz{at}ifn-magdeburg.de.
The abbreviations used are: PSD, postsynaptic density; CaBP, calcium-binding protein; DAB, diaminobenzidine; HEK, human embryonic kidney; NCS, neuronal calcium sensor; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
2 C. I. Seidenbecher, E. D. Gundelfinger, and M. R. Kreutz, unpublished observations.
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REFERENCES |
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