EF-hand Calcium-binding Motifs Research Articles

Who's Got the Calcium?

The human cardiac voltage-gated sodium channel (hH1) is essential for normal heart function, and mutations in the alpha subunit of the channel cause deadly arrhythmias. Some of these mutations are in a region that confers critical regulation of channel function by calcium, but exactly how calcium regulates the channel has been unclear. The hH1 protein contains an EF-hand calcium-binding motif that may bind calcium directly and an IQ-type calmodulin-binding motif that could confer calcium-dependent changes through changes in calmodulin. Shah et al. explored interactions between these domains to help clarify the roles of these direct and indirect responses to calcium in channel function. The authors characterized the binding properties of various channel domains and mutants by monitoring 15N-3H heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectra. The EF-hand domain alone appeared to have a very low affinity for calcium (0.6 mM) that would be unlikely to have a physiological role. However, interaction of the IQ motif with the EF-hand domain enhanced calcium-binding affinity into the micromolar range. Contrary to expectations, a mutation in the EF-hand domain that causes arrhythmias in humans didn't alter calcium affinity but rather reduced affinity of the channel for binding calmodulin. On the other hand, a mutation in the IQ calmodulin-binding motif had little effect on affinity for calmodulin but reduced calcium-binding affinity. Also unusual was the finding that calcium-loaded calmodulin actually had lower affinity for an IQ motif-containing peptide than did calcium-free calmodulin. The authors propose that the effects of calcium can be explained as follows: In basal conditions, calmodulin is bound to the channel through the IQ motif. Increased concentrations of intracellular calcium weaken this interaction such that calmodulin is released, enabling the IQ motif to interact with the EF-hand domain, thus favoring calcium-dependent modulation of the channel through direct calcium binding. Further understanding of the intricate nature of these calcium-modulated domain interactions may reveal new therapeutic strategies to manage cardiac arrhythmias and other diseases associated with altered function of other members of this channel family in other cell types. V. N. Shah, T. L. Wingo, K. L. Weiss, C. K. Williams, J. R. Balser, W. J. Chazin, Calcium-dependent regulation of the voltage-gated sodium channel hH1: Intrinsic and extrinsic sensors use a common molecular switch. Proc. Natl. Acad. Sci. U.S.A. 103 , 3592-3597 (2006). [Abstract] [Full Text]

Redistribution of STIM Proteins to the Peripheral Structures in Store-Operated Calcium Influx

Stimuli that trigger release of calcium from the endoplasmic reticulum (ER) through inositol trisphosphate (IP 3 ) receptors can deplete this intracellular calcium store and trigger store-operated calcium (SOC) influx from the plasma membrane. Liou et al . designed a siRNA screen against 2304 proteins selected on the basis of the presence of signaling domains in their primary sequence. These were tested to identify proteins that, when depleted, reduced the plateau phase of calcium influx after exposure of cells to histamine (which promotes calcium release through IP 3 receptors) in the presence of thapsigargin (to inhibit calcium reloading of the ER). siRNA for stromal interaction molecules 1 and 2 (STIM1 and STIM2), which have a single transmembrane domain and a single EF-hand calcium-binding motif, suppressed the sustained calcium signal without decreasing the initial transient calcium signal. siRNA for STIM1 or STIM2 reduced the calcium influx observed after extracellular calcium was added after stores were depleted in the absence of extracellular calcium (calcium add-back assay). Overexpression of a yellow fluorescent protein (YFP)-STIM1 resulted in increased opening of the SOC influx channel, based on a Mn 2+ quench assay, in which Mn 2+ entering through the SOC influx channel quenches the Fura-2 signal. YFP-STIM1 was localized to the ER, and store depletion caused redistribution of this signal to punctate structures near the cell periphery even in the absence of extracellular calcium, which suggests that store depletion (rather than subsequent calcium influx through the plasma membrane) may be the cause of the redistribution. Mutation of the EF-hand domain caused redistribution of YFP-STIM1 to the punctate structures in the absence of store depletion and promoted calcium influx in the absence of store depletion, and store depletion did not cause any additional influx. In cells transfected to express a wild-type cyan fluorescent protein (CFP)-STIM1 and the EF-hand YFP-STIM1, store depletion triggered redistribution of the CFP-STIM1 to the YFP-STIM1-positive punctate structures. Thus, it appears that SOC influx may involve the rapid redistribution of ER proteins to a punctate intracellular structure in close proximity to the membrane, which may mediate a direct coupling between proteins in these intracellular structures with the channels in the plasma membrane. J. Liou, M. L. Kim, W. D. Heo, J. T. Jones, J. W. Myers, J. E. Ferrell Jr., T. Meyer, STIM is a Ca 2+ sensor essential for Ca 2+ -store-depletion-triggered Ca 2+ influx. Current Biol. 15 , 1235-1241 (2005). [PubMed]

Solution structure and backbone dynamics of Calsensin, an invertebrate neuronal calcium‐binding protein

Calsensin is an EF-hand calcium-binding protein expressed by a subset of peripheral sensory neurons that fasciculate into a single tract in the leech central nervous system. Calsensin is a 9-kD protein with two EF-hand calcium-binding motifs. Using multidimensional NMR spectroscopy we have determined the solution structure and backbone dynamics of calcium-bound Calsensin. Calsensin consists of four helices forming a unicornate-type four-helix bundle. The residues in the third helix undergo slow conformational exchange indicating that the motion of this helix is associated with calciumbinding. The backbone dynamics of the protein as measured by (15)N relaxation rates and heteronuclear NOEs correlate well with the three-dimensional structure. Furthermore, comparison of the structure of Calsensin with other members of the EF-hand calcium-binding protein family provides insight into plausible mechanisms of calcium and target protein binding.

Chromosome 1 open reading frame 10 ( C1orf10) gene is frequently down-regulated and inhibits cell proliferation in oral squamous cell carcinoma

Chromosome 1 open reading frame 10 ( C1orf10) is a recently identified gene encoding a protein with an S100 EF-hand calcium-binding motif, and its expression is known to be down-regulated in esophageal squamous cell carcinoma. In this study, to determine whether the loss of C1orf10 gene function could contribute to the development of oral squamous cell carcinoma (OSCC), we have evaluated the expression status of this gene by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and quantitative real-time PCR analysis. A high frequency of decrease in C1orf10 gene was detected not only in OSCC-derived cell lines but also in tumor tissues. Next, to define biological function of this gene in oral carcinogenesis, we transfected Clorf10 with an Ecdysone-inducible system in OSCC cell lines and analyzed the effects of its overexpression. Induction of C1orf10 expression resulted in a significant decline in the rate of cell proliferation, and in an arrest in the G 1 phase of the cell cycle, with a down-regulation of Cyclin D1 expression. However, we could not detect significant difference in the percentage of apoptotic cells. Thus, our results suggest that the down-regulation of C1orf10 gene plays a role in oral carcinogenesis, and that its expression may negatively regulate OSCC cell proliferation by arresting the cell cycle.

αPIX Associates with Calpain 4, the Small Subunit of Calpain, and Has a Dual Role in Integrin-mediated Cell Spreading

Binding of integrins to the extracellular matrix results in actin cytoskeletal rearrangements, e.g. during cell spreading, by regulating the activity of Rho GTP-ases. We have shown previously that alphaPIX (Cool-2 or ARHGEF6), a Rac1/Cdc42-specific guanine nucleotide exchange factor (GEF), binds to beta-parvin/affixin and colocalizes with integrin-linked kinase in actively spreading cells, suggesting that alphaPIX is involved in integrin-induced signaling leading to activation of Rac1/Cdc42. Here we report calpain 4, the small subunit of the proteases mu-calpain and m-calpain, as a novel binding partner of alphaPIX. This association was identified by the CytoTrap system and confirmed by coimmunoprecipitation and glutathione S-transferase pull-down assays. The alphaPIX triple domain SH3-DH-PH was found to be required for calpain 4 binding. During integrin-dependent spreading of CHO-K1 cells, alphaPIX colocalized with mu- and m-calpain, integrin-linked kinase, and beta1 integrin in early integrin-containing clusters. Overexpression of alphaPIX wild type but not the GEF-deficient mutant (L386R/L387S) resulted in enhanced formation of characteristic cellular protrusions during cell spreading, suggesting that alphaPIX GEF activity is necessary for this specific actin cytoskeletal reorganization. The calpain inhibitors calpeptin and calpain inhibitor IV significantly inhibited integrin-dependent cell spreading. However, concomitant overexpression of alphaPIX wild type or the L386R/L387S mutant restored cell spreading. Together, these data suggest that alphaPIX is a component of early integrin clusters and plays a dual role in integrin-dependent cell spreading. Whereas alphaPIX GEF activity contributes to enhanced formation of cellular protrusions, the GEF-independent association with calpain 4 leads to induction of a yet unknown signaling cascade resulting in cell spreading.

Identification of RIZ1 Targets Involvedin Erythroid Differentiation of K562 Human Erythroleukemia Cells Using Surface-Enhanced Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (SELDI).

RIZ1 (PRDM2) is a member of the nuclear protein methyltransferase superfamily involved in chromatin remodeling. RIZ1 functions as a tumor suppressor gene in a number of human cancers and is down regulated in some human acute myeloid leukemias. We previously found RIZ-1 to be silenced in K562 erythroleukemia cells by promoter hypermethylation. Furthermore, expression of RIZ1 in K562 promotes erythroid differentiation and also potentiates TGF-β1 mediated differentiation. To investigate similarities between genes altered by RIZ1 expression and the TGF-β1 pathway, we used SELDI to compare the protein profiles of K562 against K562 + RIZ1 and K562 + TGF-β1. Protein extracts for SELDI profiling were separated into six fractions according to their isoelectric points. Proteins from each fraction were then bound to two different protein chip surfaces (H50-hydrophoboic and CM10-cation exhange) and their mass/charge determined using SELDI. We analyzed four replicates from each sample and classified proteins as differentially expressed if their P-values were below 0.05. In total, we observed 104 differentially expressed proteins (60 upregulated and 44 down regulated) between K562 and K562 + RIZ1 and 176 proteins (96 upregulated and 80 down regulated) between K562 and K562 + TGF-β1. We used 2D-PAGE to identify differentially expressed proteins identified by SELDI analysis and located 48 proteins that were over expressed in K562 + RIZ1 and K562 + TGF-β1 relative to K562. To establish whether these proteins were the same proteins observed using SELDI, we determined if the proteins had the same pI and molecular weight and if the gel-eluted proteins bound to the same protein chip surface with the same mass/charge. 15 of 48 proteins passed the above criteria and we determined their identities using Trypsin-based peptide mapping strategies with molecular weight and pI restrictions. We identified two candidate proteins (14-3-3ε and S100/A13) that are similarly over expressed in K562 + RIZ1 and K562 + TGF-β1. These proteins have been shown to be associated with TGF-β1 signaling. Schistosomal 14-3-3ε interacts with SmRK1, a divergent type I transforming growth factor β1 receptor (TR-I) present on the surface of adult parasites and also binds to and activates human TR-I. S100/A13 belongs to a family of low molecular weight proteins characterized by the presence of two calcium-binding EF-hand motifs that includes S100C/A11, a member recently shown to play a key role in a PKCα mediated pathway essential for the growth inhibition of normal human keratinocytes by TGF-β1. In summary, we demonstrate the potential for using SELDI to identify novel proteins involved in regulating and connecting cellular growth and differentiation pathways.

Yeast Miro GTPase, Gem1p, regulates mitochondrial morphology via a novel pathway.

Cell signaling events elicit changes in mitochondrial shape and activity. However, few mitochondrial proteins that interact with signaling pathways have been identified. Candidates include the conserved mitochondrial Rho (Miro) family of proteins, which contain two GTPase domains flanking a pair of calcium-binding EF-hand motifs. We show that Gem1p (yeast Miro; encoded by YAL048C) is a tail-anchored outer mitochondrial membrane protein. Cells lacking Gem1p contain collapsed, globular, or grape-like mitochondria. We demonstrate that Gem1p is not an essential component of characterized pathways that regulate mitochondrial dynamics. Genetic studies indicate both GTPase domains and EF-hand motifs, which are exposed to the cytoplasm, are required for Gem1p function. Although overexpression of a mutant human Miro protein caused increased apoptotic activity in cultured cells (Fransson et al., 2003. J. Biol. Chem. 278:6495–6502), Gem1p is not required for pheromone-induced yeast cell death. Thus, Gem1p defines a novel mitochondrial morphology pathway which may integrate cell signaling events with mitochondrial dynamics.

Molecular cloning, characterization, tissue-specific and phytohormone-induced expression of calcium-dependent protein kinase gene in cucumber (Cucumis sativus L.)

A cucumber cDNA designated CsCPK5 and encoding a calcium-dependent protein kinase (CsCDPK5) was isolated and characterized. An open reading frame of 1542 bp was detected that could encode a protein of 514 amino acid residues with a calculated molecular mass of 56.5kDa. Comparison of the deduced amino acid sequence of CsCDPK5 with sequences of other CDPKs revealed the highest similarity (85%) to AtCDPK6. As described for other CDPKs, CsCDPK5 has a long variable domain preceding a catalytic domain, an autoinhibitory function domain, and a C-terminal calmodulin-domain containing 4 EF-hand calcium-binding motifs. The N-terminal long variable domain of CsCDPK5 does not contain the N-myristoylation motif, which is found in many CDPKs. The relative expression level of the CsCPK genes in various organs of cucumber plants and seedlings and in etiolated, excised cotyledons and hypocotyls following treatments with light and/or benzyladenine (BA), abscisic acid (ABA), gibberellic acid (GA) or indole acetic acid (IAA) was determined by northern analysis using the CsCPK5 cDNA probe. The CsCPK transcripts are most abundant in cucumber plant Leaves with less accumulation in cucumber seedling roots and hypocotyls and lowest Levels in cucumber plant flowers and seedling hooks and cotyledons. All phytohormones tested enhanced the accumulation of the transcripts 2-3-fold in etiolated cotyledons. On the other hand, levels of the transcripts increased to a lesser extent in both light and BA- or IAA-treated cotyledons and no effect was noted in response to light treatment with GA. In hypocotyls, no major changes in the relative levels of CsCPK transcripts were observed in the phytohormone-treated etiolated and light-exposed tissues, except an up-regulatory effect with IAA treatment in the etiolated and IAA, ABA and GA treatments in light-exposed hypocotyls. These observations suggest that exogenous phytohormones can up-regulate the CsCPK transcript levels in tissue-specific, and light-dependent and independent manners.

S100 proteins in the epidermis.

The S100 proteins comprise a family of 21 low molecular weight (9-13 kDa) proteins that are characterized by the presence of two calcium-binding EF-hand motifs. Fourteen S100 protein genes are located within the epidermal differentiation complex on human chromosome 1q21 and 13 S100 proteins (S100A2, S100A3, S100A4, S100A6, S100A7, S100A8, S100A9, S100A10, S100A11, S100A12, S100A15, S100B, and S100P) are expressed in normal and/or diseased epidermis. S100 proteins exist in cells as anti-parallel hetero- and homodimers and upon calcium binding interact with target proteins to regulate cell function. S100 proteins are of interest as mediators of calcium-associated signal transduction and undergo changes in subcellular distribution in response to extracellular stimuli. They also function as chemotactic agents and may play a role in the pathogenesis of epidermal disease, as selected S100 proteins are markedly overexpressed in psoriasis, wound healing, skin cancer, inflammation, cellular stress, and other epidermal states.

PINOID-mediated signaling involves calcium-binding proteins.

The plant hormone auxin is a central regulator of plant development. In Arabidopsis, the PINOID (PID) protein serine/threonine kinase is a key component in the signaling of this phytohormone. To further investigate the biological function of PID, we performed a screen for PID-interacting proteins using the yeast two-hybrid system. Here, we show that PID interacts with two calcium-binding proteins: TOUCH3 (TCH3), a calmodulin-related protein, and PID-BINDING PROTEIN 1 (PBP1), a previously uncharacterized protein containing putative EF-hand calcium-binding motifs. The interaction between PID and the calcium-binding proteins is significant because it is calcium dependent and requires an intact PID protein. Furthermore, the expression of all three genes (PID, TCH3, and PBP1) is up-regulated by auxin. TCH3 and PBP1 are not targets for phosphorylation by PID, suggesting that these proteins act upstream of PID. PBP1 was found to stimulate the autophosphorylation activity of PID, and calcium influx and calmodulin inhibitors where found to enhance the activity of PID in vivo. Our results indicate that TCH3 and PBP1 interact with the PID protein kinase and regulate the activity of this protein in response to changes in calcium levels. This work provides the first molecular evidence for the involvement of calcium in auxin-regulated plant development.

Molecular characterization of a cDNA encoding putative calcium binding protein, HvCaBP1, induced during kernel development in barley (Hordeum vulgare L.).

Seed development is known to involve complex physiological and molecular events. In order to gain information on the molecular events that occur in the grains of barley during kernel development, we conducted suppression subtractive hybridization (SSH) using grains of barley cv. Karl at 14 days after fertilization (DAF) as the tester and grains at 5 DAF as the driver. We isolated an SSH clone that showed homology with a specific calcium binding protein in rice called EFA27. Screening the cDNA library, we identified two clones as a calcium binding protein. These clones, each carrying one calcium-binding EF-hand motif, were designated HvCaBP1 (Hordeum vulgare Calcium Binding Protein 1). HvCaBP1 possesses an N-terminal region with a conserved single Ca(2+)-binding EF-hand motif and one transmembrane helix. Northern hybridization showed that the highest expression occurred in grains and that expression increased in kernels at 8 DAF. As shown in situ hybridization, the HvCaBP1 gene was highly expressed in the embryo and tissues of the endosperm near the embryo and was detected in the vascular tissues of the glume in the kernel at 8 DAF. Accumulation of HvCaBP1 mRNAs subsequently increased in vegetative tissues for up to 48 h after abscisic acid (ABA) treatment. Transcripts of HvCaBP1 mRNAs may be regulated by endogenous ABA in the grains during kernel development.

Atypical Rho GTPases Have Roles in Mitochondrial Homeostasis and Apoptosis

The human genomic sequencing effort has revealed the presence of a large number of Rho GTPases encoded by the human genome. Here we report the characterization of a new family of Rho GTPases with atypical features. These proteins, which were called Miro-1 and Miro-2 (for mitochondrial Rho), have tandem GTP-binding domains separated by a linker region containing putative calcium-binding EF hand motifs. Genes encoding Miro-like proteins were found in several eukaryotic organisms from Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster to mammals, indicating that these genes evolved early during evolution. Immunolocalization experiments, in which transfected NIH3T3 and COS 7 cells were stained for ectopically expressed Miro as well as for the endogenous Miro-1 protein, showed that Miro was present in mitochondria. Interestingly, overexpression of a constitutively active mutant of Miro-1 (Miro-1/Val-13) induced an aggregation of the mitochondrial network and resulted in an increased apoptotic rate of the cells expressing activated Miro-1. These data indicate a novel role for Rho-like GTPases in mitochondrial homeostasis and apoptosis.

The bacterial scaffoldin: structure, function and potential applications in the nanosciences.

Natural protein complexes may provide the best templates for nanometer-scale technology and new biomaterials. The bacterial cellulosome is becoming a well-understood multi-protein complex found in cellulolytic microorganisms. The scaffoldin subunits of the bacterial cellulosome function to organize and position other protein subunits into the complex. The scaffoldins can also serve as an attachment device for harnessing the cellulosome onto the cell surface and/or for its targeting to substrate. Biochemical and molecular biological evidence have identified a receptor/adaptor type of protein domain pair, called "cohesin and dockerin," which is responsible for cellulosome self-assembly. The recognition between cohesin and dockerin is generally type and/or species specific. More than 80 cohesin and 100 dockerin sequences have been found, mostly from anaerobic bacteria. X-ray crystallography and NMR have been used to determine the three-dimensional structures of representative cohesin and dockerin domains, respectively. The cohesin peptide is about 140 amino acids in length and highly conserved in sequence and domain structure. The dockerin domain comprises about 70 amino acids and contains two 22 amino acid duplicated regions, each of which includes an "F-hand" modification of the EF-hand calcium-binding motif. Biochemical evidence and site-directed mutagenesis have confirmed that the two F-hand motifs are required for function and calcium dependence; at least two amino acids from each motif are critical for cohesin-dockerin recognition. In this report, we review the structure and function of the scaffoldin of the bacterial cellulosome and emphasize a detailed sequence analysis of the cohesin and dockerin domains. We also speculate about potential applications in nanoscience that may be based on cohesin-dockerin recognition.

Zinc-dependent Interaction between Dishevelled and the Drosophila Wnt Antagonist Naked Cuticle

During Drosophila development, the naked cuticle (nkd) gene attenuates wingless/Wnt signaling through a negative feedback loop mechanism. Fly and vertebrate Nkd proteins contain a putative calcium-binding EF-hand motif, the EFX domain, that interacts with the basic/PDZ region of the Wnt signal transducer, dishevelled (Dsh). Here we show that Dsh binding by Drosophila Nkd in vitro is mediated by the EFX domain as well as an adjacent C-terminal sequence. In vivo data suggest that both of these regions contribute to the ability of Nkd to antagonize Wnt signaling. Mutations in the Nkd EF-hand designed to eliminate potential ion binding affected Nkd-Dsh interactions in the yeast two-hybrid assay but not in the glutathione S-transferase pull-down assay. Addition of the chelating agent EDTA abolished the in vitro Nkd-Dsh interaction. Surprisingly zinc, but not calcium, was able to restore Nkd-Dsh binding, suggesting a zinc-mediated interaction. Calcium 45- and zinc 65-blotting experiments show that Nkd is a zinc-binding metalloprotein. The results further clarify how Nkd may antagonize Wnt signaling via interaction with Dsh, and identify a novel zinc-binding domain in Drosophila Nkd that collaborates with the conserved EFX domain to bind Dsh.

Characterization of a strawberry cDNA clone homologous to calcium-dependent protein kinases that is expressed during fruit ripening and affected by low temperature.

A full-length cDNA clone (FaCDPK1) encoding a calcium-dependent protein kinase (CDPK) has been isolated from a strawberry fruit cDNA library. FaCDPK1 contains the basic features of CDPKs: a catalytic kinase domain linked to a regulatory calmodulin-like domain by a junction sequence that has been shown to act as an autoinhibitory pseudosubstrate. Although the calmodulin-like domain of CDPKs typically contains four EF-hand calcium-binding motifs, FaCDPK1 was predicted to contain only three EF-hand motifs. FaCDPK1 gene expression was observed in roots, stolons, meristems, flowers, and leaves. FaCDPK1 mRNA was not detected in young fruits, but accumulated as fruit turned to white, suggesting a role for this gene in the developing strawberry fruit. In ripe fruit the levels of transcript increased in response to low temperature.

The Calcium Sensor Protein Visinin-like Protein-1 Modulates the Surface Expression and Agonist Sensitivity of the α4β2 Nicotinic Acetylcholine Receptor

The calcium sensor protein visinin-like protein-1 (VILIP-1) was isolated from a brain cDNA yeast two-hybrid library using the large cytoplasmic domain of the alpha4 subunit as a bait. VILIP-1 is a myristoylated calcium sensor protein that contains three functional calcium binding EF-hand motifs. The alpha4 subunit residues 302-339 were found to be essential for the interaction with VILIP-1. VILIP-1 coimmunopurified with detergent-solubilized recombinant alpha4beta2 acetylcholine receptors (AChRs) expressed in tsA201 cells and with native alpha4 AChRs isolated from brain. Coexpression of VILIP-1 with recombinant alpha4beta2 AChRs up-regulated their surface expression levels approximately 2-fold and increased their agonist sensitivity to acetylcholine approximately 3-fold. The modulation of the recombinant alpha4beta2 AChRs by VILIP-1 was attenuated in VILIP-1 mutants that lacked the ability to be myristoylated or to bind calcium. Collectively, these results suggest that VILIP-1 represents a novel modulator of alpha4beta2 AChRs that increases their surface expression levels and agonist sensitivity in response to changes in the intracellular levels of calcium.

Characterization of Mouse Profilaggrin: Evidence for Nuclear Engulfment and Translocation of the Profilaggrin B-Domain during Epidermal Differentiation

Filaggrin is a keratin filament associated protein that is expressed in granular layer keratinocytes and derived by sequential proteolysis from a polyprotein precursor termed profilaggrin. Depending on the species, each profilaggrin molecule contains between 10 and 20 filaggrin subunits organized as tandem repeats with a calcium-binding domain at the N- terminal end. We now report the characterization of the complete mouse gene. The structural organization of the mouse gene is identical to the human profilaggrin gene and consists of three exons with a 4 kb intron within the 5' noncoding region and a 1.7 kb intron separating the sequences encoding the calcium-binding EF-hand motifs. A processed pseudogene was found embedded within the second intron. The third and largest exon encodes the second EF-hand, a basic domain (designated the B-domain) followed by 12 filaggrin repeats and a unique C-terminal tail domain. A polyclonal antibody raised against the conceptually translated sequence of the B-domain specifically stained keratohyalin granules and colocalized with a filaggrin antibody in granular layer cells. In upper granular layer cells, B-domain containing keratohyalin granules were in close apposition to the nucleus and, in some cells, appeared to be completely engulfed by the nucleus. In transition layer cells, B-domain staining was evident in the nucleus whereas filaggrin staining remained cytoplasmic. Nuclear staining of the B-domain was also observed in primary mouse keratinocytes induced to differentiate. This study has also revealed significant sequence homology between the mouse and human promoter sequences and in the calcium-binding domain but the remainder of the protein-coding region shows substantial divergence.

ATP selectively suppresses the synthesis of the inflammatory protein microglial response factor (MRF)-1 through Ca 2+ influx via P2X 7 receptors in cultured microglia

Microglia are known to express purinergic receptors for extracellular ATP of both P2Y and P2X subtypes. In the CNS, ATP is released from neurons and acts as a signal between neurons and glia. The mrf-1 gene encodes a 17-kDa protein with a single calcium-binding (EF-hand) motif and is expressed specifically in microglia. The gene was isolated from activated microglia in response to apoptosis of cerebellar granule neurons in culture and is upregulated in response to neuronal death and degeneration in vivo. We have found that ATP suppresses the synthesis of the inflammation-related protein MRF-1 in cultured rat microglia. When microglia were treated with ATP (1 mM) for 6 h, mrf-1 mRNA levels decreased to approximately 50% compared to those in the control. This effect was dependent on both the treatment period and the dose of ATP. After ATP (1 mM) treatment for 16 h, levels of mrf-1 mRNA decreased to 37.3% and MRF-1 levels decreased to 55.0% compared to those in the control. A decrease in MRF-1 or its mRNA was also induced by benzoylbenzoyl-ATP (0.1 mM), a P2X 7 receptor-selective agonist, and by the Ca 2+ ionophore A23187 (2 μM), dependent on extracellular Ca 2+. Moreover, ATP modified neither the MRF-1 degradation rate nor total protein synthesis. These results indicate that ATP selectively suppresses MRF-1 synthesis at the transcription level via Ca 2+ influx through P2X 7 receptors.

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.

Isolation of a cDNA encoding a putative SPARC from the brine shrimp, Artemiafranciscana

SPARC (Secreted protein, acidic, rich in cysteine) is an extracellular matrix-associated and anti-adhesive glycoprotein extensively studied in vertebrates. Its presence among invertebrates has been reported in nematodes and flies. We cloned a cDNA containing a complete open reading frame for SPARC from the brine shrimp, Artemiafranciscana. The amino acid sequence identity between the Artemia and the fly SPARCs was 55%, whereas that of the Artemia and the nematode proteins was 45%. Artemia and vertebrates exhibited a sequence identity of 30% in the predicted aa sequences. The SPARC consisted of four domains commonly found among reported SPARCs. The protein comprised 291 amino acids, having a signal peptide, a follistatin-like domain, one N-glycosylation site and one calcium-binding EF-hand motif. Fourteen cysteine residues conserved among all the secreted forms of SPARCs were present in the Artemia SPARC, and four extra cysteine residues were also found in it. The extra residues were conserved among SPARCs of the arthropods and the nematode. Phylogenetic analyses showed that the sequences of SPARCs were grouped into those of vertebrates and invertebrates. Though the structural organization of SPARC was conserved among all the species studied, SPARC within a group was highly conserved within that group, but divergent between the two. Northern blots revealed the presence of a 1.1 kb mRNA, which was faintly expressed in embryos and considerably detected in prenauplii and nauplii. The isolation of a SPARC cDNA from Artemiafranciscana provides intriguing features of the divergent protein, SPARC.