A novel 15 kDa Ca2+-binding protein present in the eggs of the sea urchin, Hemicentrotus pulcherrimus

A novel Ca2+-binding protein, different from calmodulin, has been purified to homogeneity from the soluble cytoplasmic protein fraction of the egg of the sea urchin, Hemicentrotus pulcherrimus. This protein, designated as 15 kDa protein, shows a Ca2+-dependent mobility shift upon SDS-gel electrophoresis and has Ca2+-binding ability. This protein did not resemble the sea urchin egg calmodulin in either molecular mass or amino acid composition. The 15 kDa protein could not activate cyclic adenosine 3',5'-monophosphate-dependent phosphodiesterase from bovine brain and did not bind to fluphenazine-Sepharose 6B. Antibodies against the 15 kDa protein did not react with sea urchin egg calmodulin. These results suggest that the 15 kDa protein is a novel Ca2+-binding protein in the sea urchin egg.     

p85 binds to G-actin in a Ca(2+)/calmodulin-dependent manner, thus regulating the initiation of cytokinesis in tetrahymena

Tetrahymena p85 is localized to the presumptive division plane before the formation of contractile ring microfilaments. p85 binds to calmodulin in a Ca(2+)-dependent manner and both proteins colocalize to the division furrow. Inhibition of the binding of p85 and Ca(2+)/calmodulin prevents both the localization of p85 and calmodulin to the division plane and the formation of the contractile ring, suggesting that the interaction of p85 and Ca(2+)/calmodulin is important in the formation of the contractile ring. We investigated the mechanisms of the formation of contractile ring, and the relationship among p85, CaM, and actin using co-sedimentation assay: p85 binds to G-actin in a Ca(2+)/calmodulin-dependent manner, but does not bind to F-actin. Therefore, we propose that a Ca(2+)/calmodulin signal and its target protein p85 are cooperatively involved in the recruitment of G-actin to the division plane and the formation of the contractile ring.     

The effect of calmodulin on the phosphoprotein intermediate of Mg2+-dependent Ca2+-stimulated adenosine triphosphatase in human erythrocyte membranes.

The effect of calmodulin on the formation and decomposition of the Ca2+-dependent phosphoprotein intermediate of the (Mg2+ + Ca2+)-dependent ATPase in erythrocyte membranes was investigated. In the presence of 60 microM-Ca2+ and 25 microM-MgCl2, calmodulin (0.5-1.5 microgram) did not alter the steady-state concentration of the phosphoprotein, but increased its rate of decomposition. Higher calmodulin concentrations significantly decreased the steady-state concentration of phosphoprotein. Calmodulin (0.5-1.7 microgram) increased Ca2+-transport ATPase activity by increasing the turnover rate of its phosphoprotein intermediate. Increasing the MgCl2 concentration from 25 microM to 250 microM increased the (Mg2+ + Ca2+)-dependent ATPase activity, but decreased the concentration of the phosphoprotein intermediate. Similarly to calmodulin, MgCl2 increased the turnover rate of the Ca2+-transport ATPase complex (about 3-fold). At the higher MgCl2 concentration calmodulin did not further affect the decomposition of the phosphoprotein intermediate. It was concluded that both calmodulin and MgCl2 increase the turnover of the Ca2+-pump by enhancing the decomposition of the Ca2+-dependent phosphoprotein intermediate.     

Purification and characterization of the Ca2+-ATPase of plasma membranes from Ehrlich ascites mammary carcinoma cells

Ca2+-ATPase was isolated from plasma membranes of Ehrlich ascites mammary carcinoma cells by means of calmodulin affinity chromatography. The purification procedure included removal of endogenous calmodulin from a Triton X-100 solubilizate of the membranes by DEAE ion-exchange chromatography as an essential step. With respect to its molecular mass, activation by calmodulin, Ca2+-dependent phosphorylation and highly sensitive inhibition by orthovanadate, the purified enzyme resembles the Ca2+-ATPase of erythrocyte membranes. In contrast to the strong calmodulin dependence of the isolated enzyme the Ca2+-ATPase in native Ehrlich ascites carcinoma cell membranes cannot be remarkably stimulated by added calmodulin. It is suggested that the membrane-bound Ca2+-ATPase in the presence of Ca2+ is activated by interaction with endogenously bound calmodulin.     

Isolation and characterization of calmodulin from an insulin-secreting tumour.

A major protein constituent of a rat islet cell tumour that exhibited Ca2+-dependent changes in electrophoretic mobility has been purified to homogeneity and compared in its physicochemical and biological properties with bovine brain and rat brain calmodulin (synonymous with phosphodiesterase activator protein, calcium-dependent regulator, troponin C-like protein and modulator protein). The protein, like these calmodulins, contained trimethyl-lysine, exhibited a blocked N-terminus and had an identical amino-acid composition and molecular weight on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Peptide "maps' prepared after digestion of the three proteins with trypsin, papain or Staphylococcus V-8 proteinase were virtually superimposable. Ca2+ altered the electrophoretic mobilities the enhanced the native protein fluorescence in an equivalent manner with all three proteins. Equilibrium dialysis experiments demonstrated in each case the binding of 4g-atoms of calcium/mol of protein; the binding sites were equivalent and showed Kd 0.8 microM. Tumour and brain proteins were equipotent as Ca2+-dependent activators of partially purified rat brain cyclic nucleotide phosphodiesterase, and in this action were inhibited in an identical manner by trifluoperazine. The proteins also exhibited the common property of Ca2+-dependent binding to troponin I, histone H2B and myelin basic protein. The estimated tumour content of calmodulin was 450 mg/kg fresh wt., a value similar to that reported in islets of Langerhans. These results further document the validity of the islet cell tumour as an experimental model of Ca2+-mediated molecular events associated with insulin secretion. They also suggest that brain calmodulin may be substituted for endogenous calmodulin in experimental investigations into the mechanism of insulin secretion.     

Calmodulin in mouse male germ cells: a qualitative and quantitative study

Isolated male germ cells of the mouse possess a heat-stable stimulatory activity of Ca2+-dependent, calmodulin-free phosphodiesterase. Ionic exchange chromatography allowed partial purification of the activator and the isolation of multiple forms of phosphodiesterase stimulation inhibitor. The activator has been identified as calmodulin on the basis of chromatographic behaviour and electrophoretic mobility. Quantitative analysis showed variations of calmodulin levels at different stages of spermatogenesis. Quantitative analysis of cyclic nucleotide hydrolysis in germ cell cytosol showed that the activity of Ca2+-dependent phosphodiesterase is different in meiotic and post-meiotic mouse male germ cells. These data suggest that calcium-dependent pathway and a Ca2+-dependent regulation of cyclic nucleotides are present in developing germ cells.     

The role of calmodulin on Ca2+ -dependent K+ transport regulation in the human red cell

Several lipophilic calmodulin antagonists (phenotiazines, butyrophenones and diphenylbutylpiperidines) inhibited Ca2+-induced loss of KC1 from human red cells. However, the Ki values for this effect did not bear good correlation with the Ki values reported for well-known calmodulin-dependent systems. In addition, the inhibition was strongly dependent on the haematocrit and valinomycin-induced KC1 fluxes were also affected. Added calmodulin did not have any effect on Ca2+-dependent 86Rb uptake by inside-out vesicles derived from red cell membranes whereas stimulation of Ca2+-dependent ATPase was apparent. Lipophilic anticalmodulins at high doses had all kinds of effects on 86Rb uptake by inside-out vesicles: increase, decrease or no change of the fraction of activated vesicles reached at submaximal Ca2+ concentrations, with or without modification of the relative rate of 86Rb uptake. The hydrophylic compound 48/80 decreased the fraction of activated vesicles reached at submaximal Ca2+ concentrations without affecting the relative rate of 86Rb uptake, but this effect took place only at concentrations 10-fold higher than the reported Ki for calmodulin-dependent systems. These results suggest that Ca2+-dependent K+ channels of red cells are not regulated by calmodulin.     

Cell cycle-associated changes of guanylate cyclase activity in synchronized Tetrahymena: a possible involvement of calmodulin in its regulation

Guanylate cyclase activity decreased during the division phase of heat-shock synchronized Tetrahymena pyriformis, strain GL. However, when Ca2+ was removed by EGTA to negate the effects of the Ca2+-binding protein (calmodulin), which is required for the full activity of guanylate cyclase in this organism, no significant change in the enzymatic activity was observed throughout the cell cycle. On the other hand, the reduced guanylate cyclase activity at division phase was associated with a decreased level of calmodulin content. These results suggest that fluctuations in guanylate cyclase activity during the cell cycle would be dependent on the concentration of calmodulin.     

Monitoring the total available calmodulin concentration in intact cells over the physiological range in free Ca2+

We describe the design, characterization and application of a new genetically encoded fluorescent biosensor for intracellular detection of both free Ca(2+)-calmodulin and apocalmodulin, which together comprise the available calmodulin concentration. The biosensor binds both forms of calmodulin with an apparent Kd value of 3 microM, and has kinetic properties making it suitable for monitoring dynamic changes on a subsecond time scale. It can be used in conjunction with the fluorescent Ca(2+)-indicator, indo-1, allowing the available calmodulin and free Ca2+ concentrations to be monitored concurrently. We have determined an intracellular available calmodulin concentration of 8.8 +/- 2.2 microM under resting conditions in a human kidney cell line stably expressing the biosensor. Elevation of the intracellular free Ca2+ concentration by agonist, store-operated Ca(2+)-entry or ionophore results in Ca(2+)-dependent consumption of the available calmodulin. A plot of normalized values for the available calmodulin concentration versus the free Ca2+ concentration fits a consumption curve with a cooperativity coefficient of 1.8 and a [Ca2+]50 of 850 nM. There is no detectible binding of calmodulin to the biosensor above a free Ca2+ concentration of approximately 4 microM, consistent with an available calmodulin concentration < or = 200 nM under these conditions, and an overall excess of calmodulin-binding sites.     

A high affinity, calmodulin-responsive (Ca2+ + Mg2+)-ATPase in isolated bone cells

Although acute alterations in Ca2+ fluxes may mediate the skeletal responses to certain humoral agents, the processes subserving those fluxes are not well understood. We have sought evidence for Ca2+-dependent ATPase activity in isolated osteoblast-like cells maintained in primary culture. Two Ca2+-dependent ATPase components were found in a plasma membrane fraction: a high affinity component (half-saturation constant for Ca2+ of 280 nM, Vmax of 13.5 nmol/mg per min) and a low affinity component, which was in reality a divalent cation ATPase, since Mg2+ could replace Ca2+ without loss of activity. The high affinity component exhibited a pH optimum of 7.2 and required Mg2+ for full activity. It was unaffected by potassium or sodium chloride, ouabain or sodium azide, but was inhibited by lanthanum and by the calmodulin antagonist trifluoperazine. This component was prevalent in a subcellular fraction which was also enriched in 5'-nucleotidase and adenylate cyclase activities, suggesting the plasma membrane as its principal location. Osteosarcoma cells, known to resemble osteoblasts in their biological characteristics and responses to bone-seeking hormones, contained similar ATPase activities. Inclusion of purified calmodulin in the assay system caused small non-reproducible increases in the Ca2+-dependent ATPase activity of EGTA-washed membranes. Marked, consistent calmodulin stimulation was demonstrated in membranes exposed previously to trifluoperazine and then washed in trifluoperazine-free buffer. These results indicate the presence of a high affinity, calmodulin-sensitive Ca2+-dependent ATPase in osteoblast-like bone cells. As one determinant of Ca2+ fluxes in bone cells, this enzyme may participate in the hormonal regulation of bone cell function.     

Identification of a Ca2+/calmodulin-binding domain within the carboxyl-terminus of the angiotensin II (AT1A) receptor.

To identify regulators of the type 1A angiotensin II receptor (AT1A), we investigated the interaction of cellular proteins with a fusion protein containing the rat AT1A receptor carboxyl-terminus. An approximately 20 kDa cytoplasmic protein interacted with the fusion protein in a Ca2+-dependent manner and was identified as calmodulin. A control peptide with high affinity for Ca2+/calmodulin and a peptide corresponding to a membrane proximal portion of the AT1A receptor carboxyl-terminus with analogy to known calmodulin-binding sequences were synthesised and tested for calmodulin-binding. Using in vitro binding assays combined with gel shift analysis, we demonstrated the formation of complexes between calmodulin and both peptides, which were Ca2+-dependent and of 1:1 stoichiometry. Affinity gels produced from these peptides also purified calmodulin from cell extracts. These results suggest a novel feedback regulation of the AT1A receptor by Ca2+/calmodulin and identify the membrane proximal region of the carboxyl-terminus as a focal point for interactions important for AT1A receptor function.     

The effects of several ion channel blockers and calmodulin antagonists on fertilization-induced acid release and 45Ca2+ uptake in sea urchin eggs

The effects of calcium antagonists, diltiazem and verapamil, and calmodulin antagonists, chlorpromazine, N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), were tested on two responses of the sea urchin egg to insemination: (1) H+ release; (2) Ca2+ uptake. It was found that calcium antagonists inhibited both processes, while calmodulin antagonists only inhibited H+ release but not Ca2+ uptake. Verapamil and diltiazem were effective to inhibit H+ release when added to the egg suspension up to 120 sec and W-7 was effective around 150 sec after insemination. Calcium antagonists became ineffective earlier than W-7 in inhibiting H+ release. A calmodulin-dependent step may thus occur linking the Ca2+ uptake and H+ release. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an anion channel blocker, also inhibited both Ca2+ uptake and H+ release. This result suggests that an uptake of anion(s) occurs along with Ca2+ uptake.     

Ca2+/calmodulin stimulates GTP binding to the ras-related protein ral-A.

Ral-A is a Ras-related GTP-binding protein that has been suggested to be the downstream target of Ras proteins and is involved in the tyrosine kinase-mediated, Ras-dependent activation of phospholipase D. We reported recently that Ral-A purified from human erythrocyte membrane binds to calmodulin in a Ca2+-dependent manner at a calmodulin binding domain identified near its C-terminal region (Wang, K. L., Khan, M. T., and Roufogalis, B. D. (1997) J. Biol. Chem. 272, 16002-16009). In this study we show the enhancement of GTP binding to Ral-A by Ca2+/calmodulin. The stimulation up to 3-fold by calmodulin was Ca2+-dependent, with half-maximum activation occurring at 180 nM calmodulin and 80 nM free Ca2+ concentration. The present work supports a regulatory role of Ca2+/calmodulin for the activation of Ral-A and suggests a possible direct link between signal transduction pathways of Ca2+/calmodulin and Ral-A proteins.     

The role of calcium in DNA synthesis and development of mouse preimplantation embryos in vitro.

The effect of calcium upon embryonic growth was studied using cultured mouse preimplantation embryos. Both morphological development of the embryos and embryo DNA synthesis were shown to be dependent on the Ca2+ concentration in the medium in which the embryos were grown. Reduction of the calcium concentration below 10(-5) M completely blocked cell division and blastocyst formation in the cultured embryos, but only moderately inhibited embryo DNA synthesis. Trifluoperazine, a calmodulin antagonist, strongly inhibited the Ca(2+)-dependent DNA synthesis in the embryos. On the other hand, the drug only slightly affected the morphological development of the embryos. These results demonstrate that calcium independently affects two different aspects of the embryo development, i.e. DNA synthesis and cell division. It is suggested that the former effect is calmodulin-dependent, while the latter involves the calcium-dependence of metabolite transport through the cell membranes.     

Biochemical characterization of the inhibitory effect of CsA on cytolytic T lymphocyte effector functions

We have examined the effects of cyclosporine A (CsA) on a number of CTL effector functions. CsA partially inhibited the CTL-mediated lysis of Ag-bearing target cells. Both target cell- and anti-TCR mAb-induced granule exocytosis were markedly inhibited by CsA. In addition, marked inhibition of PMA and calcium ionophore (A23187) induced granule exocytosis was produced by CsA suggesting that the inhibitory effects of CsA on granule exocytosis involve biochemical events after protein kinase C activation and increases in intracellular free Ca2+. CsA had no inhibitory effects on TCR-mediated phosphatidylinositol metabolism. The inhibitory effects of CsA were not mediated by the cAMP-dependent protein kinase inhibitory pathway and no effect of CsA on the Ca2+-induced binding of calmodulin to calmodulin-binding proteins could be demonstrated. CsA was also a potent inhibitor of IgE receptor-mediated exocytosis in rat basophil leukemia cells. CsA had no effect on receptor-mediated phosphatidylinositol hydrolysis; 400 ng/ml CsA resulted in a 90% inhibition of serotonin release but had no effect on phosphatidylinositol hydrolysis. These results indicate that CsA may inhibit some common event in Ca2+-dependent secretory cells. Taken together, these results suggest that CsA does not inhibit signal transduction but rather interferes with the biochemical events in the later stages of Ca2+-dependent reactions that follow the binding of calmodulin to cytoskeletal or cytoplasmic calmodulin binding proteins.     

Calmodulin-dependent regulation of a lipid binding domain in the v-SNARE synaptobrevin and its role in vesicular fusion

Trans SNARE complex assembly is an essential step in Ca2+-dependent membrane fusion, although the SNARE proteins do not bind Ca2+ ions. Studies to evaluate how the Ca2+sensor protein calmodulin might regulate this process led to the identification of a consensus calmodulin binding motif in the v-SNARE VAMP2. This sequence (residues 77-90) is situated precisely C-terminal to the tetanus toxin (TeNT) and botulinum B toxin cleavage site (76Q-F77) close to the transmembrane anchor. The same domain also binds acidic phospholipids and Ca2+/calmodulin or lipid binding are mutually exclusive. Directed mutagenesis of basic or hydrophobic residues within this motif reduced interactions with both Ca2+/calmodulin and phospholipids to a similar extent. The effects of these mutations on Ca2+-dependent exocytosis was explored using an hGH release assay in permeabilized pheochromocytoma PC12 cells. Treatment of cells with tetanus toxin (TeNT), which cleaves endogenous VAMP, abolished secretion. Secretion could be re-established by transfecting TeNT-resistant VAMP with mutations (Q76V,F77W) in the cleavage site. However rescue of exocytosis was abolished when additional mutations (K83A,K87V or W89A,W90A) were introduced that inhibited calmodulin and phospholipid binding to VAMP. Thus calmodulin and/or phospholipid binding to the membrane proximal region of VAMP is required for Ca2+-dependent exocytosis. We speculate that interactions between cis phospholipids at the vesicle surface and the membrane proximal region of VAMP inhibits SNARE complex assembly. Displacement of these interactions by Ca2+/calmodulin may promote SNARE complex assembly and lead to trans interactions between the membrane proximal region of VAMP and phospholipids in the plasma membrane.     

Ca2(+)-dependent regulation of the spectrin/actin interaction by calmodulin and protein 4.1

The Ca2(+)-dependent regulation of the erythroid membrane cytoskeleton was investigated. The low-salt extract of erythroid membranes, which is mainly composed of spectrin, protein 4.1, and actin, confers a Ca2+ sensitivity on its interaction with F-actin. This Ca2+ sensitivity is fortified by calmodulin and antagonized by trifluoperazine, a potent calmodulin inhibitor. Additionally, calmodulin is detected in the low-salt extract. These results suggest that calmodulin is the sole Ca2(+)-sensitive factor in the low-salt extract. The main target of calmodulin in the erythroid membrane cytoskeleton was further examined. Under native conditions, calmodulin forms a stable and equivalent complex with protein 4.1 as determined by calmodulin affinity chromatography, cross-linking experiments, and fluorescence binding assays with an apparent Kd of 5.5 x 10(-7) M irrespective of the free Ca2+ concentration. Domain mapping with chymotryptic digestion reveals that the calmodulin-binding site resides within the N-terminal 30-kDa fragment of protein 4.1. In contrast, the interaction of calmodulin with spectrin is unexpectedly weak (Kd = 1.2 x 10(-4) M). Given the content of calmodulin in erythrocytes (2-5 microM), these results imply that the major target for calmodulin in the erythroid membrane cytoskeleton is protein 4.1. Low- and high-shear viscometry and binding assays reveal that an equivalent complex of calmodulin with protein 4.1 regulates the spectrin/actin interaction in a Ca2(+)-dependent manner. At a low Ca2+ concentration, protein 4.1 potentiates the actin cross-linking and the actin binding activities of spectrin. At a high Ca2+ concentration, the protein 4.1-potentiated actin cross-linking activity but not the actin binding activity of spectrin is suppressed by Ca2+/calmodulin. The Ca2(+)-dependent regulation of the spectrin/protein 4.1/calmodulin/actin interaction is discussed.     

Calmodulin-binding proteins of Tetrahymena microsomal membranes

Tetrahymena calmodulin radioiodinated with a lactoperoxidase method retained full ability to activate Tetrahymena guanylate cyclase. Binding of [125I]calmodulin to Tetrahymena microsomal membranes was Ca2+-dependent and inhibited by excess unlabeled calmodulin or trifluoperazine. When Triton X-100-solubilized microsomes were chromatographed on calmodulin Sepharose, several proteins were found to interact with calmodulin in a Ca2+-dependent manner.     

Calmodulin-drug interaction. A fluorescence and flow dialysis study

Various Ca2+-antagonists and related compounds were probed for possible anti-calmodulin properties. Some of them efficiently inhibit calmodulin dependent activity (the plasma membrane Ca2+-ATPase and the cyclic nucleotide phosphodiesterase). The I50-values for the most potent inhibitors varied between 15 and 30 uM. Using fluorescence spectroscopy and flow dialysis methods the stoichiometry of the binding of some of the drugs to calmodulin has been investigated. The number of Ca2+-dependent high affinity binding sites has been studied on trypsin fragments of calmodulin. Compound 12-114 was bound with high affinity in a Ca2+-dependent way to both halves of calmodulin, compound 200-737 recognized one high affinity binding site only in the C-terminal half of the molecule, whereas compound 36-079 demanded the intact protein to be able to interact with high affinity in a Ca2+-dependent manner.     

Ca2+-dependent potentiation of the nonselective cation channel TRPV4 is mediated by a C-terminal calmodulin binding site

Most Ca2+-permeable ion channels are inhibited by increases in the intracellular Ca2+ concentration ([Ca2+]i), thus preventing potentially deleterious rises in [Ca2+]i. In this study, we demonstrate that currents through the osmo-, heat- and phorbol ester-sensitive, Ca2+-permeable nonselective cation channel TRPV4 are potentiated by intracellular Ca2+. Spontaneous TRPV4 currents and currents stimulated by hypotonic solutions or phorbol esters were reduced strongly at all potentials in the absence of extracellular Ca2+. The other permeant divalent cations Ba2+ and Sr2+ were less effective than Ca2+ in supporting channel activity. An intracellular site of Ca2+ action was supported by the parallel decrease in spontaneous currents and [Ca2+]i on removal of extracellular Ca2+ and the ability of Ca2+ release from intracellular stores to restore TRPV4 activity in the absence of extracellular Ca2+. During TRPV4 activation by hypotonic solutions or phorbol esters, Ca2+ entry through the channel increased the rate and extent of channel activation. Currents were also potentiated by ionomycin in the presence of extracellular Ca2+. Ca2+-dependent potentiation of TRPV4 was often followed by inhibition. By mutagenesis, we localized the structural determinant of Ca2+-dependent potentiation to an intracellular, C-terminal calmodulin binding domain. This domain binds calmodulin in a Ca2+-dependent manner. TRPV4 mutants that did not bind calmodulin lacked Ca2+-dependent potentiation. We conclude that TRPV4 activity is tightly controlled by intracellular Ca2+. Ca2+ entry increases both the rate and extent of channel activation by a calmodulin-dependent mechanism. Excessive increases in [Ca2+]i via TRPV4 are prevented by a Ca2+-dependent negative feedback mechanism.