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PubMed1989

Quantitation of calmodulin in the mammary gland of the lactating sow.

It has been suggested that calmodulin, a calcium-binding protein, has a functional role during milk secretion. High levels of calmodulin are present during lactation in rat mammary glands and a substantial increase has been observed in the bovine mammary gland prior to parturition. In the sow, regressed glands involute while suckled glands remain highly active even though they are under the same hormonal influence. In this study, tissue samples were taken from suckled and regressed glands of the same sow at both peak and late lactation. Calmodulin and total protein were measured in tissue homogenate supernatants. Residual milk was apparent in regressed glands during mid lactation but not in the same glands by late lactation. Calmodulin levels in tissue were the same for both suckled and regressed glands. There was a slight but non-significant increase in the tissue calmodulin level from peak to late lactation. Protein levels declined significantly from mid to the late stage of lactation. There was no change in protein level between the suckled and regressed glands. Calmodulin may be responsible for casein phosphorylation and/or the mediation of prolactin action on the gland. The precise regulatory mechanisms relating hormonal control to calmodulin levels during lactation need further investigation.

Animals↗
PubMed1990

Capsaicin inhibits calmodulin-mediated oxidative burst in rat macrophages.

In this study we seek to elucidate the interaction of capsaicin with the calmodulin mediated signal pathways in macrophages, by comparing its action on macrophage functions with a known calmodulin antagonist, fluphenazine. Kinetics of capsaicin uptake by macrophages (10(3) cells) revealed that a maximum of 200 microM capsaicin was taken up within 10 min. Ca2+ ionophore triggered generation of superoxide anion and hydrogen peroxide by macrophages was inhibited in a dose-dependent manner by fluphenazine (IC50, 20 microM and 12 microM, respectively) and also by capsaicin (IC50, 30 microM and 9 microM, respectively), suggesting an involvement of calmodulin in the regulation of NADPH oxidase. In vitro both fluphenazine and capsaicin inhibited Ca2(+)-Mg2+ ATPase and cAMP-phosphodiesterase from macrophages and this inhibition was reversed by exogenous addition of calmodulin. Fluorescence studies revealed a direct Ca2+ dependent interaction of capsaicin with calmodulin. From these results we suggest that capsaicin acts via calmodulin to inhibit stimulus-induced macrophage oxidative burst and also that calmodulin regulates the oxidative burst in macrophages.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Calcium, calmodulin and cell cycle progression.

Proliferation of mammalian cells both in vivo and in vitro is dependent upon physiological concentrations of extracellular Ca2+. Growth factor stimulation of quiescent cells at the G0/G1 border usually results in a rapid mobilization of Ca2+ from both intra- and extracellular pools. However, Ca2+ influx is also required for later phases of cell cycle transition, especially in the late G1 phase for initiation of DNA synthesis. Available evidence indicates that calmodulin plays the major and essential roles in the Ca(2+)-dependent regulation of cell proliferation. Ca2+ and calmodulin act at multiple points in the cell cycle, including the initiation of the S phase and both initiation and completion of the M phase. Ca2+ and calmodulin stimulate the expression of genes involved in the cell cycle progression, leading to activation of cyclin-dependent kinases p33cdk2 and p34cdc2. Ca2+ and calmodulin are also involved in activation of enzymes participating in nucleotide metabolism and DNA replication, as well as nuclear envelope breakdown and cytokinesis. Ca2+/calmodulin-dependent protein kinase II and protein phosphatase calcineurin are both involved in the Ca2+ and calmodulin-mediated signalling of growth regulation. As compared to normal cells, growth of transformed cells is independent of extracellular Ca2+ and much less sensitive to calmodulin antagonists, suggesting the existence of derangements in the Ca2+ and calmodulin-mediated growth regulation mechanisms.

Animals↗
PubMed1994

Glucocorticoid actions on synaptic plasma membranes: modulation of [125I]calmodulin binding.

The effects of corticosterone on Ca(2+)-dependent binding of [125I]calmodulin to purified synaptic plasma membranes (SPM) from rats brain were characterized. The enhancement of [125I]calmodulin binding was a sigmoidal function of steroid concentration, with the maximal increase (> 55% above control) occurring at a steroid concentration of 1 x 10(-6) M and EC50 estimated at 1-2 x 10(-7) M. Other glucocorticoids including hydrocortisone, dexamethasone and triamcinolone produced similar effects, whereas steroids without glucocorticoid activity such as 11-deoxycortisol, 11-deoxycorticosterone and cholesterol were ineffective. The steroid-induced increase of binding was correlated with an increase of membrane affinity for [125I]calmodulin as shown by Scatchard analysis, and a decrease of the rate of dissociation of [125I]calmodulin from the membranes as shown by kinetic analysis. Arrhenius analysis indicates that [125I]calmodulin binding was influenced by lipid transition of the membranes and that corticosterone resulted in a shift of membrane transition toward a higher temperature. Since a variety of biochemical processes associated with synaptic membranes are dependent upon calmodulin for their regulation, we hypothesize that the effects of glucocorticoids in promoting membrane binding of calmodulin may lead to a cascade of alterations in synaptic function.

Animals↗
PubMed2006

Calmodulin potentiates G beta gamma activation of phospholipase C-beta3.

Phospholipase C-beta (PLC-beta) isozymes (EC 3.1.4.11) hydrolyze the membrane phospholipid phosphatidylinositol-4,5-bisphosphate to generate intracellular second messenger signaling molecules inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) in response to receptor activation and other cellular stimuli. PLCbeta1 and PLCbeta3 isozymes were previously demonstrated to bind the calcium-sensitive molecule calmodulin [McCullar JS, Larsen SA, Millimaki RA, Filtz TM. Calmodulin is a phospholipase C-{beta} interacting protein. J Biol Chem 2003;278(36):33708-13]. We have now shown through fluorescence anisotropy that calmodulin/PLCbeta3 affinities increase with increasing calcium in a physiologically relevant concentration range. The bimolecular affinity constants for calmodulin interaction with PLCbeta1 or PLCbeta3 were estimated as 260 and 200 nM, respectively, from fluorescence anisotropy data. There was no effect of calmodulin on basal or G alpha q-stimulated catalytic activity for either isozyme. However, the interaction between calmodulin and PLCbeta3 leads to potentiation of activation by the G-protein beta gamma dimer in an in vitro assay. 1321N1 cells treated with calmodulin inhibitors concurrent with and post-stimulation of muscarinic receptors significantly reduced [3H]PIP hydrolysis. Together these data are suggestive of cooperative role for calmodulin in the G-protein beta gamma dimer-stimulated activity of PLCbeta3.

Calmodulin↗
PubMed2006

Arrestin binding to calmodulin: a direct interaction between two ubiquitous signaling proteins.

Arrestins serve as multi-functional regulators of G-protein coupled receptors, interacting with hundreds of different receptor subtypes and a variety of other signaling proteins. Here we identify calmodulin as a novel arrestin interaction partner using three independent methods in vitro and in cells. Arrestin preferentially binds calcium-loaded calmodulin with a Kd value of approximately 7 microM, which is within range of endogenous calmodulin concentrations. The calmodulin binding site is localized on the concave side of the C-domain and a loop in the center of the arrestin molecule, significantly overlapping with receptor and microtubule-binding sites. Using purified proteins, we found that arrestins sequester calmodulin, preventing its binding to microtubules. Nanomolar affinity of arrestins for their cognate receptors makes calmodulin an ineffective competitor for arrestin binding at relatively high receptor concentrations. The arrestin-calmodulin interaction likely regulates the localization of both proteins and their availability for other interaction partners.

Arrestins↗
PubMed2006

Complex of calmodulin with a ryanodine receptor target reveals a novel, flexible binding mode.

Calmodulin regulates ryanodine receptor-mediated Ca(2+) release through a conserved binding site. The crystal structure of Ca(2+)-calmodulin bound to this conserved site reveals that calmodulin recognizes two hydrophobic anchor residues at a novel "1-17" spacing that brings the calmodulin lobes close together but prevents them from contacting one another. NMR residual dipolar couplings demonstrate that the detailed structure of each lobe is preserved in solution but also show that the lobes experience domain motions within the complex. FRET measurements confirm the close approach of the lobes in binding the 1-17 target and show that calmodulin binds with one lobe to a peptide lacking the second anchor. We suggest that calmodulin regulates the Ca(2+) channel by switching between the contiguous binding mode seen in our crystal structure and a state where one lobe of calmodulin contacts the conserved binding site while the other interacts with a noncontiguous site on the channel.

Amino Acid Sequence↗
PubMed2005

Modulation of 6-phosphofructo-1-kinase oligomeric equilibrium by calmodulin: formation of active dimers.

Muscle 6-phospho-1-kinase (PFK) is the key regulatory enzyme of the glycolytic pathway and is a calmodulin-binding protein binding two calmodulin molecules per PFK protomer. This enzyme is characterized by a complex regulation that involves its allosteric behavior modulated by several ligands, which modulate the equilibrium between the active tetramers and the inactive dimers of the enzyme. Calmodulin is described to induce the dimerization of PFK, so inhibiting its catalytic activity. Here, we show that binding of calmodulin specifically to its higher-affinity site of PFK induce its dimerization without compromising enzyme catalytic activity forming a hitherto not described active dimmer of PFK. It is also shown that the dimerization is a Ca2+ -dependent event that responds to physiological intracellular Ca2+ concentrations and decrease the interaction of the enzyme to membrane site, which stimulate its catalytic activity. We propose that the effects of calmodulin on PFK reported here are of great physiological significance due to the response to physiological concentrations of Ca2+ and due to be in accordance to the known effects of calmodulin on cell ATP production. We also propose that calmodulin might affect the interaction of PFK to other cellular components as the cytoskeleton.

Animals↗
PubMed2001

Inhibition of T-cell invasion across cultured fibroblast monolayers by phenothiazine-related calmodulin inhibitors: impairment of lymphocyte motility by trifluoperazine and chlorpromazine, and alteration of the monolayer by pimozide.

Phenothiazines inhibit the typical shape changes displayed by activated lymphocytes and thereby their migration through polycarbonate filters. The structure activity relationship of this effect is distinct from calmodulin inhibition. Our aim was to study this effect of phenothiazines on lymphocyte migration in an environment with living solid tissue cells. We assessed the effect of trifluoperazine and chlorpromazine (TFP and CP, two strong inhibitors of lymphocyte motility) and pimozide (PIM, a much weaker inhibitor of lymphocyte motility but a strong inhibitor of calmodulin) on invasion of human Molt-4 T-cells across precultured fibroblast monolayers. As expected invasion was inhibited by TFP and CP in the micromolar range that also inhibited motility. Surprisingly, PIM inhibited monolayer invasion at least as efficiently as TFP and CP (from 2.25 microM on). Preincubation of the monolayers or the lymphoid cells show that PIM exerted this novel invasion inhibiting effect on the monolayer. TFP and CP had a much weaker effect on the monolayer. Since these three compounds inhibit calmodulin in the same order, it is likely that this effect on the monolayer was caused by inhibition of a calmodulin-dependent pathway. KN-62, a specific inhibitor of calmodulin-dependent protein kinase II acted on the monolayer like PIM, whereas ML-7, a specific inhibitor of myosin regulatory light chain kinase, inhibited lymphoid cell motility like TFP and CP. In conclusion, invasion of T-cells across cellular monolayers is inhibited both by PIM and by phenothiazines like TFP and CP, but via distinct mechanisms: TFP and CP inhibit lymphocyte motility via a calmodulin independent pathway, whereas PIM impairs the monolayer's tolerance for invasion, most likely via a calmodulin and CamKII dependent pathway.

Animals↗
PubMed1982

Interactions between calmodulin and immobilized phenothiazines.

The ability of a number of antipsychotic drugs such as phenothiazines to bind to calmodulin with high affinity in a calcium-dependent manner was applied to the study of the nature of their interactions with calmodulin. Thus, a series of phenothiazine derivatives and analogues were immobilized on agarose and examined for their binding characteristics to calmodulin. The binding of calmodulin to fluphenazine, perphenazine and 7-aminotriflupromazine involved on the one hand non-specific electrostatic interactions which are abolished by increasing the eluent salt concentration, and on the other hand, Ca2+-dependent interactions which are reversed by EGTA addition. However, the Ca2+-dependent binding of calmodulin was less specific with phenothiazine structural analogues (neutral Red, diphenylamine) and was suppressed with other phenothiazine derivatives (thionine, Azure C, Toluidine Blue) or analogues (Brilliant Cresyl Blue). It is suggested that the calcium-dependent interactions between calmodulin and drugs involve a charge transfer pi-pi interaction which may be modulated by the electron donor-acceptor properties of the substituents of the aromatic ring. Affinity chromatography using immobilized fluphenazine was also used as the basis for the purification of calmodulin from a number of tissues in a rapid one-step procedure.

Animals↗
PubMed1990

Drosophila melanogaster contains a single calmodulin gene. Further structure and expression studies.

We have previously characterized a calmodulin gene from the organism Drosophila melanogaster. In the higher vertebrates a multi-gene system for encoding calmodulin is present and, in at least one invertebrate species, genes encoding highly related calmodulin isotypes exist. We have therefore searched for additional calmodulin genes within D. melanogaster. Although our searches were sensitive enough to detect a relatively divergent gene encoding a calmodulin family protein, we were unable to detect any additional genes for calmodulin per se. Further studies of the structure and expression of the single calmodulin gene of D. melanogaster have established that the gene contains a tiny additional 5' exon encoding only 50 residues of the 5' leader. Sequencing at the 3' terminus has established that the two transcript size classes derived from the gene are produced as a result of alternative polyadenylation site usage. The relative abundance of the two size classes of mRNAs differs throughout the life cycle, indicating developmental regulation of polyadenylation site usage.

Amino Acid Sequence↗
PubMed1997

Identification of beta2-glycoprotein I as a membrane-associated protein in kidney: purification by calmodulin affinity chromatography.

Outer renal medulla calmodulin-binding proteins from a soluble protein fraction and a plasma membrane fraction solubilized in CHAPS were retained on a calmodulin-Sepharose 4B column in the presence of Ca2+, and subsequently eluted by EGTA. The calmodulin-binding proteins constituted 2.5% of the soluble protein and 0.1% of the solubilized membrane protein. beta2-glycoprotein I was identified as a calmodulin-binding protein both by N-terminal sequencing and by immunoblotting. Quantification showed that beta2-glycoprotein I constituted the major part (approx. 35%) of the calmodulin-binding membrane proteins, but only a minor part (approx. 0.1%) of the calmodulin-binding proteins in the soluble fraction. These results show for the first time that beta2-glycoprotein I binds calmodulin and that beta2-glycoprotein I may in kidney be a membrane-associated protein. Immunohistochemical studies identified beta2-glycoprotein I in several parts of the cortex and the medulla of the kidney, including Bowman's capsula, the tubular lumen and the tubular epithelium, indicating that beta2-glycoprotein I, despite its relatively high molecular mass, is filtrated in the glomerulus and subsequently reabsorbed by the tubular epithelium. This is in agreement with beta2-glycoprotein I being a marker for renal tubular disease.

Amino Acid Sequence↗
PubMed2002

Effect of calmodulin antagonists on phospholipase D activity in SH-SY5Y cells.

The aim of this study was to investigate the involvement of calmodulin in phospholipase D activation in SH-SY5Y cells. Cells prelabelled with [3H]-palmitic acid were incubated with calmodulin antagonists and/or other compounds. Phosphatidylethanol, a specific marker for phospholipase D activity, and phosphatidic acid were analysed. The calmodulin antagonists, calmidazolium and trifluoperazine, induced an extensive increase in phosphatidylethanol formation, and thus increased basal phospholipase D activity, in a dose- and time-dependent manner. The effect of calmidazolium on carbachol-induced activation of muscarinic receptors was also studied. Calmidazolium did not significantly affect the amount of phosphatidylethanol formed following carbachol addition. However, taking into account the increase in basal activity observed after calmidazolium addition, calmidazolium probably inhibits the muscarinic receptor-induced phospholipase D activation. In addition to phosphatidylethanol, basal phosphatidic acid levels were also increased after calmidazolium and trifluoperazine addition. Incubation with calmidazolium (10 microM) for 10 min induced a two-fold increase in phosphatidic acid. The calmidazolium-induced increase in basal phospholipase D activity was not affected by the protein kinase inhibitors H7 and staurosporine. On the other hand tyrosine kinase inhibitors abolished the calmidazolium-induced activation of phospholipase D. Calmidazolium also induced tyrosine phosphorylation in parallel to the phospholipase D activation. In conclusion, our data indicate that calmodulin antagonists induce phospholipase D activity in SH-SY5Y cells via a tyrosine kinase dependent pathway. This may point to a negative control of phospholipase D by calmodulin although a calmodulin-independent mechanism cannot be excluded. Calmodulin antagonists may be useful tools to further elucidate the mechanisms of phospholipase D regulation.

Calmodulin↗
PubMed2001

Immunostaining of calmodulin and aluminium in Alzheimer's disease-affected brains.

Previous in vitro studies have shown that Al(3+) binds to calmodulin, inducing alterations in its capability to interact with target proteins, accompanied by loss of immunological recognition by its conformational specific monoclonal antibody CAM1. In spite of the wealth of data of calmodulin action in vitro, little information is available on the possible involvement of this protein in the pathology typical of Alzheimer's disease. In the present study, we investigated calmodulin immunoreactivity in post-mortem human brains affected by Alzheimer's disease, compared with age-matched control brains. Conformational monoclonal antibodies raised against Ca(2+)-calmodulin, namely CAM1 and CAM4, were used in this study for the characterization of calmodulin. Calmodulin immunorecognition by monoclonal antibody CAM1 was found to be lost in cortical tissue sample from brains affected by Alzheimer's disease. This finding leads to the hypothesis of a new, possibly inactive, conformation of the molecule during the disease. On the other hand, CAM4 immunoreactivity was decreased in neurons of brains affected by Alzheimer's disease. Anti-Al(3+) monoclonal antibodies revealed instead more marked aluminium immunoreactivity in the affected brains compared to normal ones. The loss of CAM1 immunoreactivity and the occurrence of large amounts of aluminium suggest an alteration of the active conformation of calmodulin in disease-affected brains. These alterations could be involved in the development of Alzheimer's disease pathology.

Aged↗
PubMed2001

Ultrastructural localization of calmodulin in gerbil cochlea by immunogold electron microscopy.

Localization of calmodulin, a calcium binding protein, was identified in adult gerbil cochleas using paraffin section immunohistochemistry and immunogold electron microscopy with monoclonal antibody against bovine calmodulin. Immunoreactive calmodulin was abundant in inner hair cells (IHCs), outer hair cells (OHCs) and Boettcher cells of the cochleas. Other cell types containing calmodulin were marginal cells and basal cells of the stria vascularis, fibrocytes in the spiral ligament, spiral ganglion neurons and vascular smooth muscle cells. Immunogold labeling for calmodulin was observed in cuticular plate, stereocilia, and within cytoplasm of IHCs and OHCs. In OHCs the labeling was mostly observed in the region underlying lateral wall corresponding to subsurface cisterna. In IHCs the staining was diffuse in the cytoplasm and denser than that in OHCs. Boettcher cells showed dense staining along the microvillous projections facing to the intercellular spaces between Boettcher cells and Claudius cells and between the neighboring Boettcher cells. These distributions of calmodulin in the hair cells consist with the assumption that IHCs act as a true neurotransducer and OHCs as an active bi-directional mechanotransducer. The rich presence of calmodulin in Boettcher cells suggests that the cells may involve in mediating Ca(2+) regulation and play a distinctive active role in ion transport.

Animals↗
PubMed1992

Calmodulin and cell cycle control.

Previous studies have indicated a role for the calcium receptor calmodulin in the control of eukaryotic cell proliferation. Using a molecular genetic approach in the filamentous fungus Aspergillus nidulans we have shown that CaM is required for cell cycle progression at multiple points in the cell cycle. Construction of an A nidulans strain conditional for calmodulin expression reveals that this protein is required during G1/S and for the initiation of mitosis. A lack of calmodulin results in cell cycle arrest, and a failure in polar growth that accompanies germination of A nidulans spores. In addition, increased expression of calmodulin in this organism permits growth at suboptimal calcium concentrations, indicating that cell growth is coordinately regulated by calcium and calmodulin. Together these results indicate that calmodulin-dependent processes may be conserved between A nidulans and vertebrate cells, and suggest that this approach may allow us to elucidate the molecular mechanism underlying calmodulin-regulated control of cell proliferation.

Aspergillus nidulans↗
PubMed1995

Discontinuous equilibrium titrations of cooperative calcium binding to calmodulin monitored by 1-D 1H-nuclear magnetic resonance spectroscopy.

Calmodulin binds up to four calcium ions cooperatively in response to cellular signaling events. To understand the functional energetics of calcium activation of calmodulin, it is important to monitor individual Ca(2+)-binding sites and other positions at partial degrees of saturation. This study is the first use of 1-D proton NMR to monitor the equilibrium Ca(2+)-binding properties of calmodulin. Protein concentrations required for NMR experiments (approximately 1 mM) are approximately 1000-fold greater than the Kd values for calcium binding to calmodulin, preventing a direct continuous equilibrium titration of calmodulin. Thus, dialysates of calmodulin in buffers of experimentally determined [Ca2+]free were prepared to conduct discontinuous equilibrium titrations at both 92 and 152 mM KCl. For the C-terminal domain, the normalized area of the delta-protons of Y138 defined calcium binding isotherms. For N-terminal domain resonances (F16C delta H, T26C alpha H, D64C alpha H, and F65C delta H), the calcium-dependent change in chemical shift defined isotherms. These are the first residue-specific studies to monitor the energetics of Ca2+ binding to the N-terminal domain in wild-type holo calmodulin. Calcium binding to both domains appeared cooperative and binding affinity decreased in higher KCl. Isotherms resolved from the side chain resonances of F16 and F65 had a lower median ligand activity and a slightly higher degree of cooperativity than isotherms resolved from the backbone resonances of D64 and T26. Salt-dependent changes in apparent intradomain cooperativity differed for the domains: at higher salt, delta Gc increased for the C-terminal domain while remaining constant or decreasing for the N-terminal domain.

Amino Acid Sequence↗
PubMed1992

Photoaffinity labeling study of the interaction of calmodulin with the plasma membrane Ca2+ pump.

Bovine brain calmodulin was labeled with synthetic peptides corresponding to the calmodulin-binding domain of the erythrocyte plasma membrane Ca(2+)-ATPase. One 20-amino acid peptide and two 28-amino acid peptides were used, carrying L-4'-(1-azi-2,2,2-trifluoroethyl)phenylalanine residues in position 9 (peptides C20W* and C28W*) and position 25 (peptide C28WC*), respectively. The localization of the contact regions between calmodulin and the N- and C-terminal portions of the peptides was the aim of this study. The three peptides were N-terminally blocked with a 3H-labeled acetyl group to facilitate the identification of labeled fragments after isolation and digestion. The binding site for phenylalanine 25 was identified in the N-terminal domain of calmodulin while the phenylalanine derivative in position 9 labeled the C-terminal domain. Fluorescence studies using the dansylated N- and C-terminal halves of calmodulin and peptide C20W corresponding to the first 20 amino acids of the calmodulin-binding domain showed that only the C-terminal lobe of calmodulin had high affinity for the peptide (KD in the nanomolar range).

Affinity Labels↗