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PubMed1993

Localization of the multiple calmodulin messenger RNAs in differentiated PC12 cells.

Calmodulin, a ubiquitous calcium-binding protein which is involved in many biological processes, including cell proliferation and differentiation, has been shown to be encoded by three genes from which five calmodulin messenger RNAs are transcribed. In our previous studies, using the PC12 pheochromocytoma cell line as a model system for neuronal differentiation, all five calmodulin messenger RNAs were found to be present, and treatment with both nerve growth factor and dibutyryl cyclic AMP, which induce neurite outgrowth in these cells, increased the level of calmodulin and differentially increased the levels of the various calmodulin messenger RNAs. In an attempt to uncover the nature of the differential increase in the calmodulin messenger RNAs during neuronal differentiation, we examined here the subcellular distribution of the individual calmodulin messenger RNAs in PC12 cells treated with nerve growth factor and dibutyryl cyclic AMP by in situ hybridization cytochemistry, using radiolabeled oligodeoxynucleotide probes. Using an oligodeoxynucleotide probe which detects all of the calmodulin transcripts, the calmodulin messenger RNAs were found to be distributed throughout the cell bodies of differentiated PC12 cells; significant amounts of calmodulin messenger RNAs were also found in most neurites (approximately 70% of the total number). Using specific probes for the calmodulin messenger RNAs derived from each calmodulin gene, distinct patterns of localization of the different calmodulin messenger RNAs were revealed. The messenger RNAs from calmodulin genes I and II were readily detected in all cell bodies and in about one-half of the neurites. In contrast, a weak signal for the messenger RNAs from calmodulin gene III was associated with cell bodies, while no significant signal was found in neurites. A population distribution analysis of the labeling of individual PC12 cell bodies, as determined by counting autoradiographic grains, revealed differences in the relative abundance of each group of messenger RNAs derived from each of the three calmodulin genes. The order of relative abundance of the messenger RNAs in cell bodies was found to be: calmodulin gene II messenger RNA > calmodulin gene I messenger RNAs >> calmodulin gene III messenger RNAs. An analysis of the labeling density along neurites indicated a similar density of neuritic messenger RNAs from calmodulin gene I and calmodulin gene II, whereas there was no significant signal for the messenger RNAs from calmodulin gene III.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Calmodulin N-methyltransferase. Partial purification and characterization.

The distribution, properties, and substrate specificity of S-adenosylmethionine:calmodulin (lysine) N-methyltransferse (EC 2.1.1.60, calmodulin N-methyltransferase) of the rat have been studied. This enzyme is cytosolic and is found at high levels in tissues with high levels of calmodulin and at low levels in tissues with little calmodulin. In liver, heart, and skeletal muscle, which have low levels of calmodulin and very low calmodulin N-methyltransferase activity (a low ratio of calmodulin N-methyltransferase to calmodulin), calmodulin was found to be incompletely methylated, as judged by its ability to act as a substrate for purified calmodulin N-methyltransferase. Calmodulin N-methyltransferase was purified 470-fold with a 33% yield from rat testis cytosol, using ammonium sulfate precipitation and chromatography on DEAE-cellulose, CM-Sepharose, and Sephadex G-100. At pH 7.4, calmodulin N-methyltransferase did not bind to DEAE-cellulose, but bound strongly to CM-Sepharose. The enzyme eluted from Sephadex G-100 with an apparent molecular weight of 55,000. Purified calmodulin N-methyltransferase was incubated with extracts of rat tissues, and [methyl-3H]AdoMet and methylated proteins were resolved by electrophoresis in an attempt to discover substances other than calmodulin, but this enzyme only catalyzed the methylation of calmodulin, indicating a high degree of substrate specificity. Conditions were established for the in vitro preparative methylation of des(methyl)-calmodulin from Dictyostelium discoideum. Three moles of methyl/mol of calmodulin were incorporated into lysine 115 of des(methyl)calmodulin, resulting in the formation of 1 mol of trimethyllysine at the site normally methylated in calmodulins from most species. Activation of cyclic nucleotide phosphodiesterase by des(methyl)calmodulin was indistinguishable from activation by in vitro methylated or sham methylated Dictyostelium calmodulin, indicating that methylation does not affect the ability of calmodulin to activate this enzyme.

Amino Acids↗
PubMed1999

Ca2+-dependent interaction of the trpl cation channel and calmodulin.

The transient receptor potential-like ion channel from Drosophila melanogaster was originally identified as a calmodulin binding protein (Philips et al., 1992) involved in the dipterian phototransduction process. We used a series of fusion proteins and an epitope expression library of transient receptor potential-like fusion proteins to characterize calmodulin binding regions in the transient receptor potential-like channel through the use of [125I]calmodulin and biotinylated calmodulin and identified two distinct sites at the C-terminus of the transient receptor potential-like ion channel. Calmodulin binding site 1, predicted from searching of the primary structure for amphiphilic helices (Philips et al., 1992), covers a 16 amino acid sequence (S710-I725) and could only be detected through biotinylated calmodulin. Calmodulin binding site 2 comprises at least 13 amino acids (K859ETAKERFQRVAR871) and binds both [125I]calmodulin and biotinylated calmodulin. Both sites (i) bind calmodulin at least in a one to one stoichiometry, (ii) differ in their affinity for calmodulin revealing apparent Ki values of 12.3 nM (calmodulin binding site 1) and 1.7 nM (calmodulin binding site 2), respectively, (iii) bind calmodulin only in the presence of Ca2+ with 50% of site 1 and site 2, respectively, occupied by calmodulin in the presence of 0.1 microM (calmodulin binding site 1) and 3.3 microM Ca2+ (calmodulin binding site 2) and give evidence that (iv) a Ca2+-calmodulin-dependent mechanism contributes to transient receptor potential-like cation channel modulation when expressed in CHO cells.

Amino Acid Sequence↗
PubMed2004

RC3/Neurogranin and Ca2+/calmodulin-dependent protein kinase II produce opposing effects on the affinity of calmodulin for calcium.

The interaction of calmodulin with its target proteins is known to affect the kinetics and affinity of Ca(2+) binding to calmodulin. Based on thermodynamic principles, proteins that bind to Ca(2+)-calmodulin should increase the affinity of calmodulin for Ca(2+), while proteins that bind to apo-calmodulin should decrease its affinity for Ca(2+). We quantified the effects on Ca(2+)-calmodulin interaction of two neuronal calmodulin targets: RC3, which binds both Ca(2+)- and apo-calmodulin, and alphaCaM kinase II, which binds selectively to Ca(2+)-calmodulin. RC3 was found to decrease the affinity of calmodulin for Ca(2+), whereas CaM kinase II increases the calmodulin affinity for Ca(2+). Specifically, RC3 increases the rate of Ca(2+) dissociation from the C-terminal sites of calmodulin up to 60-fold while having little effect on the rate of Ca(2+) association. Conversely, CaM kinase II decreases the rates of dissociation of Ca(2+) from both lobes of calmodulin and autophosphorylation of CaM kinase II at Thr(286) induces a further decrease in the rates of Ca(2+) dissociation. RC3 dampens the effects of CaM kinase II on Ca(2+) dissociation by increasing the rate of dissociation from the C-terminal lobe of calmodulin when in the presence of CaM kinase II. This effect is not seen with phosphorylated CaM kinase II. The results are interpreted according to a kinetic scheme in which there are competing pathways for dissociation of the Ca(2+)-calmodulin target complex. This work indicates that the Ca(2+) binding properties of calmodulin are highly regulated and reveals a role for RC3 in accelerating the dissociation of Ca(2+)-calmodulin target complexes at the end of a Ca(2+) signal.

Animals↗
PubMed1994

Calmodulin expression during rat liver regeneration.

We have investigated the messenger RNAs expressed from the three calmodulin genes during rat liver regeneration. The results revealed that all the calmodulin transcripts increased from 8 hr after a partial hepatectomy, although differences in the timing and the level of expression from the three genes were observed. Calmodulin I transcripts peaked at 16 hr, whereas calmodulin II and calmodulin III progressively increased from 8 to 24 hr. At 24 hr after surgery, calmodulin I, calmodulin II and the 2.3 kb calmodulin III transcripts reached values of a 6-fold increase, whereas the 0.8 kb product of calmodulin III increased 25-fold. At 30 hr the levels of all the calmodulin transcripts were similar to those observed at 24 hr. The transcription rates of the three calmodulin genes augmented after hepatectomy (calmodulin I and calmodulin II twofold and calmodulin III fourfold), indicating that the elevation of the calmodulin transcripts could be, at least partially, the result of this increase in the transcription rates. The total calmodulin concentration also increased twofold at 24 hr after hepatectomy. We also report that the administration of the beta-adrenergic blocker, D,L-propranolol inhibited the accumulation of calmodulin protein without significantly affecting the increase of the messenger RNAs. These results indicate that the expression of calmodulin observed during liver regeneration could be regulated by cyclic AMP at the translational or posttranslational level.

Animals↗
PubMed1993

Isolation and kinetic characterization of the calmodulin methyltransferase from sheep brain.

The methyltransferase that catalyzes the trimethylation of lysine 115 in calmodulin has been purified from sheep brain. The enzyme is a monomer with an apparent molecular weight of 38,000 on the basis of gel filtration chromatography and SDS-polyacrylamide electrophoresis. In the presence of calcium the methyltransferase exhibited a Km of 100 nM for unmethylated calmodulin and a kcat of 0.0278 s-1. The enzyme was able to use calcium-depleted calmodulin as a substrate, albeit with less efficiency. The methylation of calcium-depleted calmodulin was inhibited by increases in ionic strength, whereas methylation of calcium-saturated calmodulin was not affected. This suggests a difference in the mode of interaction of calcium-saturated and calcium-depleted calmodulins with the enzyme. As with calmodulin's interactions with other calmodulin-dependent enzymes, the oxidation of the methionines of calmodulin by performic acid treatment decreases the ability of the methyltransferase to recognize and methylate calmodulin. A calmodulin-binding peptide based on the calmodulin-dependent protein kinase II sequence and the naphthalenesulfonamide W-7 inhibit the calmodulin methyltransferase-calmodulin interaction in a calcium-dependent manner. Removal of the NH2-terminal lobe (residues 1-77) does not affect the ability of the calmodulin methyltransferase to recognize and methylate lysine 115. Thus, the determinants for calmodulin methyltransferase binding reside solely in the COOH-terminal lobe of calmodulin. Further, structural features within this region, in particular, the hydrophobic cleft, that are manifested upon calcium binding may contribute to the interaction of calmodulin with the enzyme.

Amino Acid Sequence↗
PubMed1987

Purification and biochemical properties of calmodulin from Saccharomyces cerevisiae.

Calmodulin from the yeast Saccharomyces cerevisiae was purified to complete homogeneity by hydrophobic interaction chromatography and HPLC gel filtration. The biochemical properties of the purified protein as calmodulin were examined under various criteria and its similarity and dissimilarity to other calmodulins have been described. Like other calmodulins, yeast calmodulin activated bovine phosphodiesterase and pea NAD kinase in a Ca2+-dependent manner, but its concentration for half-maximal activation was 8-10 times that of bovine calmodulin. The amino acid composition of yeast calmodulin was different from those of calmodulins from other lower eukaryotes in that it contained no tyrosine, but more leucine and had a high ratio of serine to threonine. Yeast calmodulin did not contain tryptophanyl or tyrosyl residues, so its ultraviolet spectrum reflected the absorbance of phenylalanyl residues, and had a molar absorption coefficient at 259 nm of 1900 M-1 cm-1. Ca2+ ions changed the secondary structure of yeast calmodulin, causing a 3% decrease in the alpha-helical content, unlike its effect on other calmodulins. Antibody against yeast calmodulin did not cross-react with bovine calmodulin, and antibody against bovine calmodulin did not cross-react with yeast calmodulin, presumably due to differences in the amino acid sequences of the antigenic sites. It is concluded that the molecular structure of yeast calmodulin differs from those of calmodulins from other sources, but that its Ca2+-dependent regulatory functions are highly conserved and essentially similar to those of calmodulins of higher eukaryotes.

Amino Acids↗
PubMed1993

The role of polybasic compounds in determining the tyrosyl phosphorylation of calmodulin by the human insulin receptor.

A highly purified human insulin receptor preparation was shown to effect receptor autophosphorylation and the phosphorylation of poly(GluTyr) but not that of calmodulin. Addition of poly-L-lysine allowed for the stoichiometric tyrosyl phosphorylation of calmodulin in a dose-dependent fashion (EC50 approximately 83 nM) with the single target residue identified at tyr99. Higher concentrations of poly-L-lysine elicited the dose-dependent inhibition of calmodulin phosphorylation (IC50 approximately 0.7 microM) by a process which did not apparently involve either stimulation of calmodulin phosphatase activity or diminished receptor kinase activity. Polybasic substances such as poly-L-arginine, histone H1 and protamine sulphate all promoted calmodulin phosphorylation by the insulin receptor in a similar biphasic dose-dependent fashion. Poly-lysine's actions proved to lack stereo-specificity in that both the D- and L-forms were equally as effective. Reduction in the chain length of poly-L-lysine species attenuated their ability to promote calmodulin phosphorylation with L-lysine proving to be ineffective. Optimal promotion of calmodulin phosphorylation was achieved at an apparently constant ratio of calmodulin to poly-L-lysine of approximately 1:4 over a 100-fold range of calmodulin concentrations. Poly-L-lysine promoted the precipitation and subsequent resolubilization of calmodulin in a fashion whose biphasic dose-dependence paralleled that seen for its action in promoting calmodulin's phosphorylation. NaCl attenuated, in apparently identical dose-dependent fashions, poly-L-lysine's ability to both elicit the precipitation of calmodulin and to promote its phosphorylation. The presence of added Ca2+ led to a small potentiation of poly-L-lysine-dependent calmodulin phosphorylation at low concentrations, with inhibition occurring at higher concentrations where Ca2+ was shown to block calmodulin precipitation by poly-L-lysine. It is suggested that calmodulin can be phosphorylated by the insulin receptor only when it is cross-linked in a multivalent fashion to a suitable polybasic substance so that it forms large multimeric aggregates. Such a requirement for the formation of an aggregate between calmodulin and a suitable polybasic species may place specific constraints on the ability of calmodulin to serve as a substrate for receptor tyrosyl kinases within the cell.

Calmodulin↗
PubMed2001

Suramin and the suramin analogue NF307 discriminate among calmodulin-binding sites.

Calmodulin-binding sites on target proteins show considerable variation in primary sequence; hence compounds that block the access of calmodulin to these binding sites may be more selective than compounds that inactivate calmodulin. Suramin and its analogue NF307 inhibit the interaction of calmodulin with the ryanodine receptor. We have investigated whether inhibition of calmodulin binding to target proteins is a general property of these compounds. Suramin inhibited binding of [(125)I]calmodulin to porcine brain membranes and to sarcoplasmic reticulum from skeletal muscle (IC(50)=4.9+/-1.2 microM and 19.9+/-1.8 microM, respectively) and blocked the cross-linking of [(125)I]calmodulin to some, but not all, target proteins in brain membranes by [(125)I]calmodulin. Four calmodulin-binding proteins were purified [ryanodine receptor-1 (RyR1) from rabbit skeletal muscle, neuronal NO synthase (nNOS) from Sf9 cells, G-protein betagamma dimers (Gbetagamma) from porcine brain and a glutathione S-transferase-fusion protein comprising the C-terminal calmodulin-binding domain of the metabotropic glutamate receptor 7A (GST-CmGluR7A) from bacterial lysates]. Three of the proteins employed (Gbetagamma, GST-CmGluR7A and RyR1) display a comparable affinity for calmodulin (in the range of 50-70 nM). Nevertheless, suramin and NF307 only blocked the binding of Gbetagamma and RyR1 to calmodulin-Sepharose. In contrast, the association of GST-CmGluR7A and nNOS was not impaired, whereas excess calmodulin uniformly displaced all proteins from the matrix. Thus suramin and NF307 are prototypes of a new class of calmodulin antagonists that do not interact directly with calmodulin but with calmodulin-recognition sites. In addition, these compounds discriminate among calmodulin-binding motifs.

Allosteric Regulation↗
PubMed1995

The interaction of calmodulin with clathrin-coated vesicles, triskelions, and light chains. Localization of a binding site.

The binding of clathrin-coated vesicles, clathrin triskelions, and free clathrin light chains to calmodulin-Sepharose was compared. When isolated from bovine brain, all three components bound to calmodulin-Sepharose in the presence of calcium and could be eluted by its removal. In contrast, coated vesicles and triskelions isolated from bovine adrenal gland did not bind to calmodulin-Sepharose, although the free light chains from adrenal gland bound as effectively as those from brain. As distinct isoforms of the clathrin light chains are expressed by brain and adrenal gland, these results implicate the clathrin light chains as the calmodulin-binding component of coated vesicles and triskelions. Furthermore, the insertion sequences found in the neuron-specific isoforms, although not necessary for the binding of free clathrin light chains to calmodulin, must facilitate the interaction of heavy chain-associated light chains with calmodulin. Recombinant mutants of LCa, with deletions spanning the entire sequence, were tested for binding to calmodulin-Sepharose. Those mutants retaining structural integrity, as assessed by the binding of a panel of monoclonal antibodies, exhibited varying amounts of calmodulin binding activity. However, deletion of the carboxyl-terminal 20 residues abolished calmodulin interaction. Thus, the carboxyl terminus of LCa appears to constitute a calmodulin-binding site. Peptides corresponding to the carboxyl terminus of LCa or LCb inhibited the interaction of the light chains with calmodulin, suggesting that this region forms the calmodulin-binding site of both LCa and LCb. The carboxyl-terminal peptides of LCa and LCb inhibited the interaction of light chains with calmodulin approximately 10-fold less effectively than a calmodulin-binding peptide derived from smooth muscle myosin light chain kinase, but much more effectively than a calmodulin-binding peptide derived from adenylate cyclase. This comparison places the clathrin light chain-calmodulin interaction within the physiological range seen for other calmodulin-binding proteins.

Adrenal Glands↗
PubMed2003

Intracellular coupling via limiting calmodulin.

Measurements of cellular Ca2+-calmodulin concentrations have suggested that competition for limiting calmodulin may couple calmodulin-dependent activities. Here we have directly tested this hypothesis. We have found that in endothelial cells the amount of calmodulin bound to nitric-oxide synthase and the catalytic activity of the enzyme both are increased approximately 3-fold upon changes in the phosphorylation status of the enzyme. Quantitative immunoblotting indicates that the synthase can bind up to 25% of the total cellular calmodulin. Consistent with this, simultaneous determinations of the free Ca2+ and Ca2+-calmodulin concentrations in these cells performed using indo-1 and a fluorescent calmodulin biosensor (Kd = 2 nm) indicate that increased binding of calmodulin to the synthase is associated with substantial reductions in the Ca2+-calmodulin concentrations produced and an increase in the [Ca2+]50 for formation of the calmodulin-biosensor complex. The physiological significance of these effects is confirmed by a corresponding 40% reduction in calmodulin-dependent plasma membrane Ca2+ pump activity. An identical reduction in pump activity is produced by expression of a high affinity (Kd = 0.3 nm) calmodulin biosensor, and treatment to increase calmodulin binding to the synthase then has no further effect. This suggests that the observed reduction in pump activity is due specifically to reduced calmodulin availability. Increases in synthase activity thus appear to be coupled to decreases in the activities of other calmodulin targets through reductions in the size of a limiting pool of available calmodulin. This exemplifies what is likely to be a ubiquitous mechanism for coupling among diverse calmodulin-dependent activities.

Animals↗
PubMed1983

On the interrelation between calmodulin and EGTA in the regulation of the affinity to Ca2+ and the maximal activity of the erythrocyte-membrane calcium pump.

1. Calmodulin distribution in rat erythrocytes and the interrelation between calmodulin and EGTA in regulation of the rate of 45Ca accumulation by erythrocyte-membrane inside-out vesicles were studied. 2. The total content of calmodulin in rat erythrocytes is about 24 mumol/l of cells. About 60% of calmodulin is localized in cytoplasm and 40% (10 mumol/l of cells) of calmodulin is loosely bound to membranes; after subsequent washing by hypotonic solution (membranes A) the content of membrane-bound calmodulin decreases up to 0.1 mumol/l of cells. 3. The addition of exogenous calmodulin to membranes A results in increase of the maximal activity of the Ca-pump and does not influence its affinity to Ca2+. Troponin I (30 microM) completely abolishes the calmodulin effect on the Ca-pump activity without significant alterations in its basal activity. 4. The addition of EGTA in the membrane-washing solution results in decrease of the membrane-found pool calmodulin up to 0.01 mumol/l of cells (membranes B). This procedure is accompanied by decrease of the affinity of Ca-pump to Ca2+ and does not influence its maximal rate. 5. The effect of EGTA treatment (membranes B) on the affinity of Ca-pump to Ca is abolished after addition of micromolar concentrations of calmodulin or millimolar concentration of EGTA in the incubation medium. The increase of EGTA concentration in the incubation medium results in decrease of the affinity of Ca-pump to calmodulin. 6. It is assumed that two essentially different pools of calmodulin participate in the regulation of the activity of Ca-pump. (a) The pool of calmodulin which is loosely bound to membrane (this size is dependent on calmodulin concentration in cytoplasm) determines the maximal activity of Ca-pump. The effect of this calmodulin pool is blocked by troponin I. (b) The tightly bound pool of calmodulin which is removed by EGTA treatment determines the affinity of Ca-pump to Ca. 7. In this connection the reasons for contradictory data on estimation of calmodulin effect on the kinetic parameters of plasma membrane Ca-pump are discussed.

Animals↗
PubMed2002

Calmodulin binding to recombinant myosin-1c and myosin-1c IQ peptides.

BACKGROUND: Bullfrog myosin-1c contains three previously recognized calmodulin-binding IQ domains (IQ1, IQ2, and IQ3) in its neck region; we identified a fourth IQ domain (IQ4), located immediately adjacent to IQ3. How calmodulin binds to these IQ domains is the subject of this report. RESULTS: In the presence of EGTA, calmodulin bound to synthetic peptides corresponding to IQ1, IQ2, and IQ3 with Kd values of 2-4 microM at normal ionic strength; the interaction with an IQ4 peptide was much weaker. Ca2+ substantially weakened the calmodulin-peptide affinity for all of the IQ peptides except IQ3. To reveal how calmodulin bound to the linearly arranged IQ domains of the myosin-1c neck, we used hydrodynamic measurements to determine the stoichiometry of complexes of calmodulin and myosin-1c. Purified myosin-1c and T701-Myo1c (a myosin-1c fragment with all four IQ domains and the C-terminal tail) each bound 2-3 calmodulin molecules. At a physiologically relevant temperature (25 degrees C) and under low-Ca2+ conditions, T701-Myo1c bound two calmodulins in the absence and three calmodulins in the presence of 5 microM free calmodulin. Ca2+ dissociated nearly all calmodulins from T701-Myo1c at 25 degrees C; one calmodulin was retained if 5 microM free calmodulin was present. CONCLUSIONS: We inferred from these data that at 25 degrees C and normal cellular concentrations of calmodulin, calmodulin is bound to IQ1, IQ2, and IQ3 of myosin-1c when Ca2+ is low. The calmodulin bound to one of these IQ domains, probably IQ2, is only weakly associated. Upon Ca2+ elevation, all calmodulin except that bound to IQ3 should dissociate.

Amino Acid Motifs↗
PubMed1998

A novel role for calmodulin: Ca2+-independent inhibition of type-1 inositol trisphosphate receptors.

Calmodulin inhibits both inositol 1,4,5-trisphosphate (IP3) binding to, and IP3-evoked Ca2+ release by, cerebellar IP3 receptors [Patel, Morris, Adkins, O'Beirne and Taylor (1997) Proc. Natl. Acad. Sci. U. S.A. 94, 11627-11632]. In the present study, full-length rat type-1 and -3 IP3 receptors were expressed at high levels in insect Spodoptera frugiperda 9 cells and the effects of calmodulin were examined. In the absence of Ca2+, calmodulin caused a concentration-dependent and reversible inhibition of [3H]IP3 binding to type-1 IP3 receptors by decreasing their apparent affinity for IP3. The effect was not reproduced by high concentrations of troponin C, parvalbumin or S-100. Increasing the medium free [Ca2+] ([Ca2+]m) inhibited [3H]IP3 binding to type-1 receptors, but the further inhibition caused by a submaximal concentration of calmodulin was similar at each [Ca2+]m. In the absence of Ca2+, 125I-calmodulin bound to a single site on each type-1 receptor subunit and to an additional site in the presence of Ca2+. There was no detectable binding of 125I-calmodulin to type-3 receptors and binding of [3H]IP3 was insensitive to calmodulin at all [Ca2+]m. Both peptide and conventional Ca2+-calmodulin antagonists affected neither [3H]IP3 binding directly nor the inhibitory effect of calmodulin in the absence of Ca2+, but each caused a [Ca2+]m-dependent reversal of the inhibition of [3H]IP3 binding caused by calmodulin. Camstatin, a peptide that binds to calmodulin equally well in the presence or absence of Ca2+, reversed the inhibitory effects of calmodulin on [3H]IP3 binding at all [Ca2+]m. We conclude that calmodulin specifically inhibits [3H]IP3 binding to type-1 IP3 receptors: the first example of a protein regulated by calmodulin in an entirely Ca2+-independent manner. Inhibition of type-1 IP3 receptors by calmodulin may dynamically regulate their sensitivity to IP3 in response to the changes in cytosolic free calmodulin concentration thought to accompany stimulation of neurones.

Amino Acid Sequence↗
PubMed1999

Interaction of a kinesin-like protein with calmodulin isoforms from Arabidopsis.

In Arabidopsis and other plants there are multiple calmodulin isoforms. However, the role of these isoforms in regulating the activity of target proteins is obscure. Here, we analyzed the interaction between a kinesin-like calmodulin-binding motor protein (Reddy, A. S. N., Safadi, F., Narasimhulu, S. B., Golovkin, M., and Hu, X. (1996) J. Biol. Chem. 271, 7052-7060) and three calmodulin isoforms (calmodulin-2, -4, and -6) from Arabidopsis using different approaches. Gel mobility and fluorescence shift assays revealed that the motor binds to all calmodulin isoforms in a calcium-dependent manner. Furthermore, all calmodulin isoforms were able to activate bovine calcium/calmodulin-dependent phosphodiesterase. However, the concentration of calmodulin-2 required for half-maximal activation of phosphodiesterase is 2- and 6-fold lower compared with calmodulin-4 and -6, respectively. The dissociation constants of the motor to calmodulin-2, -4, and -6 are 12.8, 27.0, and 27.8 nM, respectively, indicating that calmodulin-2 has 2-fold higher affinity for the motor than calmodulin-4 and -6. Similar results were obtained using another assay that involves the binding of (35)S-labeled calmodulin isoforms to the motor. The binding saturation curves of the motor with calmodulin isoforms have confirmed that calmodulin-2 has 2-fold higher affinity to the motor. However, the affinity of calmodulin-4 and -6 isoforms for the motor was about the same. Based on these studies, we conclude that all calmodulin isoforms bind to the motor protein but with different affinities.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1993

Interaction sites on phosphorylase kinase for calmodulin.

Holophosphorylase kinase was digested with Glu-C specific protease; from the peptide mixture calmodulin binding peptides were isolated by affinity chromatography and identified by N-terminal sequence analysis. Two peptides originating from the alpha subunit, having a high tendency to form a positively charged amphiphilic helix and containing tryptophane, were synthesized. Additionally, a homologous region of the beta subunit and a peptide from the alpha subunit present in a region deleted in the alpha' isoform were also selected for synthesis. Binding stoichiometry and affinity were determined by following the enhancement in tryptophane fluorescence occurring upon 1:1 complex formation between these peptides and calmodulin. Finally, Ca2+ binding to calmodulin in presence of peptides was measured. By this way, the peptides alpha 542-566, alpha 547-571, alpha 660-677 and beta 597-614 have been found to bind specifically to calmodulin. Together with previously predicted and synthesized calmodulin binding peptides four calmodulin binding regions have been characterized on each the alpha and beta subunits. It can be concluded that endogenous calmodulin can bind to two calmodulin binding regions in gamma as well as to two regions in alpha and beta. Exogenous calmodulin can bind to two regions in alpha and in beta. A binding stoichiometry of 0.8 mol of calmodulin/alpha beta gamma delta promoter of phosphorylase kinase has been determined by inhibiting the ubiquitination of calmodulin with phosphorylase kinase. Phosphorylase kinase is half maximally activated by 23 nM calmodulin which is in the affinity range of calmodulin binding peptides from beta to calmodulin. Therefore, binding of exogenous calmodulin to beta activates the enzyme. A model for switching endogenous calmodulin between alpha, beta and gamma and modulation of ATP binding to alpha as well as Mg2+/ADP binding to beta by calmodulin is presented.

Amino Acid Sequence↗
PubMed1984

Isolation and characterization of calmodulin from the motile green alga Chlamydomonas reinhardtii.

Calmodulin, a calcium-binding protein with no known enzymatic activity but multiple, in vitro effector activities, has been purified to apparent homogeneity from the unicellular green alga Chlamydomonas reinhardtii and compared to calmodulin from vertebrates and higher plants. Chlamydomonas calmodulin was characterized in terms of electrophoretic mobility, amino acid composition, limited amino acid sequence analysis, immunoreactivity, and phosphodiesterase activation. Chlamydomonas calmodulin has two histidine residues similar to calmodulin from the protozoan Tetrahymena. However, unlike the protozoan calmodulin, only one of the histidinyl residues of Chlamydomonas calmodulin is found in the COOH-terminal third of the molecule. Chlamydomonas calmodulin lacks trimethyllysine but does have a lysine residue at the amino acid sequence position corresponding to the trimethyllysine residue in bovine brain and spinach calmodulins. The lack of this post-translational modification does not prevent Chlamydomonas calmodulin from quantitatively activating bovine brain phosphodiesterase. These studies also demonstrate that this unique calmodulin from a phylogenetically earlier eukaryote may be as similar to vertebrate calmodulin as it is to higher plant calmodulins, and suggest that Chlamydomonas calmodulin may more closely approximate the characteristics of a putative precursor of the calmodulin family than any calmodulin characterized to date.

Amino Acid Sequence↗
PubMedDate not supplied

Mutational analysis of calmodulin in Saccharomyces cerevisiae.

Calmodulin is well characterized as an intracellular Ca2+ receptor in nonproliferating tissues such as muscle and brain. Several observations indicate that calmodulin is also required for cellular growth and division. Deletion of the calmodulin gene is a lethal mutation in Saccharomyces cerevisiae, Schizosaccharomyces pombe and Aspergillus nidulans. Expression of calmodulin antisense RNA in mouse C127 cells causes a transient arrest at G1 and metaphase. Although these results indicate calmodulin plays a critical function during proliferation, they do not reveal the function. S. cerevisiae offers an excellent system for identifying calmodulin functions. Because calmodulin mutants can be readily constructed by gene replacement the consequences of mutations in calmodulin can be directly examined in vivo without interference from wild-type calmodulin. The available wealth of information concerning all aspects of the yeast life cycle provides a large framework for interpretation of new results. The recent dissection of cell cycle regulation is just the latest example of the important insights provided by analyzing basic cellular processes in yeast. Whether studies of calmodulin in yeast will reveal a universal function is unknown. One encouraging result is that yeast cells relying on vertebrate calmodulin as their only source of calmodulin survive and grow well, even if the amount of vertebrate calmodulin is equivalent to the normal steady state levels of yeast calmodulin. This review discusses the varied techniques we are using to identify the functions of calmodulin in yeast. As part of the analysis, we are defining the essential elements of calmodulin structure.

Calmodulin↗