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A G Filoteo

Publications and source records attributed to A G Filoteo.

At least 37 records · Page 2Linked to original sources
PubMed1996

Protein kinase C activates the plasma membrane Ca2+ pump isoform 4b by phosphorylation of an inhibitory region downstream of the calmodulin-binding domain.

The carboxyl-terminal region of the plasma membrane Ca2+ pump isoform 4b contains two autoinhibitory regions which keep the pump inactive in the absence of activators such as calmodulin. One of these regions is approximately coterminous with the calmodulin-binding domain, while the second region is downstream (Verma, A. K., Enyedi, A., Filoteo, A. G., and Penniston, J. T. (1994) J. Biol. Chem. 269, 1687-1691). The carboxyl-terminal region has also been identified as the site for phosphorylation of this isoform by protein kinase C (Wang, K. K. W., Wright, L. C., Machan, C. L., Allen, B. G., Conigrave, A. D., and Roufogalis, B. D. (1991) J. Biol. Chem. 266, 9078-9085). Using constructs lacking various numbers of residues at the carboxyl terminus, we studied the degree of phosphorylation by protein kinase C and the resultant activation of Ca2+ transport. The results showed that the most specific and easy phosphorylation occurred in a region of about 20 residues which is downstream of the calmodulin-binding domain, and that the downstream inhibitory domain had also about the same size and location. Phosphorylation partially activated the pump by removing only the inhibition due to this region. Binding of calmodulin to the calmodulin-binding domain activated the pump more fully by removing the inhibition due to both regions, regardless of the state of phosphorylation at the downstream inhibitory region.

Amino Acid Sequence↗
PubMed1996

The plasma membrane Ca2+ pump mutant lysine591 --> arginine retains some activity, but is still inactivated by fluorescein isothiocyanate.

Inactivation of the wild-type human plasma membrane Ca2+ pump (isoform 4b) by fluorescein isothiocyanate is accompanied by covalent modification of Lys591. The mutation of Lys591 to arginine reduced the Ca2+ transport activity to 35% of the wild-type, and diminished the amount of acylphosphate formed from ATP by a corresponding amount. When this mutant was treated with fluorescein isothiocyanate; the enzyme was still irreversibly inactivated, even though no reactive residue was available at position 591. The results show that, although Ca2+ pump function is sensitive to the residue at position 591, Lys591 is not essential for enzyme activity. They also demonstrate that irreversible inhibition of the plasma membrane Ca2+ pump by fluorescein isothiocyanate does not require the covalent modification of Lys591. This indicates that fluorescein isothiocyanate reacts with lysine residues at other positions in addition to Lys591.

Adenosine Triphosphate↗
PubMed1996

Detection of isoform 4 of the plasma membrane calcium pump in human tissues by using isoform-specific monoclonal antibodies.

The epitope location and specificity of monoclonal antibodies JA9, 5F10 and JA3, raised against the human plasma membrane Ca2+ pump (hPMCA), were analysed by using synthetic peptides of the corresponding epitopes as well as the complete isoforms, hPMCA4b, hPMCA4a and hPMCA1b, expressed in COS-1 cells. The experiments with the peptides showed that JA9 reacted specifically with a region containing residues 51-75 of hPMCA4 (a or b), but not with the same region of isoforms 1, 2 or 3. JA3 reacted with residues 1156-1180, a region unique to hPMCA4b. 5F10 reacted in the region of residues 719-738, which is highly conserved in all PMCA isoforms. Indeed, 5F10 recognized all three isoforms expressed in COS-1 cells. JA9, in contrast, reacted with both variants a and b of hPMCA4 but not with hPMCA1, and JA3 recognized exclusively hPMCA4b. We used these antibodies to discern the distribution of hPMCA4a and hPMCA4b in human brain, heart, kidney and lung. In Western blots of human brain samples, we could identify both hPMCA4a and hPMCA4b. Heart tissue also showed isoform 4b, and probably 4a. In contrast, kidney and lung showed primarily hPMCA4b. In brain, overlapping bands that did not correspond to either variant of hPMCA4 were detected, and in kidney a band migrating in the same position as hPMCA1b was observed. The distribution of the a and b forms of hPMCA4 at the protein level, as analysed by these antibodies, is consistent with the available data about the abundance of mRNAs for the hPMCA isoforms. The presence of hPMCA4b in all the samples supports the proposed role of this isoenzyme as a constitutive form of the pump.

Alternative Splicing↗
PubMed1996

Plasma membrane calcium pump isoform 4a has a longer calmodulin-binding domain than 4b.

Alternate splicing of human plasma membrane calcium pump isoform 4 (hPMCA4) transcripts causes the expression of two variants, hPMCA4a and hPMCA4b, which have different downstream regulatory regions. Of the two, hPMCA4a has a lower affinity for calmodulin and a lower effective affinity for Ca2+ (Enyedi, A., Verma, A. K., Heim, R., Adamo, H. P., Filoteo, A. G., Strehler, E. E., and Penniston, J. T. (1994) J. Biol. Chem. 269, 41-43). Additional consequences of the alternate splice were studied by analyzing the characteristics of constructs (expressed in COS-1 cells) containing different portions of the carboxyl terminus of hPMCA4a. Our results show striking differences in the structure of the calmodulin-binding and autoinhibitory domains of the two variants. The calmodulin-binding region of hPMCA4b is a region of about 28 residues, whereas that of hPMCA4a is about 49 residues long and is probably interrupted by a region not involved in the binding. The autoinhibitory region of hPMCA4b (a part of the downstream region that keeps the molecule inactive in the absence of Ca2+-calmodulin) is divided between the 28-residue calmodulin-binding region and a downstream region, whereas in hPMCA4a, all of it is contained within the 49-residue calmodulin-binding region.

Adenosine Triphosphate↗
PubMed1995

Mutants in the putative nucleotide-binding region of the plasma membrane Ca(2+)-pump. A reduction in activity due to slow dephosphorylation.

Mutants of individual residues of the plasma membrane Ca(2+)-pump were made in the highly conserved region that (in related P-type ATPases) has been associated with nucleotide binding. Alteration of the strictly conserved Asp672 to Glu nearly eliminated the ability of the pump to transport Ca2+, while alteration at Val674, Arg675, and Lys686 reduced the activity. High levels of ATP (25 mM) did not overcome the reduced activity, indicating that it could not be due to a reduction in the affinity for ATP. Effects not directly related to ATP binding seemed to result from mutations in this area. For instance, the amount of phosphorylated intermediate in the most severely inhibited mutant, Asp672-->Glu, was nearly as high as that in the wild type, a much larger amount of phosphorylated intermediate than was expected from its low activity. However, the rate of decomposition of this intermediate was much slower than that of the wild type, indicating that the inhibition of this mutant resulted from an inhibition of the E approximately P-->E step in the enzyme cycle.

Adenosine Triphosphatases↗
PubMed1995

Two residues that may ligate Ca2+ in transmembrane domain six of the plasma membrane Ca(2+)-ATPase.

In order to identify Ca2+ ligands in the putative transmembrane domain 6 of the plasma membrane Ca2+ pump, amino acids Asn879, Met882, Asp883, and Ser887 were singly altered. Asn879, Met882, and Asp883 were chosen because the corresponding amino acids have been proposed as Ca2+ ligands in the sarcoplasmic reticulum Ca2+ pump (Clarke, D. M., Loo, T. W., and MacLennan, D. H. (1990) J. Biol. Chem. 265, 6262-6267). For the alterations, a fully active truncated version of the pump was used, because the interaction of Ca2+ with the pump could be studied without interference from calmodulin binding. The mutants at Asn and Asp did not carry out ATP-supported Ca2+ uptake and formed no acylphosphate from [gamma-32P]ATP, suggesting that, like the corresponding amino acids in the sarcoplasmic reticulum Ca2+ pump, these two are Ca2+ ligands. However, all the mutants at the position of Met882 showed some activity. Indeed, the Met882--> Ile mutant was fully active at a saturating Ca2+ concentration and only the K1/2 for Ca2+ activation was shifted slightly upward. Converting the Met to Thr (which is the corresponding residue in the sarcoplasmic reticulum Ca2+ pump) reduced the activity to 20% of the wild type, further emphasizing the differences between the two Ca2+ pumps. The mutant Ser887--> Ala was expressed in greater amounts than, and had a specific activity about 50% higher than, the wild type, indicating that this serine also could not be a Ca2+ ligand and could not replace the missing Thr at position Met882.

Amino Acid Sequence↗
PubMed1994

Regulatory region of plasma membrane Ca2+ pump. 28 residues suffice to bind calmodulin but more are needed for full auto-inhibition of the activity.

The Ca(2+)-pumping activity of constructs containing various portions of the putative 28-residue calmodulin-binding domain (C domain) of hPMCA4b were compared. As the length of the C domain in the pump increased, the pump's activity decreased and the ability of calmodulin to stimulate the activity increased. Study of the calmodulin dependence of activity showed that the construct containing all 28 residues of the C domain had a K1/2 for calmodulin equal to that of the complete molecule; the constructs containing less of the C domain interacted less strongly with calmodulin. On the other hand, incorporation of all 28 residues of the C domain did not decrease the activity of the pump (in the absence of calmodulin) as low as the activity of the complete molecule. This indicates that other segments of the molecule, further toward the COOH terminus, are also required for the degree of inhibition seen in the complete molecule.

Amino Acid Sequence↗
PubMed1994

The Ca2+ affinity of the plasma membrane Ca2+ pump is controlled by alternative splicing.

The plasma membrane Ca2+ pump is a calmodulin-regulated P-type ATPase that is an essential element in controlling intracellular Ca2+ concentration. Studies on the gene structure of this pump have revealed an alternate splice option that changes the structure of the calmodulin-binding domain. This change in the structure of the enzyme results in a reduced calmodulin affinity. Tests of the enzyme's activity in the presence of a high calmodulin concentration, approximating that found inside living cells, show that this reduced calmodulin affinity causes a reduced apparent affinity of the enzyme for Ca2+. This shift in the Ca2+ activation occurs in a Ca2+ concentration range crucial to cellular function and is probably the physiologically important consequence of the alternate splice.

Alternative Splicing↗
PubMed1993

A highly active 120-kDa truncated mutant of the plasma membrane Ca2+ pump.

A truncated mutant (hPMCA4b(ct120)) of the plasma membrane Ca2+ pump was expressed in COS-1 cells. The full-length pump (hPMCA4b) consisted of 1205 residues, and the mutant lacked 120 residues (including the 28-residue calmodulin-binding inhibitory domain) at the COOH terminus. To characterize this construct, Ca2+ transport was determined in a microsomal fraction. Phosphate was added to increase Ca2+ uptake, and specificity was enhanced by adding thapsigargin to inhibit the endoplasmic reticulum Ca2+ pump. The mutant showed a similar level of expression as hPMCA4b and a Ca2+ affinity and Ca2+ transport activity about equal to that of hPMCA4b when hPMCA4b was activated. Addition of the synthetic peptide C28R2, corresponding to the calmodulin binding region of the pump, inhibited the mutant and restored the non-activated state of the enzyme. In these respects, the truncated mutant acted like hPMCA4b, when hPMCA4b had been proteolyzed to cleave a bond between the calmodulin binding region and the NH2-terminal portion of the molecule. This indicates that the effects of proteolysis are due to removal of the COOH terminus and not to rearrangement of the two fragments. Since the truncated mutant was fully active and its tryptic digestion resulted in the appearance of the expected 81- and 76-kDa active fragments, we concluded that the COOH-terminal portion which is missing cannot be important in synthesis or proper folding of the enzyme.

Alternative Splicing↗
PubMed1992

The lipid-binding peptide from the plasma membrane Ca2+ pump binds calmodulin, and the primary calmodulin-binding domain interacts with lipid.

Peptide G25 (KKAVKVPKKEKSVLQGKLTRLAVQI) representing the putative lipid-binding region (G region) of the erythrocyte Ca2+ pump was synthesized. This peptide interacted with acidic lipids, as shown by the increase in size of phosphatidylserine liposomes in its presence. This lipid interaction is consistent with the previous evidence suggesting that the portion of the pump from which this peptide was taken is responsible for the activation of the pump by acidic lipid. G25 also bound to calmodulin, as was shown by its cause of a shift in the fluorescence of 5-dimethylamino naphthalene-1-sulfonyl- (dansyl)-calmodulin, and by its competition with Ca2+ pump for calmodulin. Its Kd for dansyl-calmodulin was much higher (0.8 microM) than that of the peptides representing the primary calmodulin-binding region (C region) of the Ca2+ pump. Although the presence of the G region provided the possibility of a second calmodulin-binding site, activation of the pump by calmodulin always could be fitted by simple saturation kinetics. The calmodulin-binding peptide from the C region of the pump, C28R2, also interacted with lipid with even greater effectiveness than G25. When the C region of the pump was saturated with calmodulin, acidic lipid activation of the pump followed simple saturation kinetics. However, when calmodulin was omitted, a higher concentration of lipid was needed for saturation and the kinetics became complex. The data are consistent with the idea that calmodulin activates the pump only by interaction at the C region, but that acidic lipid activates by interaction at both of the C and G regions.

Animals↗
PubMed1991

Calmodulin-binding domains from isozymes of the plasma membrane Ca2+ pump have different regulatory properties.

Peptides C28R2 and C28R1A, representing the two main alternative classes of calmodulin-binding domains from the plasma membrane Ca2+ pump, were tested for their calmodulin-binding properties and for their capacity to interact with pump from which the calmodulin-binding domain had been removed by chymotryptic proteolysis. Peptide C28R2 was more effective in both capacities. Binding of peptide to calmodulin was measured by competition experiments. Such experiments indicated that Ki for C28R2 as an inhibitor of the pump-calmodulin interaction was 0.1 nM, whereas C28R1A had a Ki of 1 nM. Interaction of peptide with chymotryptically activated Ca2+ pump was measured by observing the inhibition by peptide of active Ca2+ transport into inside-out membrane vesicles at low Ca2+. Those experiments showed that C28R2 interacted relatively strongly (an IC50 of 1 microM), whereas C28R1A had an IC50 of 15 microM. The calmodulin-binding peptides had effects on both the K1/2 for Ca2+ and the Vmax of the proteolyzed pump. The effects on the K1/2 for Ca2+ were related to the net plus charge on the peptide, with the most positive peptides being most effective in competing with Ca2+. The substantial differences between C28R2 and C28R1A suggest that Ca2+ pumps containing calmodulin-binding domains like C28R1A have lower calmodulin affinities and higher activities in the absence of activator.

Amino Acid Sequence↗
PubMed1991

Identification of a peptide inhibitor of the cardiac sarcolemmal Na(+)-Ca2+ exchanger.

The deduced amino acid sequence of the cardiac sarcolemmal Na(+)-Ca2+ exchanger has a region which could represent a calmodulin binding site. As calmodulin binding regions of proteins often have an autoinhibitory role, a synthetic peptide with this sequence was tested for functional effects on Na(+)-Ca2+ exchange activity. The peptide inhibits the Na(+)-dependent Ca2+ uptake (KI approximately 1.5 microM) and the Nao(+)-dependent Ca2+ efflux of sarcolemmal vesicles in a noncompetitive manner with respect to both Na+ and Ca2+. The peptide is also a potent inhibitor (KI approximately 0.1 microM) of the Na(+)-Ca2+ exchange current of excised sarcolemmal patches. The binding site for the peptide on the exchanger is on the cytoplasmic surface of the membrane. The exchanger inhibitory peptide binds calmodulin with a moderately high affinity. From the characteristics of the inhibition of the exchange of sarcolemmal vesicles, we deduce that only inside-out sarcolemmal vesicles participate in the usual Na(+)-Ca2+ exchange assay. This contrasts with the common assumption that both inside-out and right-side-out vesicles exhibit exchange activity.

Amino Acid Sequence↗
PubMed1990

Peptide sequence analysis and molecular cloning reveal two calcium pump isoforms in the human erythrocyte membrane.

The sequence of more than 1,000 amino acid residues, derived from two different isoforms, has been determined from peptides generated from purified human erythrocyte membrane Ca2(+)-ATPase (hPMCA). Several of these peptide sequences correspond to the previously reported, cDNA deduced sequence of the "teratoma" Ca2+ pump isoform hPMCA1 (Verma, A. K., Filoteo, A. G., Stanford, D. R., Wieben, E. D., Penniston, J. T., Strehler, E. E., Fischer, R., Heim, R., Vogel, G., Matthews, S., Strehler-Page, M.-A., James, P., Vorherr, T., Krebs, J., and Carafoli, E. (1988) J. Biol. Chem. 263, 14152-14159). The complete primary structure of a novel isoform (hPMCA3) has been determined by molecular cloning and nucleotide sequencing of its corresponding cDNA. This new member of the plasma membrane Ca2+ pump family consists of 1,205 amino acid residues with a calculated Mr of 133,930, and it shows 88% similarity (75% identity) with the previously sequenced pump isoform. Specific probes detect major mRNA species of 5.6 kilobases for hPMCA1, and of 7.5 kilobases for hPMCA3, on Northern blots of human K562 erythroleukemic cell RNA. A large number of peptide sequences match perfectly with only one or the other of these isoforms and all peptides (with 6 exceptions corresponding to a contaminant protein or to a third minor Ca2+ pump isoform) are found in either only one or in both of the isoforms. The two erythrocyte Ca2+ pumps display high sequence divergence in a few localized regions that may determine isoform-specific functional specializations; for example, the putative extracellular loop separating transmembrane domains 1 and 2, the highly negatively charged region previously suggested to be involved in Ca2+ binding, and the site of cAMP-dependent protein kinase phosphorylation.

Amino Acid Sequence↗
PubMed1989

The calmodulin binding domain of the plasma membrane Ca2+ pump interacts both with calmodulin and with another part of the pump.

Synthetic peptides corresponding to the calmodulin-binding domain of the human erythrocyte Ca2+ pump were prepared representing residues 2-29 (C28W), 2-21 (C20W), 2-16 (C15W), and 16-29 (C14) of the sequence (James, P., Maeda, M., Fisher, R., Verma, A. K., Krebs, J., Penniston, J. T., and Carafoli, E. (1988) J. Biol. Chem. 263, 2905-2910). Peptides C28W, C20W, and C15W bound to calmodulin with an apparent 1:1 stoichiometry in the presence of Ca2+ and inhibited the activation of the Ca2+ pump by calmodulin, while C14 was ineffective. Substituting tyrosine (C28Y) or alanine (C28A) for the tryptophan residue lowered the affinity for calmodulin. The estimated Kd values for the calmodulin-peptide complexes were 0.1 nM for C28W, 5-15 nM for C20W, C28Y, and C28A, and 700-1700 nM for C15W. The Ca2+ pump in inside-out erythrocyte membrane vesicles was activated by proteolytic removal of the endogenous calmodulin-binding domain. Addition of C20W or C28W then inhibited calmodulin-independent Ca2+ transport, while a calmodulin-binding peptide from another enzyme had no effect. The inhibition of the pump by C20W was purely competitive with Ca2+, while C28W decreased the Vmax and increased the K1/2 for Ca2+, restoring the pump activity nearly to its low basal level. The results suggest that a calmodulin-binding peptide from any enzyme has two kinds of specificity: it shares with peptides from other enzymes the ability to bind to calmodulin, but only it has the specificity to interact with its own (proteolytically activated) enzyme.

Amino Acid Sequence↗
PubMed1988

Complete primary structure of a human plasma membrane Ca2+ pump.

cDNAs coding for a plasma membrane Ca2+ pump were isolated from a human teratoma library and sequenced. The translated sequence contained 1,220 amino acids with a calculated molecular weight of 134,683. All regions of functional importance known from other ion-transporting ATPases could be identified. The translated sequence also contained, near the carboxyl terminus, the calmodulin-binding domain and two domains which are very rich in glutamic acid and aspartic acid. These two domains resemble calmodulin somewhat and one of them may play a role in the binding of Ca2+. The enzyme also contains domains rich in serine and threonine, one of which has a sequence matching those of good cAMP-dependent protein kinase substrates. The carboxyl-terminal region is important for regulation by calmodulin, proteolysis, and phosphorylation. Near the amino terminus are two domains which are very rich in lysine and glutamic acid, as well as two domains resembling EF hands, one of which also has some resemblance to calmodulin. Comparison of the cloned sequence with peptide sequences from the erythrocyte Ca2+ pump showed that the two proteins have a very high proportion of identical residues but are not 100% identical, indicating that they represent different isozymes.

Amino Acid Sequence↗
PubMed1987

The ATP-binding site of the erythrocyte membrane Ca2+ pump. Amino acid sequence of the fluorescein isothiocyanate-reactive region.

The erythrocyte plasma membrane Ca2+-pumping ATPase is known to form an acyl-phosphate catalytic intermediate, but there is otherwise little structural information linking it to the other mammalian ion-pumping ATPases which also form phosphorylated intermediates (the Na+, K+-ATPase of plasma membranes, the Ca2+-ATPase of sarcoplasmic reticulum, and the H+, K+-ATPase of gastric mucosa). We show here that this enzyme possesses a fluorescein isothiocyanate-reactive region similar to that possessed by these other ATPases. Low concentrations (10 microM) of fluorescein isothiocyanate inhibit the ATPase activity of this pump, and this inhibition is prevented by 4 mM ATP. ATP also inhibits the reaction of fluorescein isothiocyanate with a single amino acid residue on the 138-kDa polypeptide chain. A tryptic fragment containing the fluorescein-conjugated residue was isolated by high pressure liquid chromatography. The sequence of this peptide was determined to be NH2-Met1-Tyr2-Ser3-Lys4-Gly5-Ala6-Ser7-Glu8++ +-Ile9-Ile10-Leu11-Arg12-COOH; fluorescein isothiocyanate reacts with the lysine residue. The identities of residues 4-8 are the same as those in a sequence common to the other ATPases mentioned above, except that serine-7 of this sequence is changed to a proline in those ATPases. This substitution, sometimes not considered a homologous one, is not expected to have a major effect on the secondary structure or polarity of this region. Outside of this 5-residue core region of the fluorescein isothiocyanate-reactive site, the homologies among the different ion-pumping ATPases are limited.

Adenosine Triphosphatases↗
PubMed1984

Regulation of plasma membrane Ca2+ ATPases by lipids of the phosphatidylinositol cycle.

When the erythrocyte plasma membrane Ca2+ pump is reconstituted into phosphatidylcholine liposomes, the inclusion of small amounts of phosphatidic acid or phosphatidylinositol 4,5-bisphosphate stimulates the enzyme's activity. Other lipids of the phosphatidylinositol cycle (diacylglycerol, phosphatidylinositol) have little effect. The stimulatory effect of phosphatidylinositol 4,5-bisphosphate is greater than that of calmodulin; this lipid also stimulates the plasma membrane Ca2+ ATPase from rat brain.

Animals↗