©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Tissue Distribution of the Four Gene Products of the Plasma Membrane Ca Pump
A STUDY USING SPECIFIC ANTIBODIES(*)

Thomas P. Stauffer , Danilo Guerini , Ernesto Carafoli (§)

From the (1) Laboratory for Biochemistry, Swiss Federal Institute of Technology (ETH), 8092 Zurich, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Antibodies against the four isoforms of the human plasma membrane Ca-ATPase (PMCA) were raised using an N-terminal sequence of the pump as epitope. The antibodies against PMCA isoforms 1, 2, and 3 were not species-specific, e.g. they also recognized the corresponding proteins in rat, whereas that against the human PMCA isoform 4 failed to do so.

The tissue distribution of the four isoforms was estimated by Western blot analysis. Two, PMCA1 and PMCA4, were expressed in all tissues tested (with the exception of the choroid plexus, where the former was not detected). In most tissues the signal from the PMCA1 protein exceeded that of PMCA4, the exception being the erythrocyte. The PMCA2 and PMCA3 proteins were only found in neuronal tissues; the PMCA2 protein was present in high concentrations in the cerebellum and in the cerebral cortex. At variance with previous results on mRNA (e.g. the kidney) no other tissues contained the PMCA2 protein. PMCA3 was the other tissue-specific isoform; in agreement with results in the rat, the protein was found in human neuronal tissues, particularly in the choroid plexus, but was practically absent in all other tissues tested.


INTRODUCTION

The Ca pump of the plasma membrane (PMCA)() (Schatzmann, 1966) is widely expressed in human tissues (Carafoli and Stauffer, 1994). Given its very high affinity for Ca, the enzyme is generally assumed to be responsible for the fine tuning of cellular Ca. Most of the biochemical data on the pump have been obtained on erythrocytes (Carafoli, 1992) although the enzyme has also been studied in detail in heart sarcolemma (Caroni and Carafoli, 1981). The biochemical study of the pump is made difficult by its very low amount in most plasma membranes (e.g. in the erythrocytes the pump represents less than 0.1% of the total membrane protein (Knauf et al., 1974)). Shortly after the sequence of the pump was deduced, it became clear that the enzyme is encoded by different genes. After the cloning of two cDNAs (PMCA1 and PMCA2) in the rat (Shull and Greeb, 1988) and of one in human (PMCA1) (Verma et al., 1988), two other cDNAs became available: rat PMCA3 (Greeb and Shull, 1989) and human PMCA4 (Strehler et al., 1990). It is now accepted that four genes encode the PMCA in humans and in the rat (Keeton et al., 1993; Stauffer et al., 1993). They have been located to human chromosomes 12 (isoform 1) (Olson et al., 1991), 3 (isoform 2) (Brandt et al., 1992a; Latif et al., 1993), X (isoform 3) (Wang et al., 1994), and 1 (isoform 4) (Olson et al., 1991). The number of possible PMCA transcripts of the four genes increased after the finding of alternative splicing at three independent sites (Carafoli and Guerini, 1993; Howard et al., 1993); the number of potential transcripts would now correspond to more than 30 independent proteins. Extensive studies on the mRNA level have resulted in the detailed description of all possible splicing options and products (Brandt et al., 1992b; Keeton et al., 1993; Stauffer et al., 1993). Quantitative information on the distribution of the mRNA of the four different genes has been obtained in humans, leading to the proposal that PMCA1 and PMCA4 are the housekeeping pump isoforms, whereas PMCA2 and PMCA3 are more specialized (i.e. they have a restricted tissue transcription pattern (Stauffer et al., 1993)). In situ hybridization work in rat brain has shown that the transcription of three PMCA isoforms (1, 2, and 3) (Stahl et al., 1992) has a striking regional distribution pattern. However, the data on the mRNA level have not yet been confirmed on the protein level.

A comparison of the results on the mRNA and those on the protein level requires antibodies recognizing the different isoforms. In this study they were raised using as epitopes the N-terminal regions of the four different isoforms, since the latter differ substantially in their first 80-90 amino acids. The antibodies have revealed that PMCA2 was expressed at very high levels in the brain, particularly in the cerebellum, where it probably represented the largest portion of the pump protein. In humans, PMCA3 was only detected in neuronal tissues, particularly high amounts of it being present in the choroid plexus. In all human tissues tested PMCA1 was present in higher concentration than PMCA4; the erythrocyte membranes were the exception (i.e. in them PMCA4 was much more abundant than PMCA1).


EXPERIMENTAL PROCEDURES

Materials

The tissues were obtained from one 45-year-old and one 50-year-old human. All tissues were from autopsy material not older than 12 h after death and were provided by Dr. C. Moll (Division of Neuropathology, University Hospital, Zurich, Switzerland). The tissues were frozen in liquid nitrogen and kept at -80 °C until used.

The organs of white Wistar rats were removed after decapitation, frozen in liquid nitrogen, and kept at -80 °C until used. Cell lines were obtained from the American Type Culture Collection (ATCC; Rockville, MD).

Generation of the Isoform-specific Antibodies

Pump segments N-terminal to the first transmembrane domain (isoform 1, amino acids 1-88; isoform 2, amino acids 1-96; isoform 3, amino acids 1-83; isoform 4, amino acids 1-84) (Strehler, 1991) were expressed in bacteria as described below and used to raise antibodies (see Fig. 1and ). These domains were chosen because of their low degree of homology (58-65% identity) in the four human isoforms. They are reasonably conserved among different species (91-96% identity) and are not subjected to alternative splicing. An antibody recognizing all isoforms was also raised against a region located at the end of the second large intracellular loop of the pump (amino acids 765-835 of isoform 2) between transmembrane domains 4 and 5 (see Fig. 1).


Figure 1: A membrane topology model for the PMCA pump. , the N-terminal region chosen for the production of isoform-specific antibodies. The sequence of this region had the lowest degree of homology among the four gene products. , the portion of the second large intracellular loop of the pump that was selected to generate the antibody against all isoforms.



Cloning and Expression

The human cDNAs were cloned into pRSET expression vectors (Invitrogen, San Diego, CA) (Stueber et al., 1990). To facilitate the purification of the expressed products, the cDNAs were inserted after a sequence coding for six His residues (His-tag) and an enterokinase recognition site. The cDNAs used for the expression of the epitopes of isoforms 2 and 4 were taken from the corresponding full-length cDNA constructs, used for the overexpression in Sf9 cells, containing a BamHI site in the 5`-untranslated region (Heim et al., 1992; Hilfiker et al., 1994). They were cloned into the vector type C (isoform 2) or B (isoform 4) between the BamHI and PstI site (isoform 2) or the DpnI site (isoform 4; see ). The cDNA for isoform 1 was obtained by PCR-mediated mutagenesis using the clone t5.13 (Verma et al., 1988) as a template, with primers containing artificial restriction sites (BglII and EcoRI) (). The sequence was cloned into a vector type C. The epitope of isoform 3 (), was obtained by PCR amplification (Stauffer et al., 1993) of a partial 5`-sequence with primers containing BglII and EcoRI restriction sites. The fragment for the general antibody was obtained by restriction digestion with BamHI and NcoI (nucleotide positions 2294-2507) of the full-length cDNA of isoform 2 (Heim et al., 1992) and cloned into a vector type C (). The resulting peptides normally contained less than 20% of non-PMCA sequences (His-tag, enterokinase cleavage site) at the N-terminal part. The expression of the fusion protein was performed according to the manufacturer's protocol (Invitrogen).

Purification of the Fusion Proteins

After disrupting the bacteria with a French press (1000 bar), the pellets containing the insoluble recombinant peptides (peptides against PMCA1-4) were dissolved in 6 M guanidine hydrochloride in phosphate buffer (0.1 M NaHPO, 0.01 M Tris-HCl, pH 8.0) and loaded on nickle chelate columns (Ni-NTA, Quiagen, Chatsworth, CA). After washing with 8 M urea in phosphate buffer (0.1 M NaHPO, 0.01 Tris-HCl, pH 8.0 and 6.3; 10 volumes for each wash), the peptides were eluted at pH 5.9 with the same buffer. The peptide used for the generation of the general antibody was recovered in the supernatant after breaking the cells and loaded on the column under nondenaturing conditions (phosphate buffer: 50 mM NaHPO, pH 7.8, 300 mM NaCl). The column was washed with 20 volumes of phosphate buffer A (50 mM NaHPO, pH 6.0, 300 mM NaCl, 10% glycerol). The fusion peptide was eluted with a 0.1-0.5 M gradient of imidazole in buffer A. All of the peptides were dialyzed against a phosphate buffer (50 mM NaHPO, pH 7.8, 300 mM NaCl) in the presence of 0.5 M urea.

Immunization of the Rabbit

150 µg of the peptides were injected subcutaneously in New Zealand White rabbits. A first boosting was performed 6 weeks after the injection, and the animal was bled 2 weeks later.

Affinity Purification of the Immune Sera

The sera were purified by affinity chromatography with the corresponding peptide fragments coupled to CNBr-activated Sepharose 4B (Pharmacia Biotech Inc.) after passing them over a column of an unrelated peptide containing the His-tag and the enterokinase recognition sequence. They were diluted four times in phosphate buffer (50 mM NaHPO, pH 8.0, 340 mM NaCl). The column was washed with 10 volumes of phosphate buffer (50 mM NaHPO, pH 8.0, 500 mM NaCl). The antibodies were eluted with 0.2 M glycine, pH 2.3, and 500 mM NaCl and dialyzed overnight in phosphate buffer (50 mM NaHPO, pH 8.0, 340 mM NaCl).

SDS-polyacrylamide Gel Electrophoresis and Western Blot Analysis

The proteins were separated by SDS-polyacrylamide gel electrophoresis (Laemmli, 1970) or by a Tricine gel (Schägger and von Jagow, 1987). The proteins were transferred to PVDF membranes (Millipore, Bedford, MA) (Towbin et al., 1979). They were blocked overnight with 1% bovine serum albumin and incubated with the primary antibody (diluted 1/1000-1/500 in TBS-T (10 mM Tris-HCl, pH 7.0, 500 mM NaCl, 0.05% Tween-20, 0.1% bovine serum albumin)) for 1 h. The incubation with the secondary antibody (alkaline phosphatase coupled to an anti-rabbit antibody; Promega Corp., Madison, WI) and the staining were performed according to the manufacturer's protocol (ProtoBlot AP, Promega Corp.).

Sensitivity of the Antibodies

The detection limits were determined by transferring different quantities of the fusion proteins to PVDF membranes. The antibodies recognized as little as 5-10 ng of the fusion protein. As indicated in Fig. 5, antibodies 4N, 1N, and 2A clearly detected the PMCA in erythrocyte ghosts, where no more than 30 ng of pump could be expected if 30 µg of membrane proteins were used. To compare the relative sensitivity of the antibodies 200 ng of the fusion protein were separated by a Tricine gel, transferred to nitrocellulose membranes, and incubated with the corresponding antibody (diluted 1/500). After incubation with I-labeled secondary antibody (donkey anti-rabbit; Amersham Corp.), the nitrocellulose-bound radioactivity was quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The sensitivity was estimated from the amount of bound radioactivity. The amount of fusion protein used did not result in the saturation of the signal. Equally strong signals were found with the antibodies against isoforms 1, 3, and 4, whereas the signal was slightly weaker with antibody 2N (results not shown).


Figure 5: PMCA isoforms in human cell lines and erythrocyte ghosts. Membrane proteins of different cell lines and erythrocyte ghosts were prepared as described under ``Experimental Procedures'' and tested for the presence of the four PMCA isoforms using the specific antibodies under the same conditions as in Fig. 4. A, human erythrocyte ghosts (30 µg of protein); B, HeLa cells (60 µg of protein); C, 293 cells (transformed primary embryonic kidney) (60 µg of protein). Lane1, antibody 1N; lane2, antibody 2N; lane3, antibody 3N; lane4, antibody 4N; lane5, antibody 2A.



Isolation of Plasma Membranes from Tissues and Cells

1 g of tissue was homogenized with a Polytron homogenizer in buffer B (0.1 M KCl, 0.05 M Hepes NaOH, pH 7.0, 0.4 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol), and the homogenate was centrifuged at 800 g for 10 min at 4 °C. The supernatant was centrifuged at 8000 g for 10 min at 4 °C, and the fraction containing the plasma membrane was sedimented for 1 h at 100,000 g (4 °C). The pellet was resuspended in a small volume of buffer B, and the protein concentration was determined according to Bradford(1976). The preparation of crude membranes from Sf9 cells was performed as described previously (Heim et al., 1992). The preparation of human erythrocyte ghosts was performed according to Niggli et al.(1987).

Coupled Enzyme Assay and Formation of the Phosphoenzyme Intermediate

The coupled enzyme assay was performed as described previously (Niggli et al., 1979). For the formation of the phosphoenzyme intermediate, the membranes were resuspended in 20 mM MOPS-KOH, pH 6.8, 80 mM KCl, 200 µM CaCl with or without 200 µM LaCl. The membrane suspension was placed on ice, and the reaction was started by adding 0.3-0.4 µM [-P]ATP (300 Ci/mmol). After 30 s the reaction was stopped by adding 7% trichloroacetic acid, 1 mM potassium phosphate (P). The denatured proteins were collected by centrifugation (6000 g) and washed again with 7% trichloroacetic acid, 1 mM P. They were separated on an acidic SDS-polyacrylamide gel (Sarkadi et al., 1986), stained with Coomassie Brilliant Blue, dried, and exposed at -70 °C for 24-48 h for autoradiography.

RESULTS

Nomenclature

The isoform-specific antibodies are designated by the number of the isoform followed by an N (e.g. antibody specific for isoform 1 = 1N). The antibody recognizing all four isoforms is named 2A.

Expression and Purification of the Peptides Used for the Generation of the Antibodies

In the first attempts to express the peptides encompassing the N-terminal region of the different pump isoforms, some were cloned in the expression vector with parts of the first transmembrane domain. However, none of these constructs expressed the encoded peptide in significant amounts. The N-terminal peptides were thus truncated some amino acids upstream of the first transmembrane domain (see Fig. 1).

All peptides were expressed at high levels (see Fig. 2as an example) but were insoluble in normal buffer. They were thus dissolved in 6 M guanidinium HCl. The peptide encompassing the C-terminal region of the second intracellular loop used to produce the antibody that recognized all four isoforms (see Fig. 1) was instead soluble. As mentioned under ``Experimental Procedures,'' a His-tag was added to the N terminus of the peptides to permit their purification. The expression and purification of the 1N peptide is shown as an example in Fig. 2 ; the peptide could be purified to homogeneity, as judged by SDS-polyacrylamide gel electrophoresis (Fig. 2, lane2). All four expressed N-terminal peptides were dialyzed after purification against phosphate buffer in the presence of 0.5 M urea to prevent their precipitation.


Figure 2: Purification of the peptides. A, the unpurified (lane1) and the purified N-terminal peptide (lane2) of isoform 1 were separated on a Tricine gel and stained with Coomassie Brilliant Blue.



Characterization of the Antibodies

The antibodies were affinity-purified against the corresponding peptides. To remove antibodies against the His-tag and the other sequences not specific for the PMCA isoforms, the sera were passed before affinity purification over a column of an unrelated peptide containing all these sequences.

It was important to show that the antibodies reacted only with the corresponding human pump isoforms. PMCA1CI() (for the nomenclature see Carafoli(1994)), PMCA2CI (Hilfiker et al., 1994), and PMCA4CI (Heim et al., 1992) were expressed with the help of the baculovirus system (see ``Experimental Procedures''). Crude membranes of Sf9 cells containing 200-300 ng of protein of the pump isoforms were transferred to PVDF membranes and incubated with the affinity-purified antibodies (Fig. 3A). Antibody 2A reacted equally well with all three expressed pump isoforms, whereas the N-terminal antibodies recognized only the corresponding isoform. Since the full-length cDNA of human PMCA3 is not yet available, isoform 3 could not be expressed, i.e. a direct test of this isoform was not possible. However, as indicated in Fig. 4, 5, and 7, the pattern of the recognized protein did not fit with that of the other isoforms. The production of the antibody against PMCA3 was more difficult, and different sera were thus produced until a satisfactory signal could be obtained. The experiments in Fig. 3B demonstrate that the antibodies used in this study only recognized the corresponding isoform even when other isoforms would be present in large excess. Antibodies 1N and 2N were tested in Western blot on 2 µg of overexpressed PMCA2CI. The 2N antibody reacted only with the corresponding protein (Fig. 3B, lane1), whereas no reaction was observed with the 1N antibody (Fig. 3B, lane2) despite the 60% identity of the N-terminal sequences of the two isoforms. The additional bands detected (Fig. 3B, lane1) were due to the high amount of protein present in the Western blot but were all specific for PMCA2CI (Fig. 3B, compare lane1 with lanes2 and 3). As controls equal amounts of proteins of crude membranes of Sf9 cells expressing the SERCA pump were tested with antibody 2N. No signal was detected (Fig. 3B, lane3).


Figure 3: Isoform specificity of the antibodies. A, the isoform specificity of the affinity-purified antibodies was tested on the PMCA pump overexpressed in Sf9 cells. Lane1, 200 ng of hPMCA1; lane2, 200 ng of hPMCA2; lane3, 200 ng of hPMCA4. The antibodies (1N, 2N, 3N, 4N, and 2A) were diluted 1/1000. B, the antibody specificity was tested in the presence of larger amounts of the other isoforms. Lanes1 and 2, 10 µg of membrane proteins of Sf9 cells overexpressing hPMCA2, corresponding to 2 µg of the pump, were incubated with antibody 2N (dilution: 1/500) (lane1) and with antibody 1N (dilution: 1/500) (lane2). Lane3, 10 µg of Sf9 membranes overexpressing the SERCA pump incubated with antibody 2N (dilution: 1/500).




Figure 4: Isoforms of the pump in human neuronal tissues. Membrane proteins of different tissues (prepared as described under ``Experimental Procedures'') were tested for the presence of the four isoforms of the pump. A, cerebellum (60 µg of protein); B, cerebral cortex (lobus occipitalis; 60 µg of protein); C, choroid plexus (90 µg of protein). Lane1, antibody 1N (dilution: 1/500); lane2, antibody 2N (dilution: 1/500); lane3, antibody 3N (dilution: 1/500); lane4, antibody 4N (dilution: 1/500); lane5, antibody 2A (dilution: 1/500).



Inhibitory Effects of the Antibodies

The possible inhibitory effect of the antibodies on the activity of the pump was tested using erythrocyte ghosts or membranes from Sf9 cells overexpressing the PMCA1 isoform. 2.2 pmol of the enzyme were preincubated with an equimolar amount or with a 5-10-fold excess of the corresponding antibody (erythrocyte ghosts: 2A, 4N, 2N; membranes of Sf9 cells: 2A, 1N, 2N). As a negative control antibody 2N was used, because isoform 2 is not expressed in these cells (see Fig. 5A). The activity of the enzyme was determined using the coupled enzyme assay. None of the antibodies affected the activity or the calmodulin-stimulated ATPase activity of the pump (results not shown).

PMCA Isoforms in Human Neuronal Tissues

Earlier results (Shull and Greeb, 1988; Greeb and Shull, 1989; Brandt et al., 1992b; Keeton et al., 1993; Stauffer et al., 1993) had shown that large amounts of transcripts of pump isoforms 2 and 3 were present in neuronal tissues. Membrane proteins of human cerebellum were thus separated by SDS-polyacrylamide gel electrophoresis, transferred to PVDF membranes, and incubated with the different antibodies; Fig. 4A shows that all isoforms were clearly detected. Using antibody 1N, two bands of equal intensity and of approximate molecular mass of 130 and 135 kDa were detected (Fig. 4A, lane1). The antibody specific for isoform 2 recognized proteins of molecular masses of about 130 and 135 kDa (Fig. 4A, lane2); the two proteins were apparently present in equivalent amounts. Two bands of equal intensity of about 130 and 135 kDa were found using antibody 3N, although the signal was much weaker then that of PMCA2. Only a single band of molecular mass of about 134 kDa was on the other hand visualized by the antibody specific for PMCA4 (Fig. 4A, lane4). The general antibody (2A) detected two proteins of molecular masses similar to those detected by antibodies 1N and 2N (Fig. 4A, lane5).

All isoforms were detected also in the human occipital cerebral cortex. A single protein band of approximate molecular mass of 135 kDa was revealed in the case isoform 1 (Fig. 4B, lane1). The 2N antibody recognized three proteins of molecular masses of about 130, 135, and 138 kDa, respectively, that of about 135 kDa being the most abundant (Fig. 4B, lane2). A major protein and a minor one of about 135 or 130 kDa, respectively, were detected with the antibody specific for isoform 3 (Fig. 4B, lane3), whereas antibody 4N revealed one major protein of about 134 kDa and a minor one of about 130 kDa (Fig. 4B, lane4). The bands obtained with the general antibody corresponded to those revealed by the isoform-specific antibodies (Fig. 4B, lane5). Additional bands in the range 85-130 kDa were also visualized in the occipital cortex, very likely representing degradation products.

Since the transcript of isoform 3 had been detected by in situ hybridization in relatively high amounts in the choroid plexus (Stahl et al., 1992), human choroid plexus were examined. The tissue was positive for PMCA3 and PMCA4 (Fig. 4C, lanes3 and 4). Antibody 3N recognized proteins of approximate molecular masses of 130-135 kDa. Antibody 4N recognized three proteins of molecular masses of about 134, 130, and 116 kDa, the last probably being a proteolytic product of the 130-134-kDa bands. As expected, two proteins corresponding to those revealed by antibodies 3N and 4N were detected with the 2A antibody (Fig. 4C, lane5). No signals of the expected molecular masses were detected with the 1N and 2N antibodies (Fig. 4C, lanes1 and 2).

hPMCA Isoforms in Non-neuronal Tissues

Human erythrocyte ghosts were isolated as described previously (Niggli et al., 1987). A clear single signal was detected in them with antibodies 4N, 1N, and 2A (Fig. 5A, lanes1, 4, and 5). The bands had the same approximate molecular mass (135 kDa). No signals were detected when the membranes were incubated with the antibodies for isoforms 2 and 3 (Fig. 5A, lanes2 and 3). In HeLa cell membranes, isoforms 1 (135 kDa) and 4 (134 kDa) (but not isoforms 2 and 3) were detected (Fig. 5B, lanes1-4). The general antibody revealed a single band of about 135 kDa (Fig. 5B, lane5). In 293 cells (transformed primary human embryonic kidney cells) proteins of about 135-kDa molecular mass were only detected with antibodies 1N and 2A (Fig. 5C, lanes1 and 5). In human smooth muscle cells only a positive signal of similar molecular mass (135 kDa) was found with antibodies 1N and 2A (result not shown). The proteins of the human kidney, heart, liver, and lung were found to be already significantly degraded in the samples used for the experiments. This made the identification of the PMCA, which is a low abundance protein, very difficult. Nevertheless, two bands responsive to the antibody for isoform 4 (130 and 134 kDa) were detected in human kidney and one (134 kDa) in heart (results not shown, ). No PMCA2 or PMCA3 proteins were detected in these tissues.

The specificity of the bands detected by the antibodies in human plasma membrane fractions were further tested by preabsorbing the antibodies with the peptides toward which they had been raised. After preabsorbtion no signals could be detected.

Analysis of the Phosphoenzyme Intermediate of the Pump

The plasma membrane Ca pump is predicted to be present in unusually high amounts in neuronal tissues based on the work on its transcripts (Stauffer et al., 1993). The prediction was supported on the protein level by the results of this study. In addition, the neuronal tissue that appeared to have high amounts of PMCA was the cerebellum and the cerebral cortex (results not shown). To verify these observations by an independent assay, membranes obtained from human cerebellum, brainstem, and erythrocytes were phosphorylated under conditions favoring the detection of the phosphoenzyme intermediate of the pump (i.e. in the presence of La) (Carafoli and Guerini, 1993). As a control, membranes of erythrocytes were used because the only pump phosphoenzyme intermediate detected in them was the one of the PMCA protein. Fig. 6 shows that under the experimental conditions a specific radioactive band of 135 kDa was observed in erythrocytes (Fig. 6, lane1) and in the brainstem (Fig. 6, lane3), whereas two bands of 130-135 kDa were detected in the cerebellum (Fig. 6, lane2). The bands were not seen if the phosphorylation was carried out in the absence of La (results not shown). Other radioactive bands, however, were also observed; that of 105-110 kDa, which was absent in the erythrocytes (Fig. 6, lane1), was slightly inhibited by La, which is typical of the SERCA pump. The other radioactive bands seen in the brainstem were not specific for the PMCA. A densitometric quantification revealed that the amount of the PMCA phosphoenzyme intermediate in cerebellum (0.88 scanning units) was about 3 times higher then in the brainstem (0.33 scanning units). This was in good agreement with the findings with the antibodies.


Figure 6: Ca-dependent phosphoenzyme formation from ATP. 80 µg of protein of human erythrocyte membranes (lane1), 70 µg of protein of human cerebellum (lane2), and human brainstem (lane3) were phosphorylated in the presence of 200 µM of Ca and 200 µM of La (lanes1-3) and separated on an acidic gel as described under ``Experimental Procedures.'' The radioactive bands were visualized by autoradiography after exposure for 24 h.



Rat PMCA Isoforms

Since the human and rat pump isoforms share a high degree of sequence homology, the antibodies were also tested on rat tissues. Western blot analysis of rat brains yielded signals of the expected molecular masses with all antibodies except 4N (Fig. 7A; lane4). Antibody 1N recognized a species of 130 kDa, whereas antibody 2N recognized two of about 130 and 135 kDa (Fig. 7A, lanes1 and 2). The antibody against isoform 3 recognized a band at 130 kDa and a double band at about 100 kDa (Fig. 7A, lane3). Both bands disappeared after treating the serum with the peptide corresponding to the N-terminal part of PMCA3. Two proteins of 130 and 97 kDa were detected by the general antibody (Fig. 7A, lane5). To test whether antibody 4N was unable to recognize rat PMCA4 or whether this isoform was present only in low amounts in the neuronal tissues, rat stomach, where the highest amount of the rat PMCA4 transcript was found (Keeton et al., 1993), and rat erythrocyte ghosts were tested for the presence of the isoforms. No signals with the antibody 4N (Fig. 7, B and C; lane4) were observed in either tissue, indicating that the 4N antibody did not recognize the rat PMCA4 protein. Signals at the expected molecular mass (130 kDa) were obtained with antibody 1N (Fig. 7B, lane1), whereas no specific signals were found with antibodies 2N and 3N. A molecule of the molecular mass of that found with antibody 1N was detected with antibody 2A. Interestingly, an additional protein of 135 kDa was also recognized by this antibody (Fig. 7B, lane5). The same result was obtained using a monoclonal antibody (5F10) (Borke et al., 1989a) recognizing all isoforms (result not shown). In rat erythrocyte ghosts only the signal corresponding to PMCA1 (135 kDa) was found (Fig. 7C, lane1).


Figure 7: Isoforms of the pump in rat tissues. Membrane protein preparations of different rat tissues (as described under ``Experimental Procedures'') were tested for the presence of the four isoforms of the pump using the specific antibodies under the same conditions as in Fig. 4. A, total brain (60 µg of protein); B, stomach (60 µg of protein); C, erythrocyte ghosts (30 µg of protein). Lane1, antibody 1N; lane2, antibody 2N; lane3, antibody 3N; lane4, antibody 4N; lane5, antibody 2A.



Several other rat tissues (liver, heart, kidney) were tested for the presence of the different isoforms. In all tissues positive signals were only found with the antibody against isoform 1 and with the general antibody (results not shown, ). The proteins detected always had a molecular mass of about 135 kDa. However, additional bands with smaller molecular masses were revealed by antibodies 1N and 2A. These signals disappeared after preabsorbing the antibodies with the peptides used for their generation. Therefore, it is very likely that these products corresponded to proteolytic fragments of the PMCA protein. In rat lung the amount of PMCA was below the detection limit of the antibodies (results not shown).

DISCUSSION

None of the antibodies used in this study showed isoform cross-reactivity, even in the case of PMCA1 and PMCA2 isoforms, which have a very high degree of homology. The N-terminal region of the pump is well conserved among different organisms (Strehler, 1991); therefore, it was predictable that the antibodies could recognize also the corresponding rat isoforms. All of them were indeed able to recognize the analogous rat isoform, except that against isoform 4. The only portion of rat PMCA4 sequenced so far (C terminus) (Keeton et al., 1993) has shown an unusually low homology to the human counterpart (58% PMCA4CI; 76% PMCA4CII). Possibly an equally low homology at the N terminus of the molecule could explain the negative result, since antibody 4N failed to recognize the rat protein even in tissues where a high amount of this isoform could be predicted (e.g. stomach) (Keeton et al., 1993). The additional protein seen only with the general antibody in rat stomach could therefore represent rat PMCA4. Antibody 2A was raised to detect all isoforms. Since its epitope is located near the active site of the enzyme it was possible that it could have affected the activity of the enzyme. However, no effect on the activity of the pump was found.

The plasma membrane Ca pump plays a crucial role in the Ca extrusion that follows a Ca signal in neurons (Benham et al., 1992; Bleakman et al., 1993). That the large amount of the enzyme detected here in neuronal tissues supports earlier Northern and PCR analysis (Brandt et al., 1992; Keeton et al., 1993; Stauffer et al., 1993) was therefore predictable. Since different pump isoforms and splice forms have been shown to differ in their affinity for Ca and for regulators (Hilfiker et al., 1994; Enyedi et al., 1994), it is likely that their regional distribution in the brain reflects peculiarities in the Ca regulation demands.

The in situ hybridization (Stahl et al., 1992) and PCR analysis work (Zacharias et al., 1995) on the pump transcripts in brain has shown a clear specific regional distribution of the isoforms; PMCA2 was found to be a major isoform in cerebellum. These results are now supported by the findings in this study. Taking into account the estimated relative sensitivities of the antibodies, PMCA2 could be predicted to be the major isoform in cerebellum. Preliminary confocal laser scanning microscopy work on cerebellum slides stained with an antibody recognizing all isoforms, has shown immunoreactivity in the spines and in the membrane of the soma of the Purkinje cells (De Tolosa Talamoni et al., 1993). Immunohistochemical localization in the cerebellum using isoform specific antibodies showed that PMCA2 was practically the only isoform expressed in the dendritic spines of Purkinje cells.() Thus a dominant role of PMCA2 in the Ca extrusion mechanism of Purkinje cells could be predicted. The dendrites and especially the spines have been proposed to play a role in the transduction of the Ca signal in these cells. (Berridge, 1993; Llinas and Sugimori, 1992). Since PMCA2 has higher affinity for calmodulin than the other pump types (Hilfiker et al., 1994), it will be presumably activated at lower concentrations of Ca. It is worth mentioning that no cross-reactivity with the 2N antibody was found in kidney and 293 cells (a cell line derived from kidney). This was somewhat surprising since mRNA data (Magosci et al., 1992; Keeton et al., 1993) had clearly shown the PMCA2 transcripts in this tissue. However, PMCA transcripts are detected even if present in minute amounts, below the detection level of the antibodies.

The pattern of PMCA isoforms in the choroid plexus was equally striking. The high amounts of isoform 3 transcript detected in this tissue in the rat (Stahl et al., 1992) have now been confirmed on the protein level. As discussed for PMCA2 and the cerebellum, a high proportion of the PMCA pump in the choroid plexus can thus be expected to be isoform 3. Importantly, no PMCA1 could be detected in this tissue although the in situ hybridization work on rats had indicated transcript amounts similar to those in the cerebellum (Stahl et al., 1992). Previous immunohistochemical work with a PMCA antibody had shown evident reactivity in the apical membrane in the choroid plexus facing the cerebrospinal fluid (Borke et al., 1989b). Since it is very likely that this isoform was PMCA3, a specific function of the latter in the regulation of Ca in the cerebrospinal fluid, where its concentration is lower than in the cytoplasm of the epithelial cells, appears possible.

Apart from tissues like the choroid plexus and the cerebellum, the PMCA1 and PMCA4 isoforms were the predominant proteins in all tissues tested (), although their expression ratio was not constant (e.g. in erythrocytes the majority of the pump was isoform 4, whereas in HeLa and other cells the PMCA1 pump was more abundant). Considering that the two pumps could be differently regulated (the PMCA1 structure contains a phosphorylation site for the cAMP-kinase) (James et al., 1989; Carafoli, 1992) one could speculate that actively growing cells require more options for the regulation of the PMCA pump than the circulating dying red blood cells. In general, however, these two isoforms of the pump are likely to represent the forms involved in the ``housekeeping'' of the Ca metabolism of cells ().

Sometimes more than one protein in the range of the expected molecular mass of the PMCA (125-134 kDa) (Guerini and Carafoli, 1993) was detected by the same antibody. Since no alternative splicing has been observed in the portion of the pump sequence used to produce the antibodies (Keeton et al., 1993; Stauffer et al., 1993) it is reasonable to assume that the antibodies would recognize all alternatively spliced pump isoforms. Additionally, the mRNA expression pattern and the relative amounts of the different splice forms of an isoform agree reasonably well with the data derived with the isoform-specific antibodies. Therefore, it is very likely that these proteins may reflect differently spliced isoforms or possibly post-translational alterations.

  
Table: Cloning of the cDNA fragments of the pump: a summary of the sequences of the cDNA fragments used to construct the expression vectors

The 5` position corresponds to either the number of the first nucleotide of the primers (isoform 1 or 3) or to the number of the first nucleotide of the palindromic sequence of the corresponding restriction enzyme site (isoforms 2 and 4). The numbering of the cDNA starts with the first nucleotide of the ATG corresponding to the Met of position 1. The cDNAs were obtained from human PMCA cDNAs (hPMCA 1: Verma et al. (1988); hPMCA 2: Heim et al. (1992); hPMCA 3: H. Hilfiker, D. Guerini, and E. Carafoli, unpublished results; hPMCA 4: Strehler et al. (1990)). The modified nucleotides of the primers are indicated in boldface.


  
Table: A summary of the PMCA isoforms found in the different human and rat tissues

-, no protein found in the molecular mass range of the PMCA; x, one protein found in the molecular mass range of the PMCA; x/x, two proteins found in the molecular mass range of the PMCA; x/x/x, three proteins found in the molecular mass range of the PMCA.



FOOTNOTES

*
This work has been made possible by Swiss National Science Foundation Grant 31-30859.91. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 41-1-6323011; Fax: 41-1-6321213.

The abbreviations used are: PMCA, plasma membrane Ca-ATPase; hPMCA, human PMCA; PVDF, polyvinylidene difluoride; SERCA, sarcoplasmic reticulum Ca-ATPase; MOPS, 4-morpholineethanesulfonic acid.

D. Guerini, V. B. Pon, and E. Carafoli, manuscript in preparation.

T. Stauffer, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. C. Moll (Universitätspital, Zurich, Switzerland) for providing the samples of human tissues.


REFERENCES
  1. Benham, C. D., Evans, M. L., and McBain, C. J.(1992) J. Physiol. 455, 567-583 [Abstract]
  2. Berridge, M.(1993) Nature 365, 388-389 [CrossRef][Medline] [Order article via Infotrieve]
  3. Bleakman, D., Roback, J. D., Wainer, B. H., Miller, R. J., and Harrison, N. L.(1993) Brain Res. 600, 257-267 [Medline] [Order article via Infotrieve]
  4. Borke, J. L., Minami, J., Verma, A. K., Penniston, J. T., Kumar, R. (1989a) Amer. J. Physiol. 257, F842-F849
  5. Borke, J. L., Caride, A. J., Yaksh, T. L., Penniston, J. T., and Kumar, R. (1989b) Brain Res. 489, 355-360 [Medline] [Order article via Infotrieve]
  6. Bradford, M.(1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  7. Brandt, P., Ibrahim, E., Bruns, G. A. P., and Neve, R. L. (1992a) Genomics 14, 484-487 [Medline] [Order article via Infotrieve]
  8. Brandt, P., Neve, R. L., Kammesheidt, A., Rhoads, R. E., and Vanaman, T. C. (1992b) J. Biol. Chem. 267, 4376-4385 [Abstract/Free Full Text]
  9. Carafoli, E.(1992) J. Biol. Chem. 267, 2115-2118 [Free Full Text]
  10. Carafoli, E.(1994) FASEB J. 8, 993-1002 [Abstract/Free Full Text]
  11. Carafoli, E., and Guerini, D.(1993) Trends Cardiovasc. Med. 3, 177-183
  12. Carafoli, E., and Stauffer, T.(1994) J. Neurobiol. 25, 312-324 [Medline] [Order article via Infotrieve]
  13. Caroni, P., and Carafoli, E.(1981) J. Biol. Chem. 256, 9371-9373 [Abstract/Free Full Text]
  14. De Tolosa Talamoni, N., Smith, C. A., Wasserman, R. H., Beltramino, C., Fullmer, C. S., and Penniston, J. T.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11949-11953 [Abstract]
  15. Enyedi, A., Verma, A. K., Heim, R., Adamo, H. P., Filoteo, A. G., Strehler, E. E., and Penniston, J. P.(1994) J. Biol. Chem. 269, 41-43 [Abstract/Free Full Text]
  16. Greeb, J., and Shull, G. E.(1989) J. Biol. Chem. 264, 18569-18576 [Abstract/Free Full Text]
  17. Heim, R., Iwata, T., Zvaritch, E. I., Adamo, H. P., Rutishauser, B., Strehler, E. E., Guerini, D., and Carafoli, E.(1992) J. Biol. Chem. 267, 24476-24484 [Abstract/Free Full Text]
  18. Hilfiker, H., Guerini, D., and Carafoli, E.(1994) J. Biol. Chem. 269, 26178-26183 [Abstract/Free Full Text]
  19. Howard, A., Legon, S., and Walters, J. R. F.(1993) Am. J. Physiol. 264, F91-F93
  20. Jaffe, D. B., Fisher, S. A., and Brown, T. H.(1994) J. Neurobiol. 25, 220-233 [Medline] [Order article via Infotrieve]
  21. James, P. H., Pruschy, M., Vorherr, T. E., Penniston, J. T., and Carafoli, E.(1989) Biochemistry 28, 4253-4258 [Medline] [Order article via Infotrieve]
  22. Keeton, T. P., Burk, S. E., and Shull, G. E.(1993) J. Biol. Chem 268, 2740-2748 [Abstract/Free Full Text]
  23. Knauf, P., Proverbio, F., and Hoffmann, J.(1974) J. Gen. Physiol. 63, 324-336 [Abstract/Free Full Text]
  24. Laemmli, U. K.(1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  25. Latif, F., Duh, F.-M., Gnarra, J., Tory, K., Kuzmin, I., Yao, M., Stackhouse, T., Modi, W., Geil, L., Schmidt, L., Hus, L., Orcutt, M. L., Maher, E., Richards, F., Phipps, M., Ferguson-Smidt, M., Le Paslier, D., Linhen, W. M., Zbar, B., and Lermon, M. I.(1993) Cancer Res. 53, 861-867 [Abstract]
  26. Llinas, R. R., and Sugimori, M.(1992) in The Cerebellum Revisited (Llinas, R. R., and Sotelo, C., eds) pp. 167-181, Springer Verlag, Berlin
  27. Magosci, M., Yamaki, M., Penniston, J. T., Dousa, T. P.(1992) Am. J. Physiol. 263, F7-F14
  28. Niggli, V., Penniston, J. T., and Carafoli, E.(1979) J. Biol. Chem. 254, 9955-9958 [Abstract]
  29. Niggli, V., Zurini, M., and Carafoli, E.(1987) Methods Enzymol. 139, 791-808 [Medline] [Order article via Infotrieve]
  30. Olson, S., Wang, M. G., Carafoli, E., Strehler, E. E., and McBride, O. W.(1991) Genomics 9, 629-641 [Medline] [Order article via Infotrieve]
  31. Sarkadi, B., Enyedi, A., Földes-Papp, Z., and Gárdos, G. (1986) J. Biol. Chem. 261, 9552-9557 [Abstract/Free Full Text]
  32. Schägger, H., and von Jagow, G.(1987) Anal. Biochem. 166, 368-379 [Medline] [Order article via Infotrieve]
  33. Schatzmann, H. J.(1966) Experientia 22, 364-368 [Medline] [Order article via Infotrieve]
  34. Shull, G. E., and Greeb, J.(1988) J. Biol. Chem. 263, 8646-8657 [Abstract/Free Full Text]
  35. Stahl, W. L., Eakin, T. J., Owens, J. W. M., Breininger, J. F., Filuk, P. E., and Anderson, W. R.(1992) Mol. Brain Res. 16, 223-231 [Medline] [Order article via Infotrieve]
  36. Stauffer, T. P., Hilfiker, H., Carafoli, E., and Strehler, E. E.(1993) J. Biol. Chem. 268, 25993-26003 [Abstract/Free Full Text]
  37. Strehler, E. E.(1991) J. Membr. Biol. 120, 1-15 [Medline] [Order article via Infotrieve]
  38. Strehler, E. E., James, P., Fischer, R., Heim, R., Vorherr, T., Filoteo, A. G., Penniston, J. T., and Carafoli, E.(1990) J. Biol. Chem. 265, 2835-2842 [Abstract/Free Full Text]
  39. Stueber, D., Bannwarth, W., Pink, J. R. L., Meloen, R. H., and Matile, H.(1990) Eur. J. Immunol. 20, 819-824 [Medline] [Order article via Infotrieve]
  40. Towbin, H., Staehlin, T., and Gordon, J.(1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354 [Abstract]
  41. Verma, A. K., Filoteo, A. G., Stanford, D. R., Wieben, E. D., Penniston, J. T., Strehler, E. E., Fischer, R., Heim, R., Vogel, G., Mathews, S., Strehler-Page, M.-A., James, P., Vorherr, T., Krebs, J., and Carafoli, E.(1988) J. Biol. Chem 263, 14152-14159 [Abstract/Free Full Text]
  42. Wang, M. G., Yi, H., Hilfiker, H., Carafoli, E., Strehler, E. E., and McBride, O. W.(1994) Cytogenet. Cell Genet. 67, 41-45 [Medline] [Order article via Infotrieve]
  43. Zacharias, D. A., Dalrymple, S. J., and Strehler, E. E.,(1995) Mol. Brain Res. 28, 263-272 [CrossRef][Medline] [Order article via Infotrieve]

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