From the
Antibodies against the four isoforms of the human plasma
membrane Ca
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.
The Ca
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).
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).
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.
It was important to show that the antibodies reacted only with the
corresponding human pump isoforms. PMCA1CI
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).
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.
The plasma membrane Ca
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.
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
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
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.
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.
-, 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.
We thank Dr. C. Moll (Universitätspital, Zurich,
Switzerland) for providing the samples of human tissues.
-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.
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.
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.
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 NaH
PO
, 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
NaH
PO
, pH 7.8, 300 mM NaCl). The
column was washed with 20 volumes of phosphate buffer A (50 mM
NaH
PO
, 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
NaH
PO
, 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
NaH
PO
, 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 NaH
PO
, 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).
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.
(
)
(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).
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.
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.
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.
(
)
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.
in the
cerebrospinal fluid, where its concentration is lower than in the
cytoplasm of the epithelial cells, appears possible.
metabolism of cells ().
Table: Cloning of the cDNA fragments of the pump: a
summary of the sequences of the cDNA fragments used to construct the
expression vectors
Table: A summary of the PMCA isoforms
found in the different human and rat tissues
-ATPase; hPMCA, human PMCA; PVDF, polyvinylidene
difluoride; SERCA, sarcoplasmic reticulum Ca
-ATPase;
MOPS, 4-morpholineethanesulfonic acid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.