Alternative Splicing of the First Intracellular Loop of Plasma
Membrane Ca2+-ATPase Isoform 2 Alters Its Membrane
Targeting*
Michael C.
Chicka
and
Emanuel E.
Strehler§
From the Department of Biochemistry and Molecular Biology, Mayo
Graduate School, Mayo Clinic, Rochester, Minnesota 55905
Received for publication, February 11, 2003
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ABSTRACT |
Plasma membrane
Ca2+-ATPases (PMCAs) are involved in local
Ca2+ signaling and in the spatial control of
Ca2+ extrusion, but how different PMCA isoforms are
targeted to specific membrane domains is unknown. In polarized MDCK
epithelial cells, a green fluorescent protein-tagged PMCA4b construct
was targeted to the basolateral membrane, whereas a green fluorescent
protein-tagged PMCA2b construct was localized to both the apical and
basolateral domain. The PDZ protein-binding COOH-terminal tail of
PMCA2b was not responsible for its apical membrane localization, as a
chimeric pump made of an NH2-terminal portion from PMCA4
and a COOH-terminal tail from PMCA2b was targeted to the basolateral
domain. Deletion of the last six residues of the COOH terminus of
either PMCA2b or PMCA4b did not alter their membrane targeting,
suggesting that PDZ protein interactions are not essential for proper
membrane localization of the pumps. Instead, we found that alternative splicing affecting the first cytosolic loop determined apical membrane
targeting of PMCA2. Only the "w" form, which contains a 45-amino
acid residue insertion, showed prominent apical membrane localization.
By contrast, the x and z splice variants containing insertions
of 14 and 0 residues, respectively, localized to the basolateral
membrane. The w splice insert was the crucial determinant of
apical PMCA2 localization, and this was independent of the splice
configuration at the COOH-terminal end of the pump; both PMCA2w/b and PMCA2w/a showed prominent apical targeting, whereas PMCA2x/b, PMCA2z/b, and PMCA2z/a were confined to the basolateral membrane. These data report the first differential effect of
alternative splicing within the first cytosolic loop of PMCA2 and help
explain the selective enrichment of specific PMCA2 isoforms in
specialized membrane compartments such as stereocilia of auditory hair cells.
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INTRODUCTION |
Plasma membrane Ca2+-ATPases
(PMCAs)1 constitute the major
high affinity Ca2+ extrusion system of eukaryotic cells.
Their function is crucial for the maintenance of the normally low
cytosolic free Ca2+ levels ([Ca2+]i)
in resting cells and to counteract the transient increases in
[Ca2+]i generated during Ca2+
signaling (1, 2). The spatially and temporally distinct demands on
Ca2+ influx and efflux mandate the proper spatial
distribution and abundance of the PMCAs across the plasma membrane.
This is of particular importance in polarized cells such as neurons or
epithelial cells where Ca2+ signaling often must be
restricted to the pre- and post-synaptic membrane (neurons) or where
Ca2+ influx and efflux must be spatially segregated between
the apical and basolateral membrane (Ca2+ transporting
epithelia). However, the molecular determinants and the mechanisms by
which PMCAs are targeted to distinct membrane domains are unknown.
Mammalian PMCAs are encoded by four separate genes giving rise to PMCA
isoforms 1-4. Isoform complexity is further enhanced by alternative
splicing of the primary transcripts, such that over 20 distinct PMCA
variants exist in the mammalian proteome (2). Alternative splicing
affects two major regions of the protein (splice sites A and C): the
first intracellular loop between membrane-spanning domains 2 and 3, and
the COOH-terminal tail which corresponds to a major regulatory region
of the pump and contains its calmodulin-binding site (Fig.
1).

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Fig. 1.
Scheme of PMCA2 and its major splice
variants. A model of the PMCA is shown on top, with its
membrane-spanning domains indicated by gray boxes and the
lipid bilayer as two horizontal bars. The NH2-
(N) and COOH terminus (C) is indicated, as is the
first cytosolic loop with a phospholipid-sensitive region
(PL), the large intracellular catalytic loop, and the two
major splice sites A and C. The alternatively
spliced exons are shown as separate boxes, and their sizes in
nucleotides (nt) are indicated in a scheme of the genomic
organization on the bottom. The exon configuration and
nomenclature of the major human PMCA2 splice variants are also
shown.
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Alternative splicing at site A does not change the overall reading
frame of the pump; instead, varying numbers of amino acids (from 14 to
45 in human PMCA2) are optionally inserted into the intracellular loop
separating membrane-spanning segments 2 and 3 (Fig. 1). Splicing at
site A is most complex in PMCA2 where the optional insertion of three
separate exons leads to splice variant 2w (all three exons included),
2x (only the third exon included), 2y (only the first two exons
included), and 2z (none of the alternative exons included) (2). The
functional effect of alternative splicing at site A is unknown,
although it has been noted that the changes occur close to a
phospholipid-binding domain of the pump (3, 4). The first intracellular
loop also participates in the pump mechanism as the so-called A
(actuator) domain (5).
The main splice variants (termed "a" and "b") generated at the
COOH-terminal site differ in their primary amino acid sequence due to a
change in reading frame. Functionally, the most notable consequence of
COOH-terminal splicing is a difference in calmodulin regulation of the
respective pump variants (2, 6). In addition, the extreme COOH-terminal
sequence of the b splice variants (but not of the a-forms) has been
shown recently to interact with several PDZ proteins including members
of the synapse-associated protein family (7), NO synthase I (8), and
Na+/H+ exchanger regulatory factor-2 (NHERF2)
(9). Differential interaction with specific PDZ proteins may be one
mechanism for the membrane targeting or retention of specific
transporters or receptors. We recently reported (7) that PMCA4b is
almost exclusively localized in the basolateral membrane of polarized
Madin-Darby canine kidney (MDCK) epithelial cells where it may interact
with the PDZ protein synapse-associated protein 97. By contrast, we found that a GFP-tagged PMCA2b isoform was mainly targeted to the
apical membrane where it co-localized with NHERF2 as a specific interaction partner (9).
Here we first set out to test the hypothesis that alternative splicing
affecting the COOH-terminal tail may lead to differential membrane
targeting of PMCAs. By using recombinant protein expression and
confocal fluorescence microscopy, we find that the PDZ binding tail is
dispensable for basolateral or apical membrane targeting, suggesting
that COOH-terminal splicing plays no primary role for the proper
localization of PMCA isoforms. Surprisingly, however, alternative
splicing at site A influences apical or basolateral localization of
PMCA2. PMCA2w, but not -2x or -2z, is targeted to the apical membrane,
and this is true regardless of whether the COOH-terminal splice
corresponds to the a or b variant. Our data suggest for the first time
a role for alternative splicing at site A of PMCA2, indicating the
importance of this splicing event for differential membrane targeting
of the calcium pump in polarized cells.
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EXPERIMENTAL PROCEDURES |
Construction of PMCA Expression Vectors--
GFP-PMCA4b encoding
human PMCA4x/b fused at its NH2 terminus to GFP was
generated by cloning an XhoI fragment carrying the full-length PMCA4x/b sequence into pEGFP-C2
(Clontech). The PMCA4x/b sequence was PCR-amplified
using pMM2-PMCA4b (3) as template together with primers 4b-start
(5'-ggg ggc tcg aga acg aac cca tca
gac cgt gtc ttg cc-3') and 4b-stop (5'-ccc cct
cga gtc aaa ctg atg tct cta ggc tct gta g-3') containing
XhoI recognition sequences (underlined). The full-length
coding sequence of PMCA2x/b was first assembled as a NotI
fragment in pBluescript-KS using reverse transcriptase-PCR and
cDNAs cloned previously (4). To generate full-length cDNAs for
PMCA2w/b and PMCA2z/b, a BstEII-SphI fragment
encompassing the alternative splice site A region in PMCA2x was
replaced by corresponding cassettes containing the 2w and 2z sequence,
respectively. The NotI fragments carrying the full-length
PMCA2w/b, -2x/b, or -2z/b coding sequence were then excised from
pBluescript-KS and subcloned into the NotI site of pSPORT-1
(Invitrogen). Finally, the PMCA2 sequences were cloned as
MluI-SalI (2w/b and 2x/b) or
MluI-KpnI fragments (2z/b) into the modified
pMM2 expression vector (3) to generate
pMM2-PMCA2w/b, pMM2-PMCA2x/b, and
pMM2-PMCA2z/b. To create constructs for GFP-PMCA2w/b and
GFP-PMCA2x/b, the full-length PMCA2w/b and PMCA2x/b sequences were
released as SacII/XbaI fragments from the
original pBluescript-KS vectors and cloned into a modified pEGFP-C1
expression vector (Clontech) containing consecutive
hemagglutinin and His6 tags (10) between the GFP and the
respective PMCA sequences. To make constructs GFP-PMCA2w/b
6 and
GFP-PMCA2x/b
6, a GFP-PMCA2b plasmid described previously (9) was
used as starting material because it allowed access to a convenient
downstream restriction site. First, an ~250-bp
EcoRI-SalI fragment encoding the COOH-terminal 72 residues was replaced by a corresponding PCR-generated
EcoRI-SalI fragment carrying a translational stop
codon six residues upstream of the original terminus. An ~2.3-kb
BglII fragment containing the NH2-terminal
~750 codons was then excised from this plasmid and replaced by
corresponding BglII fragments from GFP-PMCA2w/b and
GFP-PMC2x/b to create the constructs GFP-PMCA2w/b
6 and
GFP-PMCA2x/b
6, respectively. The expression vector
pMM2-PMCA2z/a has been described previously (11). The
expression vector pMM2-PMCA2w/a was generated by a
three-way ligation combining SalI-KpnI-digested
pMM2 plasmid DNA, an ~1.3-kb
SalI-ScaI fragment from GFP-PMCA2w/b (containing the 5' end coding sequence including the "w" insert at splice site
A), and an ~2.4-kb ScaI-KpnI fragment from
pMM2-PMCA2z/a (containing the 3' region and a-tail
sequence at splice site C). The chimeric PMCA construct
GFP-PMCA4x/2btail was made by replacing an ~350-bp BamHI
fragment specifying the COOH-terminal residues from 1100 to 1205 in
GFP-PMCA4b with a BamHI fragment specifying the
corresponding COOH-terminal sequence from plasmid GFP-PMCA2b (9). The
final construct encodes a GFP-tagged chimeric PMCA consisting of
residues 1-1100 of PMCA4 (x splice variant) fused to the COOH-terminal
112 residues of PMCA2 (b splice variant). The integrity of all
constructs was confirmed by DNA sequencing in the Mayo Molecular
Biology Core Facility. A schematic representation of all constructs is
shown in Fig. 2.

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Fig. 2.
Scheme of PMCA constructs. The
recombinant PMCAs used in this study are shown schematically and are
labeled on the left. The green fluorescent protein moiety is
represented by a black box; PMCA2 is gray, and
PMCA4 is white. Splice sites A and C are indicated by
arrows. Sequences encoded by alternatively spliced exon are
shown as differently stippled boxes. Deletion of the six
COOH-terminal residues is indicated by a small black
box.
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Expression of Recombinant PMCAs and Immunoblotting--
COS-1
cells were grown to ~80% confluence on 6-well plates (Costar) in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, non-essential amino acids, 1 mM sodium pyruvate, glutamine, and antibiotic/antimycotic mixture (all cell culture reagents from Invitrogen). Cells were transfected with 2 µg
of plasmid DNA using LipofectAMINE PlusTM according to the
manufacturer's instructions (Invitrogen). After ~48 h, the cells
were rinsed, lysed as described (7), and the protein concentration in
the lysates determined by the BCA assay (Pierce). About 30 µg of the
lysates from untransfected (control) and transfected cells were
separated on 7.5% SDS-polyacrylamide gels and transferred to
nitrocellulose following standard Western blotting procedures (12).
Nitrocellulose membranes were blocked in 50 mM Tris-HCl (pH
7.4), 150 mM NaCl, 0.1% Tween + 5% milk before detection
of the PMCAs using specific antibodies. Monoclonal Pan-PMCA antibody
5F10, monoclonal anti-PMCA4 antibody JA9, and affinity-purified
polyclonal anti-PMCA2 antibody NR-2 were obtained from John Penniston
and Adelaida Filoteo (Mayo Clinic) and used at dilutions of 1:2000,
1:600, and 1:5000, respectively. Secondary goat anti-mouse or goat
anti-rabbit antibodies coupled to horseradish peroxidase were purchased
from Sigma and used at 1:5000 dilution. Incubation with primary and
secondary antibodies, washing, and detection of the signals was done as
described previously (13).
Confocal Fluorescence Microscopy--
Type I MDCK epithelial
cells (ATCC number CCL-34, Manassas, VA) were grown to confluence on
glass coverslips in Dulbecco's modified Eagle's medium containing
10% fetal bovine serum. Cells were transfected with a total of 2 µg
of plasmid DNA using LipofectAMINE 2000TM (Invitrogen).
48 h after transfection, the cells were fixed for 15 min at room
temperature in 4% paraformaldehyde (Tousimis, Rockville, MD) diluted
in DPBS + Ca2+/Mg2+ (DPBS + CM,
Invitrogen). After five brief washes in DPBS + CM, coverslips were
further fixed and permeabilized in pre-chilled methanol for 15 min at
20 °C. The cells were blocked in DPBS + CM containing 5% normal
goat serum and 1% bovine serum albumin (blocking buffer) and were then
incubated for 1 h at room temperature with affinity-purified
polyclonal anti-PMCA2 antibody NR-2 (1:1000, final concentration 0.8 µg/ml) or monoclonal Pan-anti-PMCA antibody 5F10 (1:800). After
washing 3 times for 5 min in DPBS + CM, the cells were incubated for
1 h at room temperature with the appropriate anti-rabbit or
anti-mouse secondary antibodies coupled to Alexa 488 or Alexa 594 (Molecular Probes, Eugene, OR). The secondary antibodies were used at a
dilution of 1:800, and all antibodies for immunofluorescence were
diluted in blocking buffer. 4',6'-Diamidino-2-phenylindole dihydrochloride (DAPI, Molecular Probes, Eugene, OR) was also added to
the secondary antibody application at a dilution of 1:500 (final
concentration 20 µg/ml) to stain nuclei. After final washing, coverslips were mounted in Prolong mounting media (Molecular Probes). Confocal micrographs were taken on a Zeiss LSM510 microscope using an
Apochromat 63× oil immersion objective and captured using LSM510 software version 2.8 (Zeiss). Images were imported and edited using
Adobe Photoshop 5.0.
Quantification of Apical Membrane Fluorescence--
MDCK cells
were grown to confluence as a monolayer on glass coverslips,
transfected with PMCA2 expression constructs and prepared for
immunofluorescence as described above using anti-PMCA2 antibody NR-2
and Pan-PMCA antibody 5F10 followed by Alexa 488 and Alexa 594 secondary antibodies, respectively. Confocal images were captured on a
Zeiss LSM510 microscope as mentioned above. Images of the apical most
2-µm section of 20 representative cells from each transfection were
collected, applying the same detector gain, zoom, amplitude offset and
gain, transmission percent, pinhole size, and scan speed for each image
collected within the frame mode and using an average from two images.
By using the circular-shaped fixed area tool, an arbitrary area was
selected that encompassed a majority of the collected apical images of
each cell without incorporating any of the area outside of the apical
domain (such as the lateral "rim" of the cell). This fixed
circle-shaped area was then placed on the apical domain of the apical
images in order to collect the mean fluorescence intensity (using the
green channel) per given surface area for each of the 20 images from
each of the different transfections. Choosing a circle that encompasses most of the membrane is essential as it yields an unbiased sampling of
the entire apical domain as a whole and negates any effects (such as
sampling microdomains with exceptionally high or low abundance of
apical protein expression) that would skew the overall mean intensity
for a given image. Each cell could then be represented by a number
equal to the mean apical fluorescence intensity of that cell. These
numbers were used to construct a line graph showing the mean intensity
for each of the 20 cells from a given transfection and were also
displayed in a bar graph showing the averages of the 20 mean
fluorescence intensities for each of the PMCA2 transfections.
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RESULTS |
Differential Targeting of PMCA Isoforms in Polarized MDCK
Cells--
We noted previously that endogenous PMCA4b was almost
exclusively expressed in the basolateral membrane of polarized MDCK cells (7, 14), whereas a significant amount of GFP-tagged PMCA2b was
found at the apical membrane where it co-localized with its binding
partner NHERF2 (9). To confirm that the observed difference in membrane
targeting is not due to the NH2-terminally added GFP
moiety, we expressed GFP-PMCA4b in MDCK cells and compared its
localization to that of GFP-PMCA2b. Both GFP-tagged proteins were
readily expressed in transiently transfected COS cells as determined by
Western analysis using isoform-specific antibodies (Fig.
3). When expressed in polarized
Madin-Darby canine kidney cells, GFP-PMCA4b was still targeted to the
basolateral membrane (Fig.
4A), whereas the GFP-PMCA2b
construct was prominently present at the apical membrane in addition to
labeling the basolateral membrane (Fig. 4B). Thus, the
difference in membrane localization of these two PMCA isoforms is not
due to the presence of the GFP moiety at their NH2
terminus.

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Fig. 3.
Expression of recombinant PMCA
constructs. Western blots are shown of protein lysates from COS
cells transfected with various GFP-tagged (top panel) or
untagged (bottom panel) PMCA constructs as indicated on
top of each lane and as illustrated in Fig. 2. Equal amounts
of cell lysates (~30 µg) were separated on 7.5% denaturing
polyacrylamide gels, and the proteins were transferred to
nitrocellulose membranes. The membranes were probed with antibodies
against PMCA2 to detect GFP-tagged PMCA2w/b, -2w/b 6, and -2x/b 6
as well as untagged PMCA2w/a and -2z/a, with antibodies against PMCA4
to detect GFP-tagged PMCA4x/b, or with a Pan-PMCA antibody to detect
the GFP-PMCA4x/2btail chimera, the non-tagged PMCA2w/b, -2x/b, and
-2z/b as well as the endogenous COS-cell PMCAs (untransfected control).
The position of an ~130-kDa marker protein is indicated on the
left. All constructs expressed a recombinant protein of the
expected size, and all constructs were expressed at comparable
levels.
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Fig. 4.
Different membrane localization of PMCA
isoforms. GFP-tagged PMCA4b (A) and PMCA2b
(B) constructs were transiently expressed in MDCK cells, and
their localization was determined by confocal fluorescence microscopy.
A, GFP-PMCA4x/b is localized to the (baso-) lateral membrane
(right panel, en face view of section at middle height) with
virtually no staining of the apical membrane (left panel,
apical section). The basolateral localization of the
GFP-PMCA4b isoform is obvious from the x:z section shown below the en
face views. B, GFP-PMCA2w/b shows prominent apical
localization (left panel, apical section) in
addition to lateral staining (right panel, middle section).
The apical and lateral localization of this GFP-PMCA2b isoform is
easily seen in the x:z section on the bottom. The
white arrows indicate where the apical and middle sections
were taken for the en face views. Nuclei were stained with DAPI
(blue fluorescence). Scale bar, 30 µm in en
face images in A, 15 µm in en face views in B,
and 10 µm in x:z sections in A and B.
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Differential Membrane Targeting of PMCA2b and PMCA4b Is Not Due to
Their COOH-terminal Tails--
Different PMCA b splice variants
interact promiscuously with several PDZ proteins but also recognize
specific PDZ protein partners (7, 9). For example, PMCA2b, but not
PMCA4b, binds to NHERF2 which is a member of a small subfamily of PDZ
proteins involved in membrane trafficking, anchoring, and regulation of ion transporters and receptors including Na+/H+
exchanger-3 (15),
2-adrenergic receptor (16), and the
cystic fibrosis transmembrane conductance regulator (17, 18). The differential localization of PMCA2b and PMCA4b might therefore be
dependent upon different protein-protein interactions by their COOH-terminal tails. To test this hypothesis, we generated a chimeric construct (GFP-PMCA4x/2btail, see Fig. 2) in which the GFP-PMCA4b COOH-terminal tail downstream of residue 1100 was replaced by the
corresponding tail of PMCA2b. When expressed in polarized MDCK cells,
the localization of this chimera was indistinguishable from GFP-PMCA4b,
i.e. the chimeric pump was exclusively found in the
basolateral membrane (Fig. 5). Thus, the
presence of the PMCA2b COOH-tail does not impart apical targeting on a
chimeric protein with its NH2-terminal segment derived from
PMCA4.

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Fig. 5.
Basolateral localization of a PMCA4-PMCA2b
chimera. MDCK cells were transfected with the GFP-PMCA4x/2b-tail
construct encoding a GFP-tagged chimeric PMCA consisting of a PMCA4
NH2-terminal portion fused to the COOH-terminal portion
from PMCA2b. After fixing, the cells were incubated with Pan-PMCA
antibody 5F10 and an Alexa 594-labeled secondary antibody to visualize
all PMCAs (including endogenous PMCA in the non-transfected cells).
Nuclei were stained by DAPI. The corresponding x:z section is shown
below the en face view. The arrow indicates the
level at which the en face section was taken. Note the prominent
localization of the chimeric PMCA in the basolateral membrane.
Bar = 10 µm.
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Alternative Splicing at Site A Determines Differential Membrane
Localization of PMCA2b Variants--
Although PMCA2 and PMCA4 differ
at many sequence positions throughout their length (their overall
identity amounts to about 75%), the most obvious divergence between
these isoforms occurs at splice site A. Indeed, we noted that our
basolaterally targeted GFP-PMCA4b construct corresponded to the 4x/b
variant, whereas the apically targeted GFP-PMCA2b construct
corresponded to the 2w/b form (see Fig. 1 for a scheme of the splice
variants). To test if the splice site A configuration might be involved
directly in the different membrane targeting, we constructed expression vectors for each of the known human splice site A variants of PMCA2b,
i.e. hPMCA2w/b, -2x/b, and -2z/b (Fig. 2). To rule out any
possible effect of the NH2-terminal GFP moiety, these
vectors were made without any tag to represent the wild-type
full-length PMCA2 variants. Western blots of equal amounts of total
cell lysate from transfected COS cells showed that all constructs were
expressed at comparable levels (Fig. 3). When expressed in polarized
MDCK cells, PMCA2x/b and PMCA2z/b were primarily localized in the
basolateral membrane (Fig. 6,
middle and right panels), whereas PMCA2w/b showed prominent apical localization in addition to basolateral staining (Fig.
6, left panel). This difference in membrane localization was
readily seen by taking confocal "en face" sections at different depths of the cells and is best illustrated in the x:z images shown in
Fig. 6. Because all three constructs correspond to the b splice variant
at their COOH-terminal end and are identical except for the insertion
of 45 (w-form), 14 (x-form), or 0 (z-form) amino acid residues in their
first intracellular loop, the observed differences in membrane
localization must be due to the changes that occurred at splice site A,
specifically to the insertion of 31 extra residues in the w splice
form.

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Fig. 6.
Differential membrane localization of splice
site A variants of PMCA2b. Full-length PMCA2w/b, PMCA2x/b, and
PMCA2z/b were expressed in polarized MDCK cells, and their localization
was studied by confocal fluorescence microscopy. Recombinant PMCA2 was
detected with isoform-specific polyclonal antibody NR-2 followed by
anti-rabbit Alexa 488 (green fluorescence). Nuclei were
stained with DAPI and appear blue. PMCA2w/b shows prominent
apical localization in addition to basolateral staining (left
panel), whereas PMCA2x/b and PMCA2z/b are exclusively localized to
the basolateral membrane (middle and right
panel). Corresponding x:z sections are shown below the
en face views. White arrows indicate the height at which the
en face sections were captured. Bar, 20 µm in en face
views and 10 µm in x:z sections.
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The PDZ Protein-binding COOH-terminal Sequence Is Not Required for
Differential Membrane Targeting of PMCA2 Splice Site A
Variants--
The experiment using a chimeric GFP-PMCA4x/2b-tail
construct (see Fig. 5) had already shown that the COOH-terminal tail is not the major determinant for apical versus basolateral
targeting of the PMCAs. However, the presence of a functional
PDZ-binding sequence may still be required for faithful membrane
targeting of the pumps. To test if this is the case, we expressed
GFP-PMCA2w/b and GFP-PMCA2x/b constructs lacking their six
COOH-terminal residues (Figs. 2 and 3), thus rendering them
unable to interact with PDZ proteins. As shown in Fig.
7, GFP-PMCA2w/b
6 and GFP-PMCA2x/b
6 were still faithfully targeted to the apical and basolateral membranes, respectively, as predicted from their splice site A configuration. Similarly, GFP-PMCA4x/b
6 was targeted only to the basolateral membrane (not shown), as had been observed for the corresponding full-length PMCA4x/b isoform (Fig. 4A). We conclude that a
functional PDZ-binding domain at the COOH terminus is not required for
targeting of the PMCAs and hence that PDZ protein interactions are
dispensable for directing the pumps to their membrane destination.

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Fig. 7.
The PDZ binding COOH-terminal sequence is not
required for differential localization of PMCA2b splice site A
variants. GFP-tagged PMCA2w/b 6 and PMCA2x/b 6 were expressed
in MDCK cells, and their localization was determined by confocal
fluorescence microscopy. Nuclei were stained with DAPI. Note the
prominent apical targeting of PMCA2w/b 6 (left panel)
compared with the mostly basolateral localization of PMCA2x/b 6
(right panel). Corresponding x:z sections are shown
below each en face view. Arrows indicate the
height at which the en face images were captured.
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The w Insertion at Splice Site A Determines Apical Membrane
Targeting of PMCA2 Isoforms--
If the insertion of the 31 extra
amino acids of the w splice variant were indeed a major determinant of
apical targeting of PMCA2, we would expect all w variants to be
targeted to the apical membrane irrespective of the splice
configuration at their COOH termini. To test this hypothesis, we
generated expression vectors for full-length PMCA2w/a and PMCA2z/a
(Figs. 2 and 3). When these constructs were transfected into MDCK
cells, PMCA2z/a localized to the basolateral membrane, whereas PMCA2w/a
showed prominent apical localization (Fig.
8). Thus, the targeting of PMCA2w/a and
PMCA2z/a is identical to that of PMCA2w/b and PMCA2z/b, respectively. To obtain a more quantitative assessment of the apical/basolateral targeting of the different splice site A variants, we determined the
apical membrane fluorescence intensity in multiple randomly selected
cells transfected with either PMCA2w/b, 2x/b, 2z/b, 2w/a, or 2z/a as
described under "Experimental Procedures." A plot of the relative
fluorescence intensity in a fixed area of the apical membrane is shown
in Fig. 9A for 20 individual
cells from each transfection experiment, and the averaged apical
fluorescence intensity for each PMCA2 construct is displayed in Fig.
9B. The data illustrate the negligible localization of
PMCA2x/b, -2z/b, and -2z/a in the apical membrane, in contrast to the
abundant presence of PMCA2w/b and -2w/a in this membrane compartment.
The results also show that PMCA2w/b and PMCA2w/a are about equally abundant in the apical membrane, further supporting the primary importance of the w splice configuration in determining PMCA2 membrane
distribution.

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Fig. 8.
The splice site A configuration determines
the membrane localization of PMCA2 irrespective of the splice site C
configuration. Full-length PMCA2w/a and PMCA2z/a were expressed in
MDCK cells, and their localization was studied by confocal fluorescence
microscopy. Detection of recombinant PMCAs was as described in the
legend to Fig. 6. DAPI-stained nuclei appear blue. Note that
the w splice form of PMCA2a shows prominent localization in the apical
membrane (left panel), whereas the z splice form is confined
to the basolateral membrane (right panel) as was found for
the localization of PMCA2w/b and PMCA2z/b, respectively (see Fig. 6).
Corresponding x:z sections are shown below each en face
view. Arrows indicate the height at which the en face images
were captured.
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Fig. 9.
Quantitative evaluation of apical membrane
fluorescence of PMCA2 splice site A variants expressed in polarized
MDCK cells. MDCK cells were transfected with PMCA2w/b, -2x/b,
-2z/b,- 2w/a, or -2z/a and stained with PMCA2-specific antibody NR-2
followed by anti-rabbit Alexa 488 secondary antibody. Cells were imaged
under the confocal fluorescence microscope, and images were captured of
apical membrane sections of multiple randomly selected transfected
cells. The fluorescence intensity per area was determined as described
under "Experimental Procedures," and the data for 20 individual
cells from each transfection are plotted in arbitrary units in
A. The average apical fluorescence intensity for each PMCA2
construct was calculated from the data in A and is
graphically displayed in B. Note the significant apical
fluorescence for PMCA2w/b and PMCA2w/a compared with the almost
negligible apical fluorescence for PMCA2x/b, -2z/b, and -2z/a. The data
in B represent average fluorescence intensities ± S.E.
|
|
 |
DISCUSSION |
Dynamic and spatially segregated changes in Ca2+ form
the basis for the exquisite specificity of intracellular
Ca2+ signaling. This is achieved through subcellular
compartmentalization and expression of an appropriate "tool kit" of
Ca2+ transporting, buffering, and signaling molecules (19,
20). Although the PMCAs are thought to be primarily responsible for the
global maintenance of the low resting [Ca2+]i,
recent data suggest that they participate in local Ca2+
handling and help shape the duration and spreading of subplasmalemmal Ca2+ signals (8, 21-24). Accordingly, many
PMCA isoforms and splice variants are differentially expressed and
localized in several tissues and cell types. For example, auditory and
vestibular hair cells of the inner ear almost exclusively express
PMCA2a in their apical stereocilia. In the basolateral membrane, these
same cells express mainly PMCA1b (25). Similarly, in the retina of the mouse, PMCA1 is highly abundant in the synaptic terminal and inner segment of photoreceptors but absent from the outer segment, and PMCA2
is concentrated in the synaptic membrane of rod bipolar cells in the
inner plexiform layer (26).
Differential localization in distinct membrane domains may be one of
the distinguishing features among PMCA isoforms and splice variants.
Because the most pronounced difference between PMCA splice variants
occurs between the a and b COOH-terminal splice variants, it was
tempting to speculate that their different tails carry the information
for differential membrane targeting. Moreover, only the b variants are
known to interact with PDZ domains (7, 27), and PDZ protein
interactions have been shown to be involved in the targeting,
retention, and/or anchoring of several membrane receptors and ion
channels (28, 29). Contrary to findings with the cystic fibrosis
transmembrane conductance regulator and other transporters (30-32),
our results show that the targeting of PMCAs in polarized MDCK cells is
independent of their splice site C configuration and does not require a
functional PDZ-interacting COOH-terminal tail. Instead, we have
uncovered an important role for changes at splice site A in directing
PMCA2 variants to the apical versus basolateral membrane.
Only the variant with the largest insert of 45 residues (PMCA2w) was
apically directed, whereas PMCA2x (14-residue insert) and PMCA2z (no
extra amino acids inserted) were localized in the basolateral membrane.
The importance of the splice site A configuration for
apical/basolateral targeting of PMCA2 is supported by recent isoform detection and cellular localization studies. The apical stereociliary membrane of auditory and vestibular hair cells is highly enriched for
the PMCA2w/a variant (25). In lactating mammary glands, PMCA2 is also
expressed at very high levels in the apical membrane of epithelial
cells where it is believed to play an essential role in transcellular
Ca2+ transport into the (milk) lumen. In this tissue, the
splice form corresponds to PMCA2w/b (33, 34). Thus, in both cases where PMCA2 shows apical localization in polarized cell types, its splice configuration corresponds to the w variant at site A. By contrast, the
splice site C configuration appears to be of lesser importance as it is
a in one cell type but b in the other. Interestingly, Dumont et
al. (25) recently identified a novel PMCA2 splice site A variant
expressed in the apical stereocilia of frog cochlear hair cells. This
variant, termed "v," encodes an even longer insert of 57 amino
acids, consisting of the 45 residues found in the w variant and an
additional 12 residues encoded by a separate exon. In mammals, however,
a PMCA2 splice site A variant corresponding to frog PMCA2v has never
been observed.
Our knowledge concerning the spatial organization of Ca2+
extrusion mechanisms at the plasma membrane is limited. Although the molecular characterization of the two major transport systems, the
PMCAs and the Na+/Ca2+ exchangers, has advanced
rapidly over the last few years (reviewed in Refs. 2, 35, and 36), the
mechanisms by which these proteins are targeted to specific membrane
domains and assembled into functional units with other Ca2+
signaling molecules remain to be elucidated. How could the w insert at
splice site A alter the membrane targeting of PMCA2? Two different but
not mutually exclusive possibilities are as follows: 1) a change in the
interaction of the pump with specific lipids, and 2) a difference in
protein-protein interactions. Interestingly, the splice site A insert
is situated immediately NH2-terminal to a
phospholipid-sensitive region in the first cytosolic loop of the pump
(37) (see Fig. 1). The 31 extra amino acids of the w splice form may
alter the structural properties of the adjacent phospholipid-sensitive
region and/or membrane-spanning domains to enable their preferential
interaction with lipids enriched in apical membranes. The w insert is
rich in hydrophobic (Ala and Leu) as well as in helix-breaking (Gly and
Pro) residues (3, 4) and may thus assume a unique structural
conformation. This structural motif may be involved in specific
protein-protein interactions, e.g. with proteins involved in
apical targeting or apical protein retention. A differential yeast
two-hybrid screen with the first cytosolic loop of the w and x (or z)
splice variants as bait may provide an attractive future approach to
this problem.
Finally, alternative splicing at site A of PMCA2 appears to be highly
regulated. In IMR32 neuroblastoma cells, a specific and rapid switch
from the 2w to the 2x variant occurs upon KCl depolarization. This
splice switch is dependent on a rise in intracellular Ca2+,
is independent of new protein synthesis, and can be observed within
30-45 min of cell stimulation (38). Although differential membrane
distribution of PMCA2x and 2w variants has not been investigated in
IMR32 cells, it is of interest that depolarization and a rise in
Ca2+ are triggers of (neuronal) differentiation. Perhaps
the alternative splice switch at site A of PMCA2 allows the cell to
quickly re-distribute PMCA2 to membrane domains where they are needed.
In neurons, this could be at newly forming synaptic sites in the
dendrites. In epithelial cells of the mammary gland or the distal
kidney, the PMCA2w splice form may be targeted to apical membranes in
response to hormonal regulation of transcellular Ca2+ flux.
Thus, alternative RNA splicing affecting the first cytosolic loop of
PMCA2 appears to be a means for the regulated re-deployment of this
Ca2+ pump to different membrane domains.
 |
ACKNOWLEDGEMENTS |
We are grateful to Billie-Jo Brown and Anne
Vrabel for help with the construction of the PMCA2b expression plasmids
and to Aida Filoteo and Dr. John Penniston (Mayo Clinic, Rochester, MN) for antibodies against the PMCAs. We thank Dr. Steve DeMarco for experimental advice and for teaching one of us (M. C.) how to operate
the confocal microscope.
 |
FOOTNOTES |
*
This work was supported by National Institutes of
Health Grant GM-58710 and by the Mayo Foundation for Medical Education
and Research.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Present address: Program in Cellular and
Molecular Biology, University of Wisconsin, Madison, WI 53706.
§
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, Mayo Clinic/Foundation, 200 First St. S.W., Rochester, MN 55905. Tel.: 507-284-9372; Fax: 507-284-2384; E-mail: strehler.emanuel@mayo.edu.
Published, JBC Papers in Press, March 6, 2003, DOI 10.1074/jbc.M301482200
 |
ABBREVIATIONS |
The abbreviations used are:
PMCAs, plasma membrane Ca2+-ATPases;
MDCK, Madin-Darby canine
kidney;
GFP, green fluorescent protein;
DAPI, 4',6'-diamidino-2-phenylindole;
NHERF2, Na+/H+ exchanger regulatory factor-2;
DPBS, Dulbecco's phosphate-buffered saline;
CM, Ca2+/Mg2+.
 |
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