From the Program in Molecular Neuroscience, Department of Biochemistry and Molecular Biology, Mayo Graduate School, Mayo Clinic, Rochester, Minnesota 55905
Received for publication, February 14, 2001, and in revised form, March 19, 2001
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ABSTRACT |
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Spatial and temporal regulation of
intracellular Ca2+ signaling depends on localized
Ca2+ microdomains containing the requisite molecular
components for Ca2+ influx, efflux, and signal
transmission. Plasma membrane Ca2+-ATPase (PMCA) isoforms
of the "b" splice type contain predicted PDZ (PSD95/Dlg/ZO-1)
interaction domains. The COOH-terminal tail of PMCA2b isolated the
membrane-associated guanylate kinase (MAGUK) protein SAP97/hDlg as a
binding partner in a yeast two-hybrid screen. The related MAGUKs
SAP90/PSD95, PSD93/chapsyn-110, SAP97, and SAP102 all bound to the
COOH-terminal tail of PMCA4b, whereas only the first three bound to the
tail of PMCA2b. Coimmunoprecipitations confirmed the interaction
selectivity between PMCA4b and SAP102 as opposed to the promiscuity of
PMCA2b and 4b in interacting with other SAPs. Confocal
immunofluorescence microscopy revealed the exclusive presence and
colocalization of PMCA4b and SAP97 in the basolateral membrane of
polarized Madin-Darby canine kidney epithelial cells. In hippocampal
neurons, PMCA2b was abundant throughout the somatodendritic compartment
and often extended into the neck and head of individual spines where it
colocalized with SAP90/PSD95. These data show that PMCA "b" splice
forms interact promiscuously but also with specificity with different
members of the PSD95 family of SAPs. PMCA-SAP interactions may play a role in the recruitment and maintenance of the PMCA at specific membrane domains involved in local Ca2+ regulation.
Calcium ion (Ca2+) homeostasis is crucial for cell
function and survival (1). A finely controlled system of
Ca2+ transporters, channels, and Ca2+-binding
proteins allows for transient increases in the intracellular free
calcium concentration
([Ca2+]i),1
while over the long term maintaining a low resting
[Ca2+]i (2). The exquisite
specificity of Ca2+ signaling mandates that both the entry
and the removal of Ca2+ are under precise temporal and
spatial control (3, 4). Accordingly, the molecular machinery involved
in local Ca2+ signaling must be assembled, maintained, and
regulated with the requisite spatial and temporal resolution.
Mechanisms that increase local
[Ca2+]i have been studied
extensively over the last few years; consequently, significant progress
has been made in understanding the regulation and targeting of calcium
channels (5). By contrast, much less is known about the spatial
organization of Ca2+ extrusion mechanisms, specifically
that provided by plasma membrane Ca2+-ATPases (PMCAs).
These primary ion pumps are essential for the long term maintenance of
low intracellular Ca2+ but more recently have also been
implicated in dynamic events such as the regulation of Ca2+
spikes and local Ca2+ signaling (for review, see Refs.
6-8).
A multigene family of four non-allelic members encodes four conserved
mammalian PMCA isoforms (designated PMCA1-4), with additional diversity generated by alternative mRNA splicing affecting the protein at two major locations (9). The four PMCA gene products do not
differ greatly in their overall tertiary structure, which includes 10 predicted transmembrane spans, intracellular NH2 and COOH
termini, and a large cytosolic catalytic loop between membrane spans 4 and 5 (10). However, there are substantial differences in their
regulation by kinases, proteases, and the Ca2+-binding
protein calmodulin. Moreover, the major PMCA variants "a" and
"b" generated by alternative splicing in the COOH-terminal coding
region differ markedly in their regulatory properties, most notably in
calmodulin sensitivity (7). The alternative splice affects the pump
protein after the last transmembrane domain and creates different
COOH-terminal amino acid sequences for the "a" and "b" variants
because of a change in translational reading frame. The last few
residues of all "b" variants are highly conserved, and the final
four residues of PMCA4b match the minimal consensus sequence
(E-T/S-X-V*, where the asterisk indicates the COOH-terminal residue) of protein ligands for type I PDZ
(PSD95/Dlg/ZO-1) domains (11).
PDZ domains are present in a large variety of different proteins and
constitute a ubiquitous protein-protein interaction motif (12, 13).
Indeed, we reported previously that PMCA4b was able to interact with
high affinity with the PDZ domains of several members of the MAGUK
(membrane-associated guanylate kinase) protein family, and that this
interaction was dependent on the presence of the T-S-V* COOH-terminal
sequence (11).
MAGUKs are multimodular PDZ domain-containing proteins implicated in
the formation and maintenance of specialized cell-cell junctions and in
signaling processes at the cell membrane (14, 15). A subfamily of the
MAGUKs are PSD95 (95-kDa protein of the postsynaptic density)-like
molecules, also referred to as SAPs
(synapse-associated proteins). SAPs
are found throughout the brain, and some are also present in other
tissues such as gut and kidney epithelium (14-19). Their precise
function is not well understood, but they are thought to act as
scaffolding and/or clustering proteins for various membrane receptors
and transporters. In addition, they recruit regulatory and signaling
proteins to the membrane and may thus be required for the formation of
multiprotein signaling complexes at specific membrane domains (for
review, see Refs. 16-21). Several ion channels and receptor proteins
have been identified as binding targets of the SAPs, including
N-methyl-D-aspartate receptor subunits and
K+ channels (16, 17, 22, 23).
Here, we used the PMCA2b COOH-terminal sequence as bait in an unbiased
yeast two-hybrid screen of a human brain cDNA library and isolated
a clone coding for SAP97 as one of the specific interactors. To address
the promiscuity versus specificity in the interaction of
PMCA2b and PMCA4b with different SAPs, we used pull-down and coimmunoprecipitation assays to analyze their interaction with the four
SAPs SAP90/PSD95, SAP93/chapsyn-110, SAP97/hDlg, and SAP102. The
results show that PMCA2b and 4b interact promiscuously with all SAPs
except for SAP102 which binds to PMCA4b but not to PMCA2b. Using
confocal immunofluorescence microscopy, we demonstrate colocalization
of PMCA4b and SAP97 at the basolateral membrane of MDCK epithelial
cells, and of PMCA2b and SAP90/PSD95 at most, but not all, synaptic
spines in hippocampal neurons. A direct, PDZ domain-mediated
interaction with SAPs may be an attractive means to localize PMCA
isoforms to specific membrane domains and to recruit them into
multiprotein Ca2+ signaling complexes.
Plasmid Constructions--
Plasmids encoding fusion proteins of
the PMCA carboxyl-terminal sequences were made by standard molecular
biology techniques using polymerase chain reaction or suitable
restriction enzyme digests. Codons for the final 72 (PMCA2b) or 71 (PMCA4b) amino acids were cloned as EcoRI-BamHI
fragments into pAS2-1 (CLONTECH, Palo Alto, CA) to
produce the DNA binding fusions (pDB-CT2b and pDB-CT4b) for yeast
two-hybrid screens and assays. The same codons were inserted into the
pGEX-2TK (Amersham Pharmacia Biotech) vector to produce GST fusions
(GST-2b and GST-4b). Plasmid pMM2-PMCA4b for expression of
full-length human PMCA4b in mammalian cells has been described
previously (24). A vector expressing full-length human PMCA2b was
assembled from overlapping partial PMCA2 cDNAs (25) using a
combination of restriction digests and polymerase chain reaction. The
final PMCA2b cDNA fragment was cloned as an SalI-MluI fragment into the modified
pMM2 expression vector (24) to create plasmid
pMM2-PMCA2b. The integrity of all final constructs was
confimed by DNA sequencing in the Mayo Clinic Molecular Biology Core
Facility. Mammalian expression constructs for SAP93/chapsyn-110 and
SAP102 were kind gifts from Morgan Sheng (Harvard Medical School,
Boston) and Craig Garner (University of Alabama, Birmingham), respectively.
Yeast Two-hybrid Assays--
Yeast two-hybrid screening was
performed according to the instructions for the Matchmaker Two-Hybrid
System 2 (CLONTECH). Yeast of strain CG1945 were
cotransformed using the lithium acetate method (26) with bait plasmid
pDB-CT2b and a human brain cDNA library
(CLONTECH) made in the vector pACT2. Approximately
5 × 106 independent cDNA clones were screened and
assayed for HIS3 and Recombinant Protein Expression and Purification--
GST and GST
fusion proteins were expressed as described (28) in E. coli
BL21(DE3) upon induction with 0.7 mM
isopropyl-1-thio- Pull-down Assays--
Brains were removed from male Harlan
Sprague-Dawley rats (~250 g) and were immediately homogenized using a
glass-Teflon homogenizer in cold 10 mM Tris-HCl, pH 7.4, containing 0.3 M sucrose, 20 mM EDTA, 10 mM EGTA, 75 mM NaCl (10 ml/brain), and a
mixture of protease inhibitors. After ~20 strokes, the homogenate was
centrifuged at 3,700 × g for 2 min. The supernatant
was subjected to high speed centrifugation at 150,000 × g to pellet membranes. Membranes were then solubilized in
SDS buffer (50 mM Tris-HCl, pH 7.4, 5 mM EDTA,
5 mM EGTA, 50 mM NaCl, and 1% SDS), for 20 min
at 55 °C. SDS in the membrane extract was neutralized in 4 volumes
of cold 1% Triton X-100, 5 mM EDTA, 5 mM EGTA,
and the extract was chilled on ice for 10 min before centrifuging at
20,000 × g for 30 min. 5 µg of GST alone or of GST
fusion proteins on agarose beads was rocked overnight with 750 µl of
cleared brain extract. The beads were pelleted and washed three times
in TBS + 1% Triton X-100. Bound proteins were eluted in Laemmli buffer
(28) and separated on 10% polyacrylamide gels followed by transfer to
nitrocellulose after standard Western blotting procedures (28).
Nitrocellulose membranes were blocked in TBST + 5% milk before
immunoblotting with appropriate primary and secondary antibodies. All
secondary antibodies on immunoblots were detected using
RenaissanceTM chemiluminescent reagent (NEN Life Science Products).
Coimmunoprecipitations--
COS-1 cells were grown to ~80%
confluence on six-well plates (Costar) in Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum, nonessential amino
acids, 1 mM sodium pyruvate, glutamine, and
antibiotic/antimycotic mixture (all cell culture reagents were
purchased from Life Technologies, Inc.). Cells were transfected with 2 µg of total DNA using LipofectAMINE according to the manufacturer's
instructions (Life Technologies, Inc.). After ~48 h, the cells were
rinsed with cold D-PBS (Ca2+- and
Mg2+-free) and lysed in a buffer containing 50 mM HEPES at pH 7.4, 150 mM NaCl, 1% Nonidet
P-40, 0.5% sodium deoxycholate, and protease inhibitors. After 10 min
on ice the cells were scraped from the plates using a rubber policeman
and were centrifuged at 13,000 × g for 10 min at
4 °C. Half of the lysate (500 µl) was used for each
immunoprecipitation, to which 2-3 µl of antibody (anti-PMCA monoclonal 5F10) was added. After 2 h of rocking at 4 °C, 50 µl of protein A/G-agarose was added to each mixture, and rocking continued overnight at 4 °C. Protein A/G-agarose was pelleted at
4,000 × g for 30 s and quickly washed three times
in cold TBST. Bound proteins were eluted in Laemmli buffer (28). All of
the bound protein and 5% of the starting lysate were separated on 10%
polyacrylamide gels followed by transfer to nitrocellulose for Western
blotting as described above.
Antibodies for Immunoblotting--
The following antibodies were
obtained from the indicated source and used at the indicated dilution
for immunoblotting. Anti-SAP90 was from Transduction Laboratories
(Lexington, KY) and used at 1:400 dilution. Anti-SAP93 was obtained
from David Bredt (University of California, San Francisco) and used at
1:1,000 dilution. Anti-SAP97 and anti-SAP102 were obtained from Craig
Garner (University of Alabama, Birmingham), and both were used at
dilutions of 1:2,000. Anti-PMCA2 (affinity-purified and concentrated
NR2) and anti-PMCA4 (JA9) were obtained from John Penniston and
Adelaida Filoteo (Mayo Clinic) and used at dilutions of 1:5,000 and
1:600, respectively. Secondary goat anti-mouse or goat anti-rabbit
antibodies were purchased from Sigma and used at 1:5,000 dilution.
Immunofluorescence--
Type I MDCK epithelial cells (ATCC
CCL-34, Manassas, VA) were grown to confluence on glass coverslips in
Dulbecco's modified Eagle's medium containing 10% fetal bovine serum
and supplemented with 1% antibiotic-antimycotic (Life Technologies,
Inc.). The cells were then fixed for 5 min at room temperature in 4%
paraformaldehyde (Tousimis, Rockville, MD) diluted in D-PBS + Ca2+/Mg2+ (D-PBS+CM). After five brief washes
in D-PBS+CM, coverslips were further fixed and permeablized in
prechilled methanol for 5 min at
Primary rat hippocampal neurons were prepared from 17/18-day-old
embryos essentially as described (29) and used at postnatal day 21-30.
Cells grown on coverslips were fixed in 4% paraformaldehyde, 4%
sucrose, D-PBS+CM for 20 min, washed, and incubated in blocking buffer
as above, and stained with anti-PMCA2 antibody NR2 (diluted 1:800 in
blocking buffer) and anti-SAP90 (1:250 dilution in blocking buffer)
each for 1 h at room temperature. After washing three times for 5 min, the cells were incubated for 1 h at room temperature with
anti-mouse Alexa 488 and anti-rabbit Alexa 594 secondary antibodies
diluted 1:800 in blocking buffer. After secondary antibody incubation,
the cells were washed five times for 10 min with D-PBS+CM.
After the final washing, coverslips were mounted in Prolong mounting
media (Molecular Probes). Confocal micrographs were taken on a Zeiss
LSM 510 using an Apochromat 63× (MDCK), or Apochromat 100× (neurons)
objective, and captured using LSM 510 software (Zeiss). Images were
imported and edited using Adobe Photoshop 5.0.
Yeast Two-hybrid Screening Using the PMCA2b COOH-terminal Tail as
Bait--
A sequence alignment of the extreme COOH termini of the PMCA
"b" forms suggested that PMCA1b, 2b, and 3b would bind the same PDZ
domains (Fig. 1A), whereas
PMCA4b would interact with perhaps a different set of PDZ domains. For
this reason, we sought to identify proteins that would interact with
the COOH-terminal sequence of PMCA1b/2b/3b, using PMCA2b as the
representative isoform in an unbiased screen. A protein consisting of
the Gal4 DNA binding domain fused to the final 72 amino acids of
hPMCA2b (Fig. 1B) was used as bait in a yeast two-hybrid
screen of a human brain cDNA library. Screening 5 × 106 independent clones yielded ~30 HIS3- and
Selectivity of the Interaction of PMCA2b and PMCA4b with Different
MAGUKs--
We speculated that some selectivity might exist in the
interaction of these PMCAs with different members of the SAP90/PSD95 family of MAGUKs. To test this, we used GST fusion proteins containing the COOH termini of PMCA2b (CT2b) and PMCA4b (CT4b) in pull-down assays
from rat brain extracts. Rat brain contains all of the SAP proteins
(SAP90, SAP93, SAP97, SAP102) as well as all isoforms and most splice
forms of the PMCAs (9, 18). As shown in Fig. 2, there is clearly promiscuity between
PMCA2b and 4b in their ability to bind different SAPs. However, there
is also a distinct degree of binding specificity of these two PMCAs for
some SAPs as illustrated most clearly for SAP102. Although the data in
Fig. 2 indicate that SAPs generally interact with higher affinity with PMCA4b than with PMCA2b, the results also show that with the exception of SAP102, all are capable of binding PMCA2b in vitro.
Coimmunoprecipitation of Full-length PMCAs and SAPs--
To
determine if the promiscuity and selectivity of the interaction of
PMCA2b and 4b with different members of the SAP family of MAGUKs are
maintained when the full-length calcium pumps are coexpressed with
these SAPs in an in vivo environment, we cotransfected COS-1
cells with plasmids encoding different combinations of the pumps and
either SAP93 or SAP102. As predicted from the pull-down assays using
the recombinant COOH-terminal tails of the PMCAs, antibodies against
the PMCA were able to coimmunoprecipitate SAP93 together with either
PMCA2b or PMCA4b (Fig. 3, left
panel). By contrast, SAP102 only coimmunoprecipitated well with
PMCA4b but not with PMCA2b (Fig. 3, right panel), confirming
the selectivity of the interaction between PMCA4b and SAP102.
In Vivo Colocalization of PMCA4b and SAP97 at the Basolateral
Membrane of Polarized Epithelial Cells--
Using the pull-down and
coimmunoprecipitation data as a predictive guide, we sought mammalian
tissues and cell types that coexpress SAP and PMCA family members to
determine their cellular localization in vivo. In intestine
and kidney epithelia, the PMCA has previously been shown to be
predominantly localized at the basolateral membrane (30, 31). We
therefore used cultured MDCK cells as a representative epithelial cell
type showing distinct apical and basolateral membrane domains. However,
the isoform composition and subcellular distribution of PMCAs in MDCK
cells had not yet been well documented. Immunoblotting of membrane
proteins from MDCK cells indicated that PMCA4 is a major isoform in
these cells. Using membrane protein from PMCA4b-overexpressing COS-1 cells as a sizing standard, we determined that the PMCA4 variant in
MDCK cells is of the "b" splice form (Fig.
4A). This was confirmed independently by sequencing reverse transcription-polymerase chain reaction products from MDCK
mRNA.2 The anti-SAP97
antibody (a gift from C. C. Garner) recognized a single major
protein of ~140 kDa in Western blots of MDCK membranes (Fig.
4A). This corresponds to the size of SAP97 reported by other authors (32), demonstrating that MDCK cells express endogenous SAP97.
Using these antibodies, we detected strong basolateral membrane
staining for both PMCA4b and SAP97 in polarized MDCK cells (Fig.
4B) by confocal microscopy. Sections through the cells in
the x-z plane confirmed that the staining was exclusively basolateral for both proteins, with essentially complete overlap of fluorescence (Fig. 4B). The same results were obtained in the human MCF7
cell line from breast epithelium (data not shown). These results
demonstrate that in two different epithelial cell lines, PMCA4b and
SAP97 coexist at the basolateral membranes.
PMCA2b and SAP90 Colocalize in Many but Not All Dendritic Spines in
Hippocampal Neurons--
We next examined neurons from rat hippocampus
because initial immunofluorescence suggested that PMCA2 was a major
Ca2+ pump isoform in these cells. This is in agreement with
previously published data on the PMCA isoform distribution in rodent
and human hippocampus (33-35). An immunoblot from primary hippocampal neurons indicated that PMCA2 was indeed expressed in these cells. Based
on the apparent size of the PMCA2 isoform we concluded that these cells
contain the "b" splice form (Fig.
5A). Additional immunoblots
did not detect any PMCA2a isoform or PMCA4 (not shown); hence, any
immunostaining on these cells with the anti-PMCA2 antibody will
represent expression of PMCA2b. PMCA2b staining was apparent throughout
the plasma membrane of the pyramidal neurons of the hippocampus (Fig.
5B). The entire boundary of the soma can be seen as well as
extensive dendritic staining. Closer examination of the dendrites
reveals dendritic spines that are rich in PMCA2b fluorescence (Fig.
5C). This is reminiscent of earlier results obtained with
Purkinje cells of rodent cerebellum, in which PMCA2 is enriched in the
dendritic spines (35). Interestingly, however, PMCA2b staining of the
dendritic spines is heterogeneous in the hippocampal cells; some spines
were completely labeled throughout their extent, including the neck and
head of the spine. In other spines, PMCA2b fluorescence seemed to be
confined to the neck of the spine and did not occupy the mushroom-like
head. This was confirmed by immunofluorescent staining with anti-SAP90
antibody in the same cells (Fig. 5C). Within certain
populations of spines, there was nearly complete colocalization of the
PMCA2b and SAP90 fluorescence, suggesting a potentially direct
interaction between these two proteins in this specific cellular
compartment. In other spines, however, we found essentially no overlap
of signals from these two proteins (Fig. 5C). This disparity
in labeling for PMCA2b and SAP90 in some spines may reflect distinct
Ca2+ handling properties of different spine types.
This study demonstrates that PMCA2b and 4b, and likely all other
PMCA "b" splice forms, are binding partners for members of the
SAP90/PSD95 family of MAGUKs. There is, however, a certain degree of
specificity in these interactions. Earlier work had shown a strong
interaction between PMCA4b and several MAGUKs (11), but no unbiased
screen for interacting proteins had yet been performed using the
COOH-terminal tail of a PMCA "b" variant as bait. We chose the
PMCA2b COOH-terminal tail as bait to screen a brain cDNA expression
library because PMCA2b is highly expressed in specific neurons of the
brain and because its carboxyl-terminal amino acid is leucine rather
than the consensus valine found in PMCA4b. Because the last four
residues of PMCA2b are identical to those in PMCA1b and 3b (E-T-S-L*),
the interacting MAGUK proteins that we identified as binding partners
of PMCA2b are likely binding to PMCA 1b and 3b as well.
Our result showing an interaction of the carboxyl-terminal portion of
PMCA2b with a member of the MAGUK family (SAP97) in an unbiased yeast
two-hybrid screen is not unexpected based on results of unbiased
screens of peptide libraries using different PDZ domains as baits (36,
37). These studies predict that PDZ domains 1-2 of murine Dlg (SAP97)
interact preferentially with the sequence -E(S/T)X(V/I/L)*. Because all
of the PMCA "b" splice forms terminate with the sequence -ETSL/V*
(Fig. 1A), it would be expected that all of them can bind to
SAP97/hDlg.
The four MAGUKs referred to as SAPs (PSD95/SAP90,
PSD93/chapsyn-110/SAP93, hDlg/SAP97, and NE-Dlg/SAP102) are the most
closely related MAGUK family members and are all mammalian homologs of the Drosophila Dlg (discs-large) protein. We used
immobilized GST fusion proteins carrying the final ~70 amino acids of
PMCA2b (CT2b) and PMCA4b (CT4b) to examine the relative strength of
interaction of these two pump isoforms with the four SAPs in rat brain
homogenates. All of the SAPs were capable of binding CT4b, whereas only
SAP90, SAP93, and SAP97 bound to CT2b. This result was confirmed by
coimmunoprecipitations of full-length proteins overexpressed in COS-1
cells. Whereas SAP93 coprecipitated equally well with full-length
PMCA2b and PMCA4b, SAP102 coprecipitated well only with PMCA4b but not
with PMCA2b. The apparent selectivity of the two Ca2+ pump
isoforms in their ability to interact with SAP102 is remarkable considering the high degree of conservation among all PMCA "b" splice variants in their consensus COOH-terminal PDZ binding region. It
is likely that the difference in SAP102 preference is a result of
additional interactions provided by residues NH2-terminal
to the canonical PDZ binding domain in the PMCAs. Indeed, there is little sequence identity between PMCA2b and 4b in the ~50 residues immediately upstream of the six-residue COOH-terminal PDZ interacting sequence (see Fig. 1B). Structural and biochemical analyses
of PDZ domain- peptide interactions have suggested previously that residues other than those constituting the minimal essential consensus sequence contribute to the specificity of a given PDZ domain-target peptide recognition (36-38). Selectivity in the interaction with different SAPs has been reported recently for another membrane protein
containing a COOH-terminal PDZ binding consensus sequence. The
Earlier reports have provided evidence for a basolateral localization
of the PMCA in kidney and intestinal epithelial cells involved in
transcellular vectorial Ca2+ transport (30, 31). The major
PMCA isoforms expressed in these tissues are PMCA1b and PMCA4b
(43-45). Using immunoblotting and immunofluorescent labeling, we
demonstrated that PMCA4b is expressed in MDCK cells and is localized to
the basolateral membrane. In this membrane compartment, PMCA4b
colocalizes with SAP97. Because PMCA4b was readily able to pull down
SAP97 from brain homogenate, and considering the previously determined
high affinity interaction (Kd ~ 2 nM)
between these two proteins (11), PMCA4b is a likely in vivo
partner for SAP97. Regulated vectorial Ca2+ movement across
epithelia is accomplished largely through transcellular transport that
in the intestine and kidney occurs in the apical to basolateral
direction. The confinement of the PMCA to the basolateral membrane is
thus physiologically relevant, yet it is not known how the pump is
targeted to, and maintained at, this membrane location. PDZ
domain-containing proteins play an important role in the establishment
and maintenance of the apicobasolateral polarity in epithelia (14, 20,
46). PDZ proteins such as ZO-1/-2/-3, Dlg, and discs-lost
(47-49) are required for the formation of tight junctions and
maintenance of polarity. A heteromultimeric protein complex consisting
of the PDZ proteins LIN-2, LIN-7, and LIN-10 acts to localize and
tether the basolateral Caenorhabditis elegans LET-23
epidermal growth factor receptor to the appropriate domain (50), and a
PDZ domain interaction reportedly retains the membrane-bound epithelial
MAGUK (PDZ domain)-mediated localization/retention of specific PMCA
isoforms can be imagined in other cell types such as neurons. As
excitable cells, neurons are particularly dependent on spatially and
temporally controlled Ca2+ influx and efflux for function.
The PMCAs are expressed abundantly in neurons, and many isoforms and
splice variants are highly enriched in specific brain structures and
cell types (33-35). PMCA2b is a major PMCA isoform in rat hippocampal
neurons, and immunocytochemistry showed that this isoform was
distributed throughout the somatodendritic membrane. PMCA2b staining
could be seen in the neck, and in some cases even the heads, of
dendritic spines. In these spines, PMCA2b immunoreactivity overlapped
with the postsynaptic MAGUK protein SAP90/PSD95. In other spines,
PMCA2b appeared to be excluded from the synaptic region but was still
present in the neck and perisynaptic area. We also observed spines
that contained SAP90/PSD95 immunoreactivity, presumably at
postsynaptic densities, but PMCA2b immunoreactivity was only present in
the neck of the spine. Because PMCA2b can interact with SAP90 as
demonstrated by pull-down assays from rat brain homogenates, it is
plausible that this Ca2+ pump may be kept in close vicinity
of certain synapses via an interaction with SAP90. SAPs can multimerize
and act as scaffolds for the clustering of a variety of different
membrane and signaling proteins, including
N-methyl-D-aspartate receptors, K+
channels, and nitric-oxide synthase. As a major Ca2+ efflux
system, PMCA2b may be recruited into such macromolecular complexes by
SAP90 to participate in the generation of Ca2+ microdomains
at subsynaptic sites (5). Maintaining or enriching the PMCA2b in the
perisynaptic membrane and/or around the neck of dendritic spines could
be a means to insulate local Ca2+ signals in individual
spines, thereby preventing signaling noise caused by subthreshold
Ca2+ influx. On the other hand, the lack of colocalization
of PMCA2b and SAP90 in some dendritic spines may indicate the presence
of synapses (and spines) in hippocampal neurons that are
heterogeneous in their Ca2+ handling requirements.
PMCA2 expression is up-regulated late in fetal brain development and
shortly after birth in rodents (56, 57). It will be interesting to
examine the distribution of PMCA2b in relation to different SAPs and
other known SAP-interacting proteins in the spines of mature, adult
hippocampal neurons as well as in neurons of other brain regions.
Cerebellar Purkinje cells are extremely rich with dendritic PMCA2 (35,
58), but in contrast to hippocampal neurons, they express little SAP90
(18, 40, 59). Instead, SAP93 is a major SAP isoform in these neurons where it localizes to the soma and dendrites (59). Given the differences in tissue, cellular, and subcellular expression and localization of different SAPs, a specific PMCA isoform such as PMCA2b
may be interacting with varying SAP partners at different membrane
locations. In the future, it will be interesting to identify signals
that might influence the PMCA-MAGUK association and dissociation. Modulating these interactions may be one mechanism by which cells can
alter the spatial and temporal control of submembranous
Ca2+ signaling. Such regulation could occur in the short
term through phosphorylation of either the PDZ domains or their ligands
(i.e. the PMCA carboxyl-terminal tail). In the case of the
PMCAs, long term regulation may also result from changes in alternative
splicing which shift the ratio of "a" to "b" splice forms and
thereby prevent or enable specific PDZ domain interactions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase expression.
Selection of initial positives was done after 7 days of growth on
SD/-Trp/-Leu/-His agar plates containing 5 mM
3-aminotriazole (Sigma). Positive clones were propagated on
SD/-Trp/-Leu medium.
-Galactosidase assays were performed after
freeze-thaw of yeast colonies on filter lifts of 3-day-old streak
plates. The bait plasmid was dropped out by 1 µg/ml cycloheximide counterselection, and growth on -Leu medium. After bait dropout, plasmid inserts from positive yeast clones were amplified directly after picking 3-day-old yeast colonies into TE (10 mM
Tris-HCl, pH 7.5, 1 mM EDTA) containing 0.25 unit/µl
lyticase (Sigma). After 30 min at 37 °C, the yeast were subjected to
one round of freeze-thaw followed by alkaline lysis and
phenol/chloroform extraction. The aqueous fraction was precipitated
with 1/10 volume of 5 M sodium acetate, pH 5.3, and 0.7 volume isopropyl alcohol; the nucleic acid pellet was washed in 70%
ethanol and dissolved in 10 mM Tris, pH 8.0. 1% of the
dissolved DNA was used as template for polymerase chain reaction with
pACT2-specific primers. Library plasmids were prepared from
counterselected yeast containing only "prey" plasmids by using the
above alkaline lysis method (27) and transformed directly into
Escherichia coli strain DH5
by electroporation.
-D-galactopyranoside for 4 h.
Cells were pelleted, resuspended in TBS (50 mM Tris-HCl, pH
7.4, 150 mM NaCl) plus protease inhibitors (0.2 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, 2 µg/ml leupeptin, 0.2 µg/ml aprotinin, 10 mM EDTA), and
30 mM
-mercaptoethanol, and lysed by the addition of
sarkosyl (Curtis-Matheson Scientific, Houston, TX) to a final concentration of 1.5%. After 15 min on ice, the lysate was cleared by
centrifugation at 10,000 × g and supplemented by the
addition of Triton X-100 to 2%. This lysate was then bound to
glutathione-Sepharose (Sigma) and washed with TBST (TBS + 0.1% Tween),
and TBS. The quantity of bound fusion protein was estimated by
Coomassie Blue staining of SDS-polyacrylamide gels of known amounts of
fusion protein-containing glutathione-Sepharose beads. All fusion
proteins were adjusted to ~0.5 mg/ml for pull-down assays.
20 °C. The cells were blocked in
D-PBS+CM containing 5% normal goat serum and 1% bovine serum albumin
and were then incubated for 1 h at room temperature with
monoclonal pan-anti-PMCA antibody 5F10 (a gift from John Penniston and
Adelaida Filoteo, Mayo Clinic) and polyclonal anti-SAP97 diluted 1:600
and 1:200, respectively, in blocking buffer. After washing 3 × 5 min in D-PBS+CM, the cells were incubated for 1 h at room
temperature with anti-mouse Alexa 488 and anti-rabbit Alexa 594 (both
from Molecular Probes, Eugene, OR), each diluted 1:600 in blocking buffer.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase-positive clones that were analyzed further.
Sequencing of the cDNA inserts revealed that five clones specified
proteins containing at least one PDZ domain. Of these, clone J5 was one
of the strongest interactors (as determined by
-galacosidase assay;
data not shown) and encoded a nearly full-length sequence (amino acids
242-926) of human SAP97/hDlg (Fig. 1C).
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Fig. 1.
Alignment of PMCA COOH-terminal sequences and
scheme of the SAP97/hDlg clone isolated by yeast two-hybrid screening
with the PMCA2b bait. A, alignment of the final 10 amino acids of the four human PMCA "b" splice forms. PMCA1-3 all
terminate in the sequence -ETSL* (*=stop), and are expected to have
similar affinities for the same PDZ domains. PMCA4b differs in that it
terminates in -ETSV* and therefore may interact with a specific subset
of PDZ domain-containing proteins. B, alignment of the
PMCA2b and PMCA4b amino acid sequence (CT2b and CT4b) included in yeast
two-hybrid bait vectors and GST fusion proteins. Note that PMCA2b and
PMCA4b differ substantially in their carboxyl-terminal tails except for
the last six amino acids, which correspond to the core of their PDZ
binding domains. C, schematic representation of the yeast
two-hybrid "prey" clone obtained by screening a human brain
cDNA library with the CT2b bait. The entire clone (double
arrowed line) contained ~2.5 kilobases of the cDNA
representing the human homolog of Dlg (hDlg), also known as SAP97. The
clone encodes amino acids 242-926 of SAP97/hDlg as well as ~400 base
pairs of 3'-untranslated sequence. A linear scheme of the protein is
shown below the cDNA, and the major domains are indicated in
gray (PDZ domains), black (SH3 domain), and
white (guanylate kinase-like domain). N,
NH2 terminus; C, COOH terminus.
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Fig. 2.
Protein pull-down experiments from rat brain
lysate showing the promiscuity and specificity of PMCA-SAP protein
interactions. Equal amounts of GST, GST-CT2b, or GST-CT4b bound to
glutathione-Sepharose beads were incubated with equal amounts of rat
brain lysate, washed, and the bound proteins separated on
SDS-polyacrylamide gels as described under "Experimental
Procedures." Immunoblots were then done with specific anti-SAP
antibodies as indicated. SAP90, SAP97, and SAP93 bind to both CT2b and
CT4b, with SAP90 and SAP97 showing some preference for CT4b over CT2b.
By contrast, SAP102 interacts only with CT4b but not with CT2b. None of
the SAPs bound to GST alone (first lane of each
panel).
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Fig. 3.
Coimmunoprecipitation of PMCA2b and 4b with
SAP93 and SAP102. COS-1 cells were cotransfected with different
protein expression constructs as indicated on the top of
each lane. After 48 h, cells were lysed and proteins
either separated directly by SDS-polyacrylamide gel electrophoresis
(left lane of each panel), or immunoprecipitated
(IP) with anti-PMCA antibody 5F10 prior to
SDS-polyacrylamide gel electrophoresis as described under
"Experimental Procedures." Western blots (WB) of the
separated proteins were then probed with antibodies against SAP93
(left panel) or SAP102 (right panels). Note that
SAP93 coimmunoprecipitates well with both PMCA2b and 4b, whereas SAP102
coimmunoprecipitates well with PMCA4b but poorly with PMCA2b.
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Fig. 4.
PMCA4b and SAP97/hDlg are expressed in
MDCK cells and colocalize at the basolateral membrane.
A, Western blot demonstrating expression of PMCA4b and
SAP97/hDlg in MDCK cells. 3 µg of membrane protein from COS-1 cells
overexpressing hPMCA4b as control (lane 1) and 10 µg of
membrane protein from MDCK cells (lane 2) were separated by
SDS-gel electrophoresis, transferred to nitrocellulose, and probed with
anti-PMCA4 antibody JA9. Lane 3, 10 µg of MDCK membranes
was separated as above and probed with anti-SAP97 antibody. The
position of a 130-kDa molecular mass standard is indicated on the
left. B, confocal micrographs of MDCK cells grown
on glass coverslips and labeled with polyclonal anti-SAP97 antibodies
(anti-SAP97, red) and monoclonal anti-PMCA antibody 5F10
(anti-PMCA, green). The red and green
channels are shown independently as well as superimposed
(merge) to show the high degree of colocalization
(yellow) of the two proteins. A confocal micrograph of two
cells in the x-z plane is shown at higher magnification in the
far right panel. Note that SAP97 and PMCA are found entirely
in the lateral membrane. Scale bar = 20 µm.
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Fig. 5.
Coexpression and partial colocalization of
PMCA2b and SAP90 in hippocampal neurons. A, 15 µg of
total protein from primary hippocampal cultures (lane 1) or
3 µg of membrane protein from COS-1 cells overexpressing hPMCA2b as
control (lane 2) was separated by polyacrylamide gel
electrophoresis and immunoblotted with affinity-purified anti-PMCA2
antibody NR2. Expression of PMCA2b can be detected in the hippocampal
neurons, whereas no signal was seen when the blot was probed with
anti-PMCA4 antibody JA9 or an antibody specific for the "a" splice
form of PMCA2 (not shown). B, confocal micrograph of
cultured hippocampal neurons immunostained for PMCA2 (red)
and SAP90 (green). Antibodies against SAP90 label the
postsynapse specifically, whereas PMCA2 labeling is seen throughout
dendrites as well as outlining the cell body. Scale bar = 20 µm. C, higher magnification view of a dendritic
section demonstrating that at certain synapses, PMCA2 labeling extends
throughout the entire dendritic spine including the neck and head
(spines 1 and 2), whereas in others, PMCA2
labeling is found only out to the neck but excluded from the head
(spine 3). Colocalization (yellow staining in the
merged picture) of PMCA2 with SAP90 at the postsynaptic density in some
but not all spines may reflect different molecular composition and
Ca2+ buffering requirements of the spines.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid
receptor GluR1 subunit (carboxyl-terminal sequence A-T-G-L*) interacted specifically with SAP97 but not with SAP102 or SAP90 in vivo
(39). The SAPs differ with respect to membrane attachment,
multimerization, and intracellular distribution (18, 21, 40-42).
Sequences outside the PDZ domains of these proteins mediate their
unique properties, e.g. via differential
palmitoylation, cysteine-mediated dimerization, and association with
4.1-type cytoskeletal proteins. Thus, selective interactions between
different PMCAs and SAPs might reflect the specific needs of differing
cell types to influence PMCA targeting, localization, or regulation differentially.
-aminobutyric acid transporter in the basolateral membrane (51).
Recent studies have shown that SAP97 and other MAGUKs are associated
with the cortical actin cytoskeleton (15, 46, 52-54). It is attractive
to imagine that MAGUK binding to the PMCAs, e.g. that of
SAP97 to PMCA4b in kidney epithelial cells, may serve to tether the
Ca2+ pump to the membrane cytoskeletal network and thereby
help retain the PMCA in the proper membrane domain. Indeed, very recent
work implicates a PDZ domain interaction in the association of PMCA4b with the membrane cytoskeleton in platelets (55). In these cells, the
cytoskeletal attachment of the pump could be disrupted by the addition
of a competitor peptide corresponding to the PMCA4b carboxyl terminus,
presumably by disrupting a PDZ domain-PMCA4b interaction.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Aida Filoteo and John Penniston (Mayo Clinic, Rochester, MN) for antibodies against the PMCAs, to David Bredt (University of California, San Francisco, CA) for antibodies against SAP93/chapsyn-110, to Craig Garner (University of Alabama, Birmingham) for antibodies against SAP97 and SAP102 as well as for the SAP102 expression plasmid, and to Morgan Sheng (Harvard Medical School) for the SAP93/chapsyn-110 expression construct. We thank Billie Jo Brown for constructing the full-length PMCA2b expression plasmid, Noah Gray for primary hippocampal neuron preparations, Anna Pinchak for help with coimmunoprecipitations, and Michael Chicka for assistance with cell cultures and immunofluorescence staining.
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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.
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 26, 2001, DOI 10.1074/jbc.M101448200
2 S. Kip and E. E. Strehler, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: [Ca2+]i, intracellular free calcium concentration; PMCA, plasma membrane Ca2+-ATPase; PSD95, 95-kDa protein of the postsynaptic density; PDZ, PSD95/Dlg/ZO-1; MAGUK, membrane-associated guanylate kinase; SAP, synapse-associated protein; GST, glutathione S-transferase; MDCK, Madin-Darby canine kidney; D-PBS, Dulbecco's phosphate-buffered saline.
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