From the Institute of Physiology,
§ Veterinary Biochemistry, and ¶ Anatomy, University of
Zürich-Irchel, CH-8057 Zürich, Switzerland
Received for publication, September 25, 2000, and in revised form, November 30, 2000
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ABSTRACT |
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The type IIa
Na+-dependent inorganic phosphate
(Na/Pi) cotransporter is localized in the apical membrane
of proximal tubular cells and is regulated by an endocytotic pathway.
Because molecular processes such as apical sorting, internalization, or
subsequent degradation might be assisted by associated proteins, a
yeast two-hybrid screen against the C-terminal, cytosolic tail of type IIa cotransporter was designed. Most of the potential proteins found
belonged to proteins with multiple PDZ modules and were either
identical/related to PDZK1 or identical to NHERF-1. Yeast trap
truncation assays confined the peptide-protein association to the
C-terminal amino acid residues TRL of type IIa cotransporter and to
single PDZ domains of each identified protein, respectively. The
specificity of these interactions were confirmed in yeast by testing
other apical localized transmembraneous proteins. Moreover, the type
IIa protein was recovered in vitro by glutathione
S-transferase-fused PDZ proteins from isolated renal brush
border membranes or from type IIa-expressing oocytes. Further, these
PDZ proteins are immunohistochemically detected either in the
microvilli or in the subapical compartment of proximal tubular cells.
Our results suggest that the type IIa Na/Pi cotransporter
interacts with various PDZ proteins that might be responsible for the
apical sorting, parathyroid hormone controlled endocytosis or the
lysosomal sorting of internalized type IIa cotransporter.
In kidney, reabsorption of filtered inorganic phosphate
(Pi) takes place along the proximal tubules and is
controlled by a variety of hormones (e.g. parathyroid
hormone, PTH)1 and other
factors (e.g. dietary intake of Pi) (1, 2).
Three structurally unrelated sodium-dependent phosphate
(Na/Pi) cotransporter families have been identified (1, 3).
By immunohistochemistry, it was apparent that members of the type I and
the type IIa Na/Pi cotransporters are located in the apical
membrane of proximal tubular cells (4, 5). Targeted inactivation of the
type IIa Na/Pi cotransporter gene (npt2)
provided strong evidence that ~70% of Na-dependent
Pi transport across the brush border membrane is mediated
by the type IIa Na/Pi cotransporter (6). Furthermore, the
type IIa cotransporter represents the major target for the many factors
described to regulate proximal tubular Pi reabsorption (2).
Additionally, reduced proximal Pi-reabsorption, as observed in X-linked hypophosphatemia, is due to a decreased abundance of the
type IIa Na/Pi cotransporter (7).
According to the current mechanistic view, inhibition of proximal
tubular Pi-reabsorption, such as by PTH or by a diet of high
Pi content (acutely given), is achieved by a removal of
type IIa cotransporters (2) from the apical membrane. Results obtained from in vivo (rats) and in vitro (OK cells)
studies indicated that internalized type IIa Na/Pi
cotransporters are subjected to degradation in the lysosomes (8, 9).
Besides Na/Pi cotransport, the activity of the brush border
Na/H exchanger, NHE-3, is regulated by PTH as well; however,
internalization of NHE-3 seems to occur after a delay and not
immediately after binding of PTH to its receptor (10). This kinetic
difference of PTH action on the type IIa protein and on the NHE-3
exchanger, respectively, points to a regulatory mechanism specific for
the type IIa Na/Pi cotransporter. Not much is known about
the molecular reactions that underlie the internalization of the type
IIa cotransporter. Although protein kinases are activated upon the
binding of PTH to its receptor (2), the target(s) for activated kinases
relevant for the internalization of the type IIa protein has(have) not
yet been identified.
The microvillar localization of the type IIa protein and its
physiologically controlled abundance in the apical membrane suggest specific interactions of the type IIa protein with other
microvillar/subapical proteins. Such interactions may be necessary for
the correct apical positioning or may be involved in the signaling
pathway that leads to internalization. To identify such candidate
proteins, a yeast two-hybrid screen was performed. As a bait, we used
the C-terminal 75 amino acid residues (563) of the type IIa
Na/Pi cotransporter for the following reasons:
(a) based on the current model of the secondary structure,
the C terminus is located at the cytoplasmic surface (11);
(b) by truncation studies, evidence was obtained that the C
terminus is important for apical expression of the type IIa
protein.2
We found that the C terminus of the type IIa Na/Pi
cotransporter is a strong interactor to various PDZ proteins. Some of
these PDZ proteins were localized in the brush border or in the
subapical compartment of proximal tubular cells and thus may be of
importance for the polar distribution and/or regulation of the type IIa
Na/Pi cotransporter.
Bait DNA Constructs--
EcoRI and SalI
restriction sites were introduced by PCR into cDNA fragments
encoding the N- or C-terminal tails of the following proteins: mouse
type IIa Na/Pi cotransporter (aa 1-109 and 563-637, accession no. AAC52361), mouse type I Na/Pi cotransporter (aa 1-23 and 443-465, accession no. CAA54459), rat Na/H exchanger
NHE-3 (aa 1-53 and 447-831, accession no. AAA41702), mouse
glycoprotein-associated amino acid transporter bo,+AT (aa
1-34 and 451-487, accession no. AJ249198), rat
sulfate/oxalate/bicarbonate anion exchanger Sat-1 (aa 1-73 and
628-703, accession no. AAA17545), rabbit sodium/glucose cotransporter
SGLT-1 (aa 1-31 and 545-662, accession no. CAA29727), rat
sodium/sulfate cotransporter NaSi-1 (aa 1-17 and 569-595, accession
no. AAA41677), mouse peptide transporter Pept-2 (aa 1-51 and 705-740,
accession no. AAF42470), and rat calcium receptor CaR (aa 861-1079,
accession no. AAC52149).
To generate LexA DNA binding domain (DNA-BD) fusions, the fragments
were inserted in-frame into the vector pBTM116 carrying the TRP1
selection marker (12). All cDNA fragments encoding for N termini
were subcloned into the vector pFBL23 (13), where the inserts had
reverted orientation relative to LexA. Site-directed mutagenesis
(QuickChange, Stratagene) was applied to generate truncations (
All bait constructs were verified by sequencing with lower and upper
primers annealing to either the LexA DNA-BD (pBTM116: lexA_upper = 5'-CCAATTGTCGTTGACTTCGTC-3'; pFBL23: lex-N_lower = 5'-CGCGTCGGCGGCATACCTG) or to the ADH promoter (pBTM116: ADH_lower = 5'-GCATGCCGGTAGAGGTGT-3'; pFBL23: ADH-Pro-LV_upper = 5'-TCGTCATTGTTCTCGTTCCC) from the plasmids.
Prey DNA Constructs--
Deletion constructs in pACT2 used for
PDZ interaction trap assays in yeast were produced by PCR. cDNA
fragments encoding single or multiple PDZ domains (listed below) were
obtained by either site-directed mutagenesis (Stratagene) inserting
stop codons downstream of PDZ domain 1, 2, and 3, or by standard
amplification, using primers harboring NcoI or
XhoI restriction sites: mouse NaPi-Cap1 (accession no.
AF220100, aa 1-110 for PDZ1, aa 108-240 for PDZ2, aa 217-359 for
PDZ3, aa 348-472 for PDZ4, aa 1-229 for PDZ1-2, aa 1-350 for
PDZ1-3); mouse NaPi-Cap2 (accession no. AF334612, aa 1-145 for
PDZ1, aa 125-270 for PDZ2, aa 243-377 for PDZ3, aa 370-498 for PDZ4,
aa 1-254 for PDZ1-2 and aa 1-373 for PDZ1-3); mouse NHERF-1
(accession no. U74079, aa 1-129 for PDZ1). cDNA fragments,
corresponding to the cytosolic N (aa 1-107) or C terminus (aa
563-637) of type IIa mouse Na/Pi cotransporter, were also PCR-amplified by means of adapter oligonucleotides carrying
EcoRI as well as XhoI restriction sites and a
stop codon in the N-terminal prey for proper termination.
All fragments were ligated to the C terminus of the GAL4 activation
domain in vector pACT2, which delivers the LEU2 nutritional gene for complementation. All constructs were confirmed by sequencing using an upper primer GAL855 (5'-TGTTTAATACCACTACAATG-3').
Yeast Transformation and Growth Selection--
Bait vectors were
transformed into Saccharomyces cerevisiae (strain L40)
containing the genotype MATa trp1 leu2 his3
LYS2::lexA-HIS3 URA3::LexA-LacZ (15). Yeast
cells were grown in YPDA medium (1% yeast extract, 2% Difco peptone,
2% glucose, 0.003% adenine hemisulfate) or the synthetic minimal Trp
dropout medium (SD-Trp) complemented with 0.003% adenine hemisulfate
(16). For transformation, a YPDA culture from L40 (20 ml) grown to an
A600 of ~1 (18 h) was centrifuged at 1200 × g for 3 min, washed with 40 ml of distilled H2O, and resuspended in 1 ml of distilled H2O.
Aliquots of 50 µl were treated with 240 µl of polyethylene glycol
(50% w/v), 36 µl of 1 M LiAc, 25 µl of single-stranded
DNA (2.0 mg/ml), 50 µl of distilled H2O, and 1 µg of
plasmid, according to Gietz and Schiestl (17). Proper expression of the
baits was confirmed by Western blotting of total cell lysates (18) with
antibodies raised against the C- and N-terminal part of type IIa
Na/Pi cotransporter (5) or against LexA. In control
experiments (data not shown), an autonomous activation of the L40
reporter genes by the baits was verified.
Library Screening--
A cDNA library of whole adult mouse
kidneys (MATCHMAKER, CLONTECH) was used. A 100-ml
culture in SD-Trp medium was set up with four single colonies, grown to
A546 > 2 (~ 36 h) at 30 °C (260 rpm)
and used to inoculate 1 liter of YPDA to 0.2 A600. The culture was propagated to ~0.8
A600, splitted up in quarters and washed with a total of
400 ml of sterile distilled H2O after centrifugation at
4200 × g (4 °C) for 15 min. The pellets were
resuspended in a total of 160 ml of distilled H2O,
transferred to 50-ml tubes, and repelleted at 1300 × g
for 10 min at 4 °C. Each pellet was transfected separately according
to Gietz and Schiestl (17), using a reaction mixture with a scaling-up
of 50 times from above and with 50 µl of cDNA (1 µg/µl).
After an incubation at 30 °C followed by a heat shock at 42 °C
(30 min at 220 rpm), cells were harvested at 1900 × g
for 3 min, washed with 80 ml of distilled H2O, recombined
in 20 ml of distilled H2O and plated onto 100 14-cm Petri
dishes. After 5 days, 4.4 × 106 Trp/Leu/His
prototrophies were tested for LacZ expression by a
colony-lift assay (19, 20), where the permeabilized cells transferred
onto Whatman filters were overlaid with 0.2 mg/ml 5-bromo-4-chloro-3-indolyl Liquid GST Fusion Constructs and Protein Expression--
The
following GST fusion constructs were made in vector pGEX-6P-2
(Amersham Pharmacia Biotech) using EcoRI/XhoI or
BamHI/XhoI restriction sites: mouse NaPi-Cap1 (aa
1-519), mouse NaPi-Cap2 (aa 1-498), mouse NHERF-1 (aa 1-355), mouse
C2PA (aa 195-375 of C2PA plus 17 N-terminal and 19 C-terminal
nonannealing aa), mouse zetin 1 (262 aa), mouse FHL-2 (aa 6-279) and
mouse 54TMp (aa 1-281). Mouse NHERF-2 was digested with
EcoRI/XhoI and the fragment corresponding to aa
1-289 directly cloned in pGEX-6P-1. Since the full-length mouse
MAST205 (1734 aa) was not expressed in Escherichia coli, a
known interacting fragment enclosed by SmaI/XhoI
restriction sites (aa 905-1830) was subcloned in pGEX-6P-1. Finally,
the C-terminal SmaI/XhoI fragment of mouse HSP84
(aa 394-724) was also in pGEX-6P-1. All constructs were checked by
sequencing using primers Gex5' (5'-CCAGCAAGTATATAGCATGG-3') and
Gex3'rev (5'-GCTTACAGACAAGCTGTGAC).
Plasmids were transformed in XL1-Blue cells (Stratagene). 50-ml
overnight cultures were diluted 1:10 in 500 ml of LB/Amp and grown to
0.8 A600 at 37 °C. Protein expression was
induced by adding 0.2 mM
isopropyl-1-thio-
Lysates were thawed at 37 °C and spun for 3 min as above to remove
insoluble debris. Equal amounts of GST fusion proteins (~2 µg) were
incubated with 25 µl of preequilibrated glutathione-agarose beads
(Sigma; 50% slurry) in a total volume of 500 µl of binding buffer
(50 mM Tris-HCl, pH 8, 120 mM NaCl, 0.5%
Igepal CA-630, 5 mM dithiothreitol) by rocking at 4 °C
for 30 min. After absorption, beads were collected by brief
centrifugation at 12,000 rpm for 10 s (4 °C) and gently washed
three times with 500 µl of binding buffer containing 0.075% SDS.
Pull-down Experiments--
Pull-down experiments were performed
either with isolated proximal tubular brush border membranes (BBMV)
from mice or with Xenopus laevis oocytes injected
with type IIa cRNA. Purified BBMVs (27) were solubilized in binding
buffer (see above) for 5 min at 4 °C and centrifuged at 16,000 × g for 3 min. Extracts of oocytes injected with IIa cRNA
(for details, see Ref. 28) were obtained as described (29). GST fusion
protein-loaded beads were incubated for 1 h at 4 °C with
solubilizates corresponding to 0.05 mg of BBMV protein or 1-2 oocytes,
washed three times with binding buffer and used for
gel-electrophoresis. Western blotting was performed with a polyclonal
anti-IIa cotransporter antibody directed against the N terminus (5),
and immunoreactive bands were detected by ECL using a secondary
HRP-coupled IgG (Amersham Pharmacia Biotech).
Northern Blotting--
Tissue distribution of mRNA
expression of identified gene products was studied by Northern blotting
using poly(A)+ RNAs of adult mice either purchased from
CLONTECH or isolated by standard procedures.
Full-length inserts were randomly labeled with [32P]dCTP
(oligolabeling kit; Amersham Pharmacia Biotech) and used as probes.
After hybridization, all blots were washed sequentially with 2× SSC,
1× SSC, and 0.5× SSC (containing 0.1% SDS) at temperatures up to
55 °C.
Immunofluorescence and Immunogold Electron
microscopy--
Immunohistochemical distribution of NaPi-Cap1 and
NaPi-Cap2 in mouse kidney was essentially performed as described (30). Polyclonal antisera were raised against synthetic peptides derived from
the N termini of mouse NaPi-Cap1 and mouse NaPi-Cap2, respectively. Identification of Type IIa Na/Pi
Cotransporter-associated Proteins--
An adult mouse kidney cDNA
library was screened in yeast against the C-terminal tail (aa 563-637)
of the murine type IIa Na/Pi cotransporter. Initially,
based on selection for growth on synthetic media and on LacZ
expression, 138 positive colonies were obtained from 4.4 × 106 Leu/Trp/His prototrophs. To confirm the initial
positives, plasmids from 104 clones were isolated and used for
retransformation of L40 cells harboring the following bait constructs:
(a) the C terminus or (b) the N terminus of the
type IIa Na/Pi cotransporter; and, as controls,
(c) a subunit of the reverse transcriptase from human immunodeficiency virus (1RTp51) or (d) the empty vector
pBTM116. 93 prey plasmids reappeared blue when cotransformed with the
original C-terminal bait, but did not show any interactions with the
other baits used; notably no interaction occurred with the N terminus of the IIa Na/Pi cotransporter which has been predicted to
be located intracellularly as well (11). Inserts (on average around 2 kilobase pairs) from all clones were partially sequenced, grouped according to their alignments, and compared with GenBankTM entries (Table I).
As listed, most of the clones (71 out of 93) coded for PDZ proteins.
The majority of them were identical or closely related to the formerly
identified rat protein diphor-1 or its human homologue PDZK1 (32-34)
and thereafter were referred as mouse NaPi-Cap1 and mouse NaPi-Cap2,
respectively. Other PDZ proteins identified were identical/similar to
NHERF-1 (35, 36), NHERF-2 (37), and a protein named C2PA composed of a
C2 as well as a PDZ domain (accession no. AJ250999). Besides, several
clones were identical to the heat shock proteins HSP84 and HSP86 (38)
or to MAST205, a microtubulin-associated serine/threonine kinase (31).
Others were found only once, such as the mouse protein FHL-2 having
four and half LIM domains (39) or as the mouse homologues of rat zetin
1 (accession no. AF245225) and of the human putative transmembrane
protein 54TMp (accession no. AF004876). Differences in the
representation of these clones could be explained by either the low
abundance of corresponding mRNAs or by the presence of diverse
truncations of inserts existing in the library.
Specific Binding of Putative Type IIa-associated Proteins in
Yeast--
To evaluate the specificity of the identified clones for
binding the C terminus of the type IIa cotransporter, various baits derived from a number of proximal tubular, apically localized membrane
proteins were constructed and tested in yeast for possible interactions
with some of the proteins obtained by the screen (Table
II). Most of the baits used did not
activate the reporter genes in the presence of the different proteins
exhibiting binding capacity for the type IIa cotransporter. Exceptional
was the C terminus of the type I Na/Pi cotransporter (40),
which was found to associate with all the PDZ proteins listed as well
as weakly with MAST205. Furthermore, the C-terminal tail of the Na/H
exchanger, NHE-3, bound NHERF-1, as shown previously (35, 41, 42), and
additionally NaPi-Cap1, but not NaPi-Cap2.
GST Precipitations for in Vitro Corroboration of
Interactions--
Some of the interactions found by the yeast
two-hybrid assay were verified by pull-down experiments using GST
fusion constructs and either solubilized mouse kidney BBMV (Fig.
1A) or lysates of X. laevis oocytes expressing the mouse type IIa Na/Pi
cotransporter (Fig. 1B). From solubilized BBMV, the mature
type IIa protein (mass ~ 85 kDa; see Ref. 5) was precipitated by
the following GST fusion constructs (Fig. 1A):
GST-NaPi-Cap1, GST fused to the PDZ domain 3 of NaPi-Cap1 (see below),
GST-NaPi-Cap2, GST-NHERF-1, GST-NHERF-2, and GST fused to the protein
similar to NHERF-2. The type IIa cotransporter was neither pulled down
by GST alone nor by GST fused to C2PA, zetin 1, MAST205 (Fig. 1) and to
FHL-2, HSP84, or 54TMp (data not shown). Additionally, from lysates of X. laevis oocytes, the type IIa cotransporter was
pulled down by GST-MAST205 and to a weak extent by GST-C2PA. Again,
GST-zetin 1 was unable to recover the cotransporter (Fig.
1B). As NaPi-Cap1 is localized in the microvilli (see
below), GST fused to the C terminus of the type IIa cotransporter was
used to precipitate NaPi-Cap1 from solubilized BBMVs. However, no
precipitation of NaPi-Cap1 was achieved (data not shown). This could be
explained by the fact that NaPi-Cap1 contains four PDZ domains, which
may be linked to cytoskeletal or membrane proteins (43, 44) and thereby
may prevent the pull-down of NaPi-Cap1.
Tissue Distribution and Immunolocalization of Mouse NaPi-Cap1 and
NaPi-Cap2--
In our further studies, we concentrated on the two
proteins NaPi-Cap1 and NaPi-Cap2, since full-length sequences were
obtained for both proteins and other proteins, such as the NHERFs,
HSPs, and MAST205, have been partially characterized previously (31, 35, 36, 38, 45, 46). Besides, by immunofluorescence, the heat shock
proteins and MAST205 were found in all cells along the nephron and were
present in the cytoplasm (data not shown). Therefore, HSPs and MAST205
were assumed not to be implicated in brush border localization and/or
regulation of the type IIa Na/Pi cotransporter.
As illustrated in Fig. 2, NaPi-Cap1
mRNA (around 2.5 kilobase pairs) was detected in liver, kidney,
small intestine (two transcripts) and testis (one transcript), but was
not detected in other organs, such as heart, brain, and lung. NaPi-Cap2
mRNA was expressed only in kidney and intestine and was absent in
all other organs looked for. The renal localization of NaPi-Cap1 and
NaPi-Cap2 was assessed by immunohistochemistry using polyclonal sera
raised against synthetic peptides derived from the corresponding N
termini. Specificity of these antisera was tested on immunoblots using
the GST fusion constructs (results not shown). In mouse kidney
sections, both NaPi-Cap1 and NaPi-Cap2 were detected exclusively in
proximal tubules. Immunostaining was absent in other nephron segments
or in glomeruli and was not observed in blood vessels or in
interstitial cells (Fig. 3A
and data not shown). Immunostaining was uniform along the entire
proximal tubules (S1, S2, and S3 segments) and was indistinguishable
between the cortical and juxtamedullary nephrons. Proximal tubular
location of NaPi-Cap1 was also confirmed by in situ
hybridization (results not shown).
In proximal tubular cells, both NaPi-Cap1 and NaPi-Cap2 were
immunodetected at the apical side. Whereas NaPi-Cap1 was strictly associated with the microvilli, NaPi-Cap2 was predominantly located in
the subapical compartment, but was not detected in the microvilli. However, faint immunostaining for NaPi-Cap2 was also found throughout the cytoplasm (Fig. 3A). By immunogold electron microscopy,
evidence was obtained that NaPi-Cap2 was associated with vesicular
structures within the subapical compartment. Again, microvillar
localization was observed only for NaPi-Cap1 (Fig. 3B).
Yeast Trap Truncation Assays to Specify the Interaction of Type IIa
Cotransporter with Mouse NaPi-Cap1, NaPi-Cap2, and NHERF-1--
Both
NaPi-Cap1 and NaPi-Cap2 encompass four PDZ domains. To analyze which of
the four PDZ domains of NaPi-Cap1 or NaPi-Cap2, respectively, was
responsible for the interaction with the C terminus of the type IIa
Na/Pi cotransporter, each of the four PDZ domains was
tested separately by trap two-hybrid assays. As depicted in Fig.
4A, solely PDZ domain 3 of
NaPi-Cap1 and NaPi-Cap2 increased significantly the activity of
The last three C-terminal amino acid residues of the type IIa
cotransporter sequence are TRL and thus may represent a PDZ-binding cassette (41). To refine if this putative PDZ binding determinant was
responsible for the interaction with identified PDZ proteins, the last
three amino acid residues (TRL) of the type IIa cotransporter were
deleted and the respective bait was assayed in yeast in the presence of
either NaPi-Cap1, NaPi-Cap2 or additionally NHERF-1 and C2PA. As
illustrated in Fig. 5A, the
removal of the three C-terminal amino acid residues, TRL, completely
abolished the interaction of the C-terminal tail of the IIa
Na/Pi cotransporter with NaPi-Cap1, NaPi-Cap2, NHERF-1, and
C2PA. Similarly, the last three amino acid residues of the type I
Na/Pi cotransporter are represented by TRL and their
deletion also blunted the interaction with the PDZ proteins mentioned
above (data not shown). On the other hand, the C terminus, as a ligand
for the heat shock proteins HSP84 or HSP86, did not depend on the last
three amino acid residues because complex formation was disrupted not
before 46 amino acid residues were removed (Fig. 5B).
Proximal tubular apical location of the type IIa Na/Pi
cotransporter and its mode of regulation (endocytosis, lysosomal
sorting in the subapical region) (2) suggest a complex interaction pattern of the type IIa Na/Pi cotransporter with
microvillar proteins as well as with proteins localized in the
subapical compartment. In microvilli, such interacting proteins may
play a role in scaffolding and also may anchor components involved in
the signaling pathways. In the subapical compartment, type IIa
cotransporter-associated proteins can be envisaged to play not only a
role in apical sorting of newly synthesized cotransporters, but also to
be important for the initial steps leading to the routing of
internalized cotransporters to the lysosomes. A priori,
every domain of the type IIa cotransporter oriented toward the
cytoplasm could represent a possible site for such interactions.
Predictions of the secondary structure and studies on the topology of
the type IIa cotransporter suggested 8-10 transmembrane segments and 5 intracellular regions including the N and the C termini (11). In this
study, we have used the whole C terminus of the type IIa cotransporter
as a bait to screen a cDNA library of adult mouse kidneys by the
yeast two-hybrid strategy.
Most of the proteins identified (71 out of 93) represented PDZ proteins
(see Table I). Two similar proteins with four modular PDZ repeats were
identified: NaPi-Cap1, being identical to the previously described
proteins diphor-1 and PDZK1 (3, 32, 33), and NaPi-Cap2, being 28%
identical to NaPi-Cap1. Other PDZ proteins found belonged either to the
NHERF family (NHERF-1, NHERF-2, and a protein similar to NHERF-2)
(35-37) or were related to the C2PA protein, which harbors a PDZ
domain and a C2 domain in addition. Further, two large groups of
proteins comprised the heat shock proteins HSP84 and HSP86 (38, 45) and
the microtubulin-associated serine/threonine kinase MAST205 (31).
Interaction of above proteins with the C terminus of the type IIa
cotransporter was specific (with one exception) since both the N
terminus of the type IIa cotransporter and the C or N termini of
various other proximal tubular, apically localized membrane proteins
failed to stimulate LacZ expression in yeast. Interestingly,
all identified PDZ proteins bound also to the C terminus of the type I
Na/Pi cotransporter, which has been described to be
localized in the apical membrane as well (4). However, if such an
interaction with the type I cotransporter reflects any physiological
significance, it remains to be shown if the C terminus of the type I
Na/Pi cotransporter is faced toward the cytoplasm and not
to the extracellular space.
Additional evidence for an interaction of the type IIa cotransporter
with identified proteins was obtained from pull-down experiments,
demonstrating that at least all PDZ proteins (NaPi-Cap1/2, C2PA, and
NHERF isoforms) were (in vitro) binding partners for the
mature type IIa Na/Pi cotransporter as present in isolated proximal tubular brush border membranes or/and in oocytes of X. laevis injected with type IIa cRNA.
A first mandatory step to establish the physiological relevance of
positive interactions in yeast was to determine the location of the
putative candidate proteins in renal tissue, i.e. such proteins were required to be expressed in microvilli and/or subapical compartment of proximal tubular cells. The proteins NaPi-Cap1 and
NaPi-Cap2 showed strict and NHERF-1 mostly proximal tubular location.
NaPi-Cap1 (see also Ref. 44) and NHERF-1 (47) were found exclusively in
microvilli, whereas NaPi-Cap2 was distributed predominantly in the
subapical compartment of proximal cells. Interestingly, as revealed by
immunogold electron microscopy, NaPi-Cap2 appeared to be associated
with vesicular structures in the subapical compartment. At present, the
nature of such vesicles is not known and vesicular proteins eventually
linked to NaPi-Cap2 remain to be determined.
Although the type IIa cotransporter was precipitated by both NHERF-2
and its likely isoform, a conclusion about this kind of association
would be premature since the renal localization of NHERF-1 isoforms
have not yet been verified by morphological techniques. With respect to
C2PA, preliminary immunohistochemical studies indicated that this
protein is barely localized in the proximal tubules (data not shown).
Furthermore, immunofluorescence data disclosed that the heat shock
proteins HSP84/86 and the microtubulin-associated serine/threonine
kinase (MAST205) were localized along the whole nephron and were
neither specifically localized in the brush border nor in the subapical
region (data not shown). Therefore, these proteins are probably not
implicated in either apical sorting or endocytosis of the type IIa
cotransporter. It can, however, not be excluded if HSP84/86 or MAST205
plays a role in protein synthesis or lysosomal routing of the type IIa
cotransporter; the latter has been shown previously to be dependent on
an intact microtubular network (48). Further studies on other proteins which have emerged from the two hybrid approach were impossible, due to
the lack of suitable antibodies. Based on the criteria discussed above
(positive yeast assay, pull-down and immunolocalization), NaPi-Cap1/2
and NHERF-1 were supposed to associate with the type IIa cotransporter.
But it remains to be shown if all these interactions also occur under
in vivo conditions.
PDZ domains are stretches of 80-90 amino acids and have been assigned
to an increasing number of proteins having physical organizer functions
in the formation of protein complexes of various sizes. Such protein
complexes may allow a proper arrangement of components involved in
signal transduction pathways and/or may allow a correct recruitment of
membrane-inserted proteins, as has been shown e.g. for
synaptosomal membrane transporters and channels (41, 42, 49). In
addition, evidence for PDZ interactions in the localization of proteins
to the apical membrane has been accumulated (50, 51). The proteins
NaPi-Cap1, NaPi-Cap2, and NHERF-1, on which our studies concentrated,
contain two or four PDZ domains. However, their interactions with the C
terminus of the type IIa cotransporter were confined to only one of
these domains, i.e. PDZ3 of NaPi-Cap1/2 and PDZ1 of NHERF-1.
As shown by others, NaPi-Cap1 also interacts with a small
transmembrane, cancer-associated protein, MAP17 (43), and with the
multidrug resistance-associated protein, MRP2 (44). The latter two
proteins are supposed to interact with PDZ domains 1 and/or 4 of
NaPi-Cap1. Presumably, other PDZ domains of NaPi-Cap1 and NaPi-Cap2 may
be occupied by cytoskeletal proteins or by members of the ERM family of
actin-binding proteins (52). As for NHERF-1, it was initially identified as a protein which binds to the Na/H exchanger NHE-3 (35,
36), but interaction of NHERF-1 with other proteins, such as the
Often, PDZ folds recognize a C-terminal amino acid motif of the
D(S/T)XV type; however, many variations thereof appeared to anchor PDZ domains as well (41, 42, 49). The last three C-terminal
amino acid residues of the type IIa cotransporter, TRL, resemble a PDZ
binding motif. Indeed, deletion of the amino acid residues TRL is
sufficient to completely abrogate the interaction with the PDZ proteins
NaPi-Cap1, Napi-Cap2, and NHERF-1.
In summary, a yeast two-hybrid screen with the C terminus of the type
IIa Na/Pi cotransporter as a bait revealed several proteins that are likely to be associated with the IIa cotransporter.
Physiological relevance of the identified proteins is met by their
proximal tubular as well as apical localization. The proteins
NaPi-Cap1, NaPi-Cap2, and NHERF-1 are of particular interest and may
mediate apical positioning and/or endocytosis of the type IIa
Na/Pi cotransporter. These multidomain PDZ proteins are
presumably cosequestered by type IIa cotransporters at the membrane and
are therefore assumed to build up a complex network consisting of the
type IIa Na/Pi cotransporter, other membrane proteins,
cytoskeletal proteins, or components of signaling pathways (33, 43). It
seems of interest that NHERF-1 recruits ezrin, which in turn functions as a protein kinase A anchoring site for conferring cAMP-mediated regulation of NHE-3 (41, 46, 55). Thus, NHERF-1 seems to serve as an
organizer for signaling complexes necessary for the regulation of a
variety of proteins, e.g. permitting phosphorylation of
NHE-3 (35). It must be determined if NHERF-1 exerts a similar function
in the regulation of the type II cotransporter.
The observation that the C terminus of the type IIa cotransporter
recognizes PDZ motifs of several proteins (NaPi-Cap1/2 and NHERF-1)
suggests that these proteins may differently affect apical sorting
and/or endocytosis. Thus, in the case of the type IIa Na/Pi cotransporter, such interactions may not be of static
nature but rather may be dynamically regulated by the activation status of specific signaling pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3;
46;
56 and
67) of the C terminus from the type IIa
Na/Pi cotransporter by introducing stop codons at the respective positions. As a control bait, the subunit of the reverse transcriptase from human immunodeficiency virus (1RTp51) in pBTM116 was
used (generously provided by Prof. U. Hübscher; see Ref. 14).
-D-galactopyranoside
(Alexis), 150 mM NaCl, 50 mM Tris-HCl (pH 7.4),
and 0.8% agarose. Putative positive colonies were restreaked on
selective medium and rescreened by colony-lift assay. Yeast DNA of true
positives was isolated (21), and prey plasmids were rescued by
transformation into KC8 cells, which carries trpC,
leuB, and hisB mutations (22). Protein-protein interactions of purified prey plasmids were reconfirmed in yeast. Insert sizes were checked by BglII digestion and subjected
to dideoxy sequencing (Microsynth, Switzerland). Identical sequences were grouped via ClustalW at Pôle Bio-Informatique (Lyonnais) or
via Pileup from Genetics Computer Group (Oxford) and overlaps connected
(Contig assembly program; Baylor College of Medicine). Searches for
protein relationships were performed at the National Center for
Biotechnology Information at the National Institutes of Health
(Bethesda, MD) using BLAST (23). The modular architecture of proteins
was determined by SMART (24).
-Galactosidase Assays--
Interactions of single
proteins and constructs thereof were determined by a liquid assay for
-galactosidase from permeabilized cells in the presence of
o-nitrophenyl-
-D-galactopyranoside (ONPG, Sigma) (25, 26). The activity of
-galactosidase
(A420) was normalized to 1.0 × 107 cells assayed (1 A600 = 1.0 × 107 cells/ml).
-D-galactoside (Axon Lab), and the
incubation was continued for 5 h at 28 °C. Cells were pelleted at 5000 × g for 10 min at 4 °C, resuspended in 4 ml
of lysis buffer (50 mM Tris-HCl, pH 8, 120 mM
NaCl, 0.5% Igepal (Sigma CA-630), 5 mM dithiothreitol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 2 mg/ml lysozyme), and immersed on ice for 15 min.
The cells were subjected three times to pulsed sonication for 30 s
on ice. Aliquots were cleared at 12,000 rpm in a tabletop microcentrifuge for 15 min (4 °C) and frozen at
80 °C.
-Actin was visualized by phalloidin-Texas Red. Anti MAST205 antibody was kindly provided by P. Walden (31), and an anti-HSP86 antibody was
purchased from Affinity Purified Reagents.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Compilation of all proteins identified by the two-hybrid screen as
putative associaters to the C terminus of the type IIa Na/Pi
cotransporter
Binding specificity of identified proteins toward other apical brush
border proteins of proximal tubular cells
-galactosidase expression: intensity of
color is represented by (++++) for very strong, (+++) for strong, (++)
for weak, (+) for very weak and (
) for none. In addition, the
following C- and N-terminal baits were used: sodium/glucose
cotransporter SGLT-1, peptide transporter Pept-2, calcium receptor CaR,
glycoprotein-associated amino acid transporter bo,+AT, and
sodium/sulfate cotransporter NaSi-1 (for further descriptions of these
proteins, see "Experimental Procedures"). All these baits as well
as the controls (the DNA-BD alone, the viral protein p51, and the
basolaterally expressed sulfate/oxalate/bicarbonate anion exchanger,
Sat-1) did not activate the reporter gene in the presence of the prey
constructs listed.
View larger version (49K):
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Fig. 1.
Type IIa cotransporter is recovered in
vitro by various proteins identified by the two-hybrid
approach. Purified GST alone or GST fusion proteins (see
"Experimental Procedures") immobilized on glutathione-agarose were
incubated in the presence of lysates from murine kidneys BBMVs
(A) or from X. laevis oocytes expressing the
mouse IIa Na/Pi cotransporter (B). After intense
washing, the final samples were denatured in the absence of a reducing
agent, and transferred material was immunoblotted using a polyclonal
antibody raised against the type IIa Na/Pi cotransporter.
The band of ~85 kDa represents the mature Na/Pi
cotransporter (see also Ref. 5). As controls for the stability of the
type IIa protein, aliquots of the lysates were analyzed as well.
View larger version (51K):
[in a new window]
Fig. 2.
Expression pattern of mouse NaPi-Cap1 and
NaPi-Cap2 mRNA. Poly(A)+ RNA from multiple mice
tissues was probed with randomly labeled inserts of NaPi-Cap1
(A) and NaPi-Cap2 (B). The multiple tissue blot
was additionally probed for -actin (C).
View larger version (70K):
[in a new window]
Fig. 3.
Localization of NaPi-Cap1 and NaPi-Cap2 in
mice proximal tubules. A, cryosections were stained
with polyclonal antibodies raised against NaPi-Cap1 and NaPi-Cap2
(upper row) or with phalloidin red to localize
-actin (middle row). Merged pictures are shown
in color. Colocalization of NaPi-Cap1 with microvillar
-actin is indicated in yellow. Bar
size, 50 µm. B, immunogold electron microscopy.
As indicated by arrowheads, NaPi-Cap1 was associated with
microvilli whereas NaPi-Cap2 was detected as attached to vesicular
structures in the subapical compartment. Eventually, NaPi-Cap1
was also present in the intermicrovillar clefts (open
arrowheads).
-galactosidase, in accordance with GST-NaPi-Cap1/PDZ3 by which the
type IIa cotransporter was pulled down (see Fig. 1). The PDZ domain 3 of both NaPi-Caps was also found to be mostly important for the
interaction with the C terminus of the type I Na/Pi
cotransporter (data not shown). Furthermore, our results indicated that
the type IIa cotransporter via its C terminus interacts predominantly
with the first PDZ domain of NHERF-1 (Fig. 4B).
View larger version (14K):
[in a new window]
Fig. 4.
Single PDZ domain interactions of mouse
proteins NaPi-Cap1, NaPi-Cap2, and NHERF-1 with the C terminus of the
mouse type IIa Na/Pi cotransporter in yeast. Yeast
cells containing the C terminus as a bait were transformed with the
indicated prey constructs, and interactions were analyzed by liquid
-galactosidase assays with permeabilized liquid cultures of total
yeast lysates. All prey constructs were also tested for possible
LacZ activation in yeast harboring either the DNA-BD from
the empty vector (pBTM116) or the control bait protein p51, as shown in
B but not in A. All these controls were negative,
indicating specific interactions of the C terminus with the PDZ preys
listed.
View larger version (12K):
[in a new window]
Fig. 5.
Implication of the C-terminal amino acid
motif (TRL) of the type IIa Na/Pi cotransporter for
interaction with identified proteins. Yeast cells, transformed
with the C terminus or with truncated C termini (CT-3, CT-46, or
CT-56), were retransformed with various PDZ proteins (A) or
the heat shock proteins HSP84/86 (B), as depicted.
LacZ activation ( -galactosidase) was determined in total
lysates of yeast. None of the prey constructs activated autonomously
the reporter genes when transformed in yeast containing either the
DNA-BD alone (empty vector) or the viral protein p51 as controls
(results not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic receptor (53), the cystic fibrosis transmembrane conductance regulator (50, 54), and the B1 subunit of
H-ATPase (47), has been reported as well.
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ACKNOWLEDGEMENT |
---|
We gratefully acknowledge the professional assistance of C. Gasser in preparing the figures for this paper. We would like to thank Dr. J. Camonis for his generous donation of the plasmid pFBL23.
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FOOTNOTES |
---|
* This work was supported by Swiss National Science Foundation Grant 31-46523.96 (to H.M) and by a grant from the "Stiftung für wissenschaftliche Forschung," University of Zürich (to J. B.).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: Inst. of
Physiology, University Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. Tel.: 41-1-635-50-32; Fax:
41-1-635-57-15; E-mail: juergbiber@access.unizh.ch.
Published, JBC Papers in Press, November 30, 2000, DOI 10.1074/jbc.M008745200
2 N. Hernando, J. Biber, and H. Murer, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: PTH, parathyroid hormone; Na/Pi, Na+-dependent inorganic phosphate; OK cell, opossum kidney cell; DNA-BD, DNA binding domain; ADH, anti-diuretic hormone; SD-Trp, synthetic minimal tryptophan dropout medium; GST, glutathione S-transferase; BBMV, brush border membrane vesicle; CT, C terminus; MAST, microtubulin-associated serine/threonine kinase; HSP, heat shock protein; aa, amino acid(s); PCR, polymerase chain reaction.
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