Polycystin-1, the PKD1 gene product, is in a complex containing E- cadherin and the catenins

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is a common human genetic disease characterized by cyst formation in kidney tubules and other ductular epithelia. Cells lining the cysts have abnormalities in cell proliferation and cell polarity. The majority of ADPKD cases are caused by mutations in the PKD1 gene, which codes for polycystin-1, a large integral membrane protein of unknown function that is expressed on the plasma membrane of renal tubular epithelial cells in fetal kidneys. Because signaling from cell-cell and cell-matrix adhesion complexes regulates cell proliferation and polarity, we speculated that polycystin-1 might interact with these complexes. We show here that polycystin-1 colocalized with the cell adhesion molecules E-cadherin and alpha-, beta-, and gamma-catenin. Polycystin-1 coprecipitated with these proteins and comigrated with them on sucrose density gradients, but it did not colocalize, coprecipitate, or comigrate with focal adhesion kinase, a component of the focal adhesion. We conclude that polycystin-1 is in a complex containing E-cadherin and alpha-, beta-, and gamma-catenin. These observations raise the question of whether the defects in cell proliferation and cell polarity observed in ADPKD are mediated by E-cadherin or the catenins.

Full text

Introduction
Autosomal dominant polycystic kidney disease (ADPKD)
is one of the most common genetic diseases in humans. It
is a multisystem disease that causes renal cysts and renal
failure as well as cysts in the liver, pancreas, and other
organs, aneurysms of the cerebral and other arteries, car-
diac valvular insufficiency, and colonic diverticula (1).
ADPKD is caused by mutations in at least 3 genes. Two
of these, PKD1 and PKD2, have been cloned and
sequenced (2–5). Families with ADPKD unlinked to
PKD1 or PKD2 have been identified, so at least 1 more
locus must exist (6–9). Both PKD1 and PKD2 code for
novel molecules whose biochemical functions are
unknown. The PKD1 gene product, polycystin-1, is a large
intrinsic membrane protein with a molecular weight of
approximately 485 kDa that is expressed in a develop-
mentally regulated manner. The amino terminal domain
of polycystin-1 is predicted to be extracytoplasmic. The
carboxyl terminal domain of polycystin-1 is predicted to
have between 7 and 11 transmembrane helices. Poly-
cystin-2, the PKD2 gene product, is an intrinsic mem-
brane protein with a molecular weight of 110 kDa. It is
predicted to have intracellular amino and carboxyl ter-
mini and has significant similarities to transient receptor
potential (Trp) channel subunits. The observation that
carboxyl terminal fragments of polycystin-1 and -2 inter-
act in vitro and in transfected cells, coupled with the sim-
ilarity of the disease caused by mutations in either PKD1
or PKD2, suggests that these molecules act together in an
uncharacterized biochemical pathway (10, 11).
The primary structure of polycystin-1 suggests that it
might be a receptor. The large extracellular domain
begins with 2 leucine-rich repeats and has 16 novel
repeats with an Ig-like fold, an LDL-A domain, a calci-
um-dependent lectin domain, and a domain of nearly
1,000 amino acids that is similar to the sea urchin recep-
tor for egg jelly. These domains mediate protein-protein
and protein-carbohydrate interactions in other proteins,
and by analogy, are likely to mediate similar interactions
in polycystin-1. We and others have shown that poly-
cystin-1 is expressed in the plasma membrane of epithe-
lial cells (12–17). This observation is consistent with the
hypothesis that polycystin-1 might interact with an
extracellular ligand or ligands.
We had observed that polycystin-1 was concentrated
on the apical and lateral aspects of epithelial cells in
developing kidneys (12). This localization pattern was
similar to that of the cell adhesion molecule E-cadherin
in developing kidneys, and suggested that polycystin-1
might interact with E-cadherin. This hypothesis was par-
ticularly intriguing because of the roles that E-cadherin
plays in orchestrating cell proliferation and polarization
of epithelial monolayers. E-cadherin modulates prolif-
eration and polarity through associated cytoplasmic
polypeptides, the catenins (18–20). E-cadherin regulates
cell proliferation and gene expression by participating in
the regulation of β-catenin metabolism (21, 22). Cyst-lin-
ing epithelial cells in ADPKD express markers for cell
proliferation in vivo and are abnormally sensitive to
growth factors in vitro, suggesting that there is a
derangement of the control of cell growth in ADPKD
(23). E-cadherin orchestrates cell polarity by directing a
cytoskeletal network containing ankyrin and spectrin to
the basolateral aspect of the epithelial cell (24, 25). Na,K-
ATPase binds to ankyrin and is retained in the basolat-
eral membrane by its interaction with the ankyrin/spec-
trin cytoskeleton (26, 27). When these complexes are
disrupted, for example by ischemia, Na,K-ATPase
The Journal of Clinical Investigation | November 1999 | Volume 104 | Number 10 1459
Polycystin-1, the
PKD1
gene product, is in a complex
containing E-cadherin and the catenins
Yonghong Huan and Janet van Adelsberg
Department of Medicine, Columbia University, New York, New York 10032, USA
Address correspondence to: Janet van Adelsberg, Department of Medicine, Columbia University, 630 West 168th Street, Box 84,
New York, New York 10032, USA. Phone: (212) 305-4476; Fax: (212) 305-3475; E-mail: jsv1@columbia.edu.
Received for publication September 2, 1998, and accepted in revised form October 5, 1999.
Autosomal dominant polycystic kidney disease (ADPKD) is a common human genetic disease characterized
by cyst formation in kidney tubules and other ductular epithelia. Cells lining the cysts have abnormalities in
cell proliferation and cell polarity. The majority of ADPKD cases are caused by mutations in the PKD1 gene,
which codes for polycystin-1, a large integral membrane protein of unknown function that is expressed on
the plasma membrane of renal tubular epithelial cells in fetal kidneys. Because signaling from cell-cell and
cell-matrix adhesion complexes regulates cell proliferation and polarity, we speculated that polycystin-1
might interact with these complexes. We show here that polycystin-1 colocalized with the cell adhesion mol-
ecules E-cadherin and α-, β-, and γ-catenin. Polycystin-1 coprecipitated with these proteins and comigrated
with them on sucrose density gradients, but it did not colocalize, coprecipitate, or comigrate with focal adhe-
sion kinase, a component of the focal adhesion. We conclude that polycystin-1 is in a complex containing
E-cadherin and α-, β-, and γ-catenin. These observations raise the question of whether the defects in cell pro-
liferation and cell polarity observed in ADPKD are mediated by E-cadherin or the catenins.
J. Clin. Invest. 104:1459–1468 (1999).

becomes depolarized and appears in both lateral and api-
cal plasma membranes (28, 29). Some, but not all, inves-
tigators have found that Na,K-ATPase is depolarized in
ADPKD epithelia (30–32). These observations suggest-
ed that polycystin-1 might modulate cell proliferation
and cell polarity by interacting with E-cadherin or the
catenins. We found that polycystin-1 is part of a complex
containing E-cadherin and the catenins, suggesting that
some of the defects observed in ADPKD might be medi-
ated by E-cadherin or the catenins.
Methods
Antibodies. Rabbit polyclonal antibody against polycystin
peptide B145 has been described previously (12). Mouse
monoclonal antibodies against human γ-catenin were
obtained from Zymed Laboratories (South San Francis-
co, California, USA) and from Transduction Laborato-
ries (Lexington, Kentucky, USA). Mouse monoclonal
antibodies against human β1-integrin was obtained from
GIBCO BRL (Gaithersburg, Maryland, USA) and Trans-
duction Laboratories. Monoclonal antibodies against
human E-cadherin, α- and β-catenin, and focal adhesion
kinase (FAK) were obtained from Transduction Labora-
tories. Cy3 and horseradish peroxidase–labeled second-
ary antibodies absorbed against human, rat, mouse, and
rabbit serum proteins were obtained from Jackson
ImmunoResearch Laboratories Inc. (West Grove, Penn-
sylvania, USA). Oregon Green 488–labeled secondary
antibodies were obtained from Molecular Probes Inc.
(Eugene, Oregon, USA).
Immunofluorescence. Indirect immunofluorescence on
frozen sections was performed as described previously
(12). Briefly, the sections were fixed in methanol and ace-
tone (1:1, vol/vol) at –20°C for 10 minutes. The sections
were air dried, rehydrated in PBS, and blocked for 15–30
minutes in 0.2% (wt/vol) BSA in PBS. Primary antibod-
ies were diluted in blocking buffer containing the fol-
lowing: polycystin-1, either 1:10 (affinity purified) or
1:50 (anti-serum); E-cadherin, 1:25; α-catenin, 1:5; β-
catenin, 1:50; γ-catenin (plakoglobin), 1:100; FAK, 1:5;
and β1-integrin, 1:5. Human fetal kidneys of 20–24 weeks
gestational age were obtained under a protocol approved
by the Institutional Review Board of Columbia Univer-
sity. The sections were postfixed in 4% paraformaldehyde
before being mounted for confocal microscopy in Pro-
long Antifade (Molecular Probes Inc.).
Confocal microscopy. Confocal microscopy was per-
formed with a Zeiss LSM 410 laser scanning on a Zeiss
Axiovert 100 microscope platform using an argon-kryp-
ton laser (Carl Zeiss North America, Thornwood, New
York, USA). The 488-nm and 568-nm laser lines were
used for Oregon Green and Cy3 detection, respectively.
Images were collected with a 515–540-nm band pass fil-
ter (green channel) and a 590-nm long pass filter (red
channel). Sections were scanned at low power on the red
channel (polycystin-1 staining) to select tubules strong-
ly stained for polycystin. These tubules were then exam-
ined at high power with both red and green channels.
Red and green images were collected simultaneously and
stored for analysis as both individual red or green images
and as the dual-scanned image. For each image, tissue
sections were viewed at ×100 or ×40. The images (512 ×
512 pixels) were projected at 600 pixels/inch using
Adobe Photoshop 4.0 (Adobe Systems Inc., Mountain
View, California, USA), which was also used to make the
montages shown in Figures 1 and 5.
Cell lines. The human pancreatic adenocarcinoma (HPAC)
cell line was obtained from the American Type Culture Col-
lection (Rockville, Maryland, USA). Cells were grown in a
1:1 mixture of DMEM and Ham’s F12 medium containing
1.2 g/L sodium bicarbonate, 15 mM HEPES, 0.002 mg/mL
insulin, 0.005 mg/mL transferrin, 40 ng/mL hydrocorti-
sone, and 10 ng/mL EGF, with 5% FBS (HyClone Labora-
tories, Logan, Utah, USA). Insulin, transferrin, and EGF
were purchased from Fisher Scientific (Atlanta, Georgia,
USA). DMEM, F12, and HEPES were purchased from
GIBCO BRL. Hydrocortisone was obtained from Sigma
Chemical Co. (St. Louis, Missouri, USA).
RT-PCR. Total RNA was prepared from cultured cells
using RNAzol B (Tel-Test Inc., Friendswood, Texas,
USA). Primers for the 3′end of the PKD1 gene were
selected using the Prime program (Wisconsin Package,
version 8.0) from Genetics Computer Group Inc. (Madi-
son, Wisconsin, USA). The forward primer (5′to 3′)
sequence was GGC TGT TAT TCT CCG CTG
(nucleotides 12,441–12,458, HSU24497). The reverse
primer (5′to 3′) sequence was GGG TGG ACC TTG TTC
TTG (nucleotides 13,034–13,017). RT-PCR was carried
out using the Access RT-PCR System from Promega
Corp. (Madison, Wisconsin, USA), according to the man-
ufacturer’s instructions, except that 10% DMSO was
included in the reaction. Human β-actin primers from 2
exons (Stratagene, La Jolla, California, USA) were used as
a positive control; RNA treated with RNase A (Sigma
Chemical Co.) was used as a negative control. RT-PCR
products were separated on a 3% NuSieve 3:1 agarose gel
(FMC BioProducts, Rockland, Maine, USA) in 1×Tris-
acetate-EDTA buffer, and were purified using QIAEX
resin (QIAGEN Inc., Valencia, California, USA) for direct
sequencing at the DNA facility at Columbia University.
Immunoprecipitation and immunoblotting. HPAC cells
were labeled overnight with [35S]methionine/cysteine
(EXPRESS label; NEN Life Science Products, Inc.,
Boston, Massachusetts, USA) at 0.1 mCi/mL in methio-
nine/cysteine–free medium with dialyzed FBS. Cells were
suspended in lysis buffer containing 1% (vol/vol) Triton
X-100, 0.5% (vol/vol) NP-40, 150 mM NaCl, 10 mM Tris-
HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 0.2 mM
Na3VO4, 1 mM PMSF, 2 µg/mL aprotinin, 1 µg/mL E-64,
10 µg/mL leupeptin, and 1 µg/mL pepstatin A. Cells
were lysed by passage through a 22-gauge needle several
times. Insoluble material was removed by centrifugation
at 10,000 gfor 30 minutes at 4°C. Lysates were pre-
cleared by incubation with preimmune rabbit serum and
protein A–agarose (Oncogene Science Inc., Uniondale,
New York, USA) for 30 minutes at 4°C. After removal of
protein A–agarose by centrifugation, lysates were incu-
bated for 1 hour at 4°C with equivalent concentrations
of protein A–purified IgG from preimmune serum,
immune serum from rabbits immunized with peptide
B145, or immune serum plus peptide B145 at 0.4
mg/mL. For most experiments, antibody to peptide
B145 was purified on a B145 peptide column as
described previously (12). Affinity-purified antibody was
1460 The Journal of Clinical Investigation | November 1999 | Volume 104 | Number 10

used at a concentration of 1–5 µg/mL. After 1 hour, pro-
tein A–agarose was added and the lysates were incubat-
ed for an additional 30 minutes. Immunoprecipitates
were washed 4 times with lysis buffer and then boiled in
SDS-PAGE sample buffer. Cleared cell lysates and
immunoprecipitates were separated on a 4–10% linear
gradient polyacrylamide gel and examined by autoradi-
ography using a PhosphorImager from Molecular
Dynamics (Sunnyvale, California, USA).
For coprecipitation experiments, unlabeled HPAC
cell lysates were prepared as above, except that affini-
ty-purified antibody to peptide
B145 was used for all experi-
ments. Cell lysates and
immunoprecipitates were sepa-
rated on a 4–15% gradient gel,
transferred to nitrocellulose,
and probed with mouse mono-
clonal antibodies against differ-
ent components of cell adhesion
complexes using an HRP-
labeled anti-mouse secondary
antibody (Jackson ImmunoRe-
search Laboratories Inc.) and
enhanced chemiluminescence
(Amersham Pharmacia AB, Upp-
sala, Sweden) to detect antibody
binding to the nitrocellulose membrane as described
previously. Antibodies were used at the following dilu-
tions: E-cadherin, 1:500; α-catenin, 1:500; β-catenin,
1:1,000; γ-catenin (plakoglobin), 1:1,000; and FAK,
1:1,000. The HRP-labeled secondary antibodies were
used at 1:10,000 to 1:50,000, depending on the lot.
For double immunoprecipitation experiments, meta-
bolically labeled HPAC cell lysates were prepared and
cleared as described above, except that both protein A and
recombinant protein G–agarose (Roche Molecular Bio-
chemicals, Mannheim, Germany) were used during the
The Journal of Clinical Investigation | November 1999 | Volume 104 | Number 10 1461
Figure 1
Confocal images of sections of human fetal kid-
ney subjected to double-stain indirect immuno-
fluorescence with antibodies to polycystin-1
(red) and to junctional proteins (green). Each
horizontal series shows, from left to right, the
green channel (junctional protein), the red
channel (polycystin-1), and the total fluores-
cence (dual-scanned image). Colocalization is
seen in the right-hand panels as yellow staining.
(a–c) Double staining for polycystin-1 and E-
cadherin, where (a) is E-cadherin, (b) is poly-
cystin-1, and (c) is the dual-scanned image.
(d–f) Double staining for α-catenin, where (d)
is α-catenin, (e) is polycystin-1, and (f) is the
dual-scanned image. (g–i) Double staining for
β-catenin, where (g) is β-catenin, (h) is poly-
cystin-1, and (i) is the dual-scanned image. (j–l)
Double staining for γ-catenin, where (j) is γ-
catenin, (k) is polycystin-1, and (l) is the dual-
scanned image. In all these images, polycystin-
1 and E-cadherin or the catenins are colocalized
(yellow) at the apical and lateral aspects of the
epithelial cells. There are areas where distinct
green or red staining can be seen, showing that
not all of the polycystin-1 colocalizes with the
junctional proteins. These images were acquired
with the ×100 lens. (m–o) Confocal images of
antibodies to polycystin-1 (red) and β-catenin
(green) incubated in the presence of the peptide
against which the polycystin-1 antibody was
raised. The peptide blocked polycystin-1 stain-
ing (n) but did not affect β-catenin staining (m,
o). Some precipitated anti-rabbit antibody is
seen as red dots (n). These images were
acquired with the ×40 lens.

clearing step. Monoclonal antibodies to E-cadherin or
β-catenin were precipitated with protein G–agarose in the
first immunoprecipitation. The precipitates were washed
3 times with 1 mL lysis buffer and the precipitated pro-
teins were released with 1% SDS at 37°C for 30 minutes.
The eluate was diluted to a final concentration of 0.1%
SDS with lysis buffer, and was reprecipitated with affin-
ity-purified antibody to peptide B145 as described above.
To estimate the molar ratio of polycystin-1 to E-cad-
herin and β-catenin in precipitated complexes, the
results of PhosphorImager analysis (Molecular Dynam-
ics) were normalized to the number of methionines and
cysteines in each molecule. The molar ratio of E-cadherin
to β-catenin obtained by this method was 1:1 (range
0.7–1.1), agreeing with previous results (33, 34).
Sucrose density gradient centrifugation. HPAC cell lysates
prepared as above were layered onto linear 5–20%
(wt/vol) sucrose gradients in lysis buffer and centrifuged
in an SW-40 rotor (Beckman Instruments Inc., Fullerton,
California, USA) at 100,000 gfor 24 hours at 4°C. Twen-
ty-four fractions (0.5 mL each) were collected from each
gradient. Ten-microliter aliquots of each fraction were
used for immunoblotting. The remainder of each frac-
tion was immunoprecipitated with affinity-purified
antibody against polycystin-1. The samples were sepa-
rated on a 7.5% gel, transferred to PVDF (Millipore
Corp., Bedford, Massachusetts, USA), and probed with
mouse monoclonal antibodies against β-catenin. The
blots were then stripped and reprobed with antibodies
to FAK. Densitometry was performed on a densitometer
from Molecular Dynamics.
To determine the distribution of polycystin-1 in gra-
dients, metabolically labeled cell lysates were subjected
to density gradient centrifugation as above. Ten-micro-
liter fractions were separated on a 10% gel and trans-
ferred to PVDF. This was cut into strips at the 66 kDa
molecular weight marker, and probed with antibodies
to β-catenin and actin (Roche Molecular Biochemicals).
The remainder of each fraction was immunoprecipitat-
ed with affinity-purified antibody to polycystin-1 and
separated on a 7.5% gel. Autoradiograms of the
immunoprecipitates were viewed with a Storm Phos-
phorImager System from Molecular Dynamics.
Results
Polycystin colocalizes with E-cadherin and the catenins. We and
others had previously found that polycystin-1 was most
highly expressed in renal epithelial cells of fetal kidney
(12, 35). By indirect immunofluorescence, polycystin-1
was inconsistently detectable in the collecting duct of
adult kidney. To test the hypothesis that polycystin inter-
acts with complexes containing E-cadherin, we stained
sections of fetal kidney with antibodies to polycystin-1
and E-cadherin. We selected tubules that were strongly
stained for polycystin and then scanned those tubules
for simultaneous localization of both polycystin-1 and
cell adhesion molecules. The majority of the tubules
were located just below the nephrogenic zone in the
developing kidneys examined. The staining pattern and
morphology of the tubules suggested that the majority
were of ureteric bud origin. Figure 1 shows that anti-
bodies to E-cadherin (Figure 1a, green) and polycystin-1
(Figure 1b, red) both stained the plasma membrane of
tubular epithelial cells. Figure 1c shows that the majori-
ty of polycystin-1 colocalized with E-cadherin (yellow),
suggesting that the 2 proteins might be part of a com-
plex. There was a significant amount of E-cadherin in
the apical plasma membrane of the tubule because this
fetal tubule is not yet completely polarized. Polycystin-1
and E-cadherin were colocalized in the apical and later-
al plasma membrane. There were areas where polycystin-
1 and E-cadherin did not colocalize, shown as distinct
green or red staining (Figure 1c). These data suggest that
there are pools of E-cadherin and polycystin-1 in the
plasma membrane that do not interact.
The cytoplasmic domain of E-cadherin interacts with
cytoplasmic molecules known as the catenins. These mol-
ecules both regulate the adhesive function of E-cadherin
and mediate signaling from E-cadherin. The observation
that polycystin-1 colocalized with E-cadherin suggested
that polycystin-1 should also colocalize with the catenins.
Distinct complexes containing E-cadherin and subsets of
the catenins have been identified in epithelial cell cultures,
suggesting that these complexes might have different sig-
naling properties. The common components of these
complexes were E-cadherin and α-catenin; β-catenin and
1462 The Journal of Clinical Investigation | November 1999 | Volume 104 | Number 10
Figure 2
Expression of polycystin-1 in HPAC cells. (a) RT-PCR with PKD1-specific
primers yielded the predicted 564-bp product (PKD1, – RNase). RT-PCR
with human β-actin primers from 2 exons yielded the mRNA-specific
product of 661 bp (actin, – RNase). The sizes of the markers on the left
are 1,000, 700, 500, and 300 bp. Treatment of the RNA template with
RNase A before RT-PCR eliminated both the actin and the PKD1 prod-
ucts (+ RNase). (b) Polycystin-1 was precipitated from [35S]methion-
ine/cysteine–labeled HPAC cells as described, displayed on a 4–10% lin-
ear gradient gel, and viewed with a PhosphorImager analysis system
(Molecular Dynamics). IM + peptide: precipitation with immune serum
in the presence of the immunizing peptide. IM: precipitation with
immune serum. PI: precipitation with preimmune serum. Note that the
polycystin polypeptide of approximately 500 kDa is precipitated only by
immune serum and only in the absence of peptide.

γ-catenin were found in a mutually exclusive manner in
these complexes. It was possible that polycystin-1 might
be identified in only 1 type of E-cadherin complex.
Accordingly, we stained sections of fetal kidney with anti-
bodies to α-, β-, and γ-catenin and to polycystin-1. We
found that polycystin-1 colocalized with α-catenin (Fig-
ure 1, d–f), β-catenin (Figure 1, g–i), and γ-catenin (Figure
1, j–l). There was no difference in the location or amount
of polycystin-1 associated with β- and α-catenin, suggest-
ing that polycystin-1 did not preferentially interact with a
subset of E-cadherin complexes. All 3 catenins localized to
the apical and lateral plasma membranes (Figure 1, d, g,
and j), paralleling the apicolateral distribution of E-cad-
herin in these immature epithelia. All 3 catenins were
expressed in mesenchymal cells as well as epithelial cells of
the developing kidney. The expression of β-and γ-catenin
was quite high and is easily seen (Figure 1, g and j), where-
as the expression of α-catenin was lower and is not well
seen in this figure (Figure 1d). As we had observed for E-
cadherin, the association of polycystin-1 with the catenins
was not complete. There were areas where polycystin-1 did
not colocalize with the catenins, seen as distinct areas of
green or red staining (Figure 1, f, i, and l).
To demonstrate the specificity of polycystin-1 staining,
we incubated peptide B145 (the antigen against which
the polycystin-1 antibody was raised) with the primary
antibodies during immunostaining. The result of a rep-
resentative experiment is shown in Figure 1, m–o. Pep-
tide abolished polycystin-1 staining (Figure 1n) but had
no effect on β-catenin staining (Figure 1, m and o). These
data show, first, that polycystin-1 staining was peptide
specific. Second, the peptide had no effect on β-catenin
staining, demonstrating that there were no nonspecific
effects of peptide on antibody reactivity. Third, these
images show that there was excellent optical separation
between the red channel (polycystin-1) and the green
channel (in this case, β-catenin), because no β-catenin
staining was found in the red channel.
HPAC pancreatic epithelial cells express polycystin-1. To
determine whether polycystin-1 interacts with E-cad-
herin and the catenins, we sought a human epithelial cell
line that expressed all the proteins of interest. Polycystin-
1 is expressed in the pancreatic ductal epithelium (van
Adelsberg, unpublished observations, and ref. 17), sug-
gesting that pancreatic ductal cell lines would be an
appropriate in vitro model system. Patients with ADPKD
develop pancreatic cysts, as do mice with a targeted
mutation in the PKD1 gene (36). Because the disease is
manifested in the pancreatic duct in vivo, cell lines
derived from the pancreatic duct should be appropriate
for studying the relevant biochemical interactions of
polycystin-1 in vitro. We identified a human pancreatic
epithelial cell line, HPAC, that grows as a monolayer and
expresses the proteins E-cadherin, α-, β-, and γ- catenin,
β1integrins, and FAK (see Figure 3). We screened this cell
line for PKD1 gene and protein expression.
To determine whether the PKD1 gene was expressed
in HPAC cells, we performed RT-PCR on total RNA
isolated from these cells. Figure 2a shows that primers
from the unique 3′end of PKD1 amplified the expect-
ed product of 564 bp. This product was purified and
sequenced; the sequence was identical to the pub-
lished PKD1 sequence. Primers from 2 exons of the
human β-actin gene amplified the expected product
of 661 bp. There was no larger product, suggesting
that there was no contamination by genomic DNA.
Treatment of the RNA template with RNase A before
RT-PCR eliminated both the PKD1 and the actin PCR
products (Figure 2a, + RNase), providing additional
The Journal of Clinical Investigation | November 1999 | Volume 104 | Number 10 1463
Figure 3
Coimmunoprecipitation of polycystin-1 with junctional complex pro-
teins. (a) Immunoprecipitation of HPAC cell lysates with affinity-purif ied
antibody to polycystin-1. Each panel is an immunoblot probed with anti-
body to the protein named at the top of the panel. In each panel, the left
lane is an aliquot of the cell lysate (lysate). The middle and right lanes
were immunoprecipitated with affinity-purified antibody against poly-
cystin-1 in the absence (–) or presence (+) of the immunizing peptide.
Each set of samples was probed with antibody to E-cadherin, α-catenin,
β-catenin, γ-catenin, β1-integrin, or FAK. Note that E-cadherin and the
catenins coprecipitated with polycystin-1 only in the absence of compet-
ing peptide. The doublet of 120 kDa and 140 kDa in the lysate probed
with antibody to β1-integrin represents reduced (140 kDa) and unre-
duced (120 kDa) β1-integrin. Results are representative of at least 5 inde-
pendent experiments. (b) Double immunoprecipitation of metabolically
labeled HPAC lysates with antibodies to E-cadherin or β-catenin (first
immunoprecipitation) followed by affinity-purified antibody to poly-
cystin-1 (second immunoprecipitation). The left pair of lanes shows the
first immunoprecipitation with monoclonal antibody to β-catenin (β-cat)
or E-cadherin (E-cad). Note the high-molecular-weight polypeptide that
is precipitated as part of the complex. The center pair of lanes shows the
reimmunoprecipitation of the complex with affinity-purified antibody to
polycystin-1. The high-molecular-weight polypeptide seen in the left pan-
els was reprecipitated by polycystin antibody and is therefore poly-
cystin-1. Note that some of the E-cadherin/catenin complex remains
associated with polycystin-1 (arrows). The right pair of lanes shows con-
trol experiments performed on aliquots of the same HPAC lysate. The
lane marked “PKD1” was precipitated with affinity-purified antibody to
polycystin-1. The lane marked “IgG” was precipitated with the same con-
centration of nonimmune rabbit anti-mouse IgG. This figure is a single
gel. The results are representative of 3 independent experiments.

evidence that the PCR product was amplified from
reverse transcribed PKD1 message.
To discover whether there was significant polycystin-1
protein expression in HPAC cells, confluent cultures
were labeled with [35S]methionine/cysteine and lysates
were immunoprecipitated with the anti-peptide anti-
body against polycystin-1. Figure 2b shows that the
immune serum precipitated a polypeptide of approxi-
mately 500 kDa that was not precipitated by the preim-
mune serum. Incubating the immune serum with lysate
in the presence of the immunizing peptide blocked pre-
cipitation of this polypeptide, showing that the
immunoprecipitation was specific for the peptide. Iden-
tical results were obtained with 2 different antibodies
raised against the extracellular domain of polycystin-1
(not shown). These results show that the HPAC cells
express polycystin-1, and that the anti-peptide antibody
specifically precipitates polycystin-1 from HPAC cell
lysates. We were unable to detect polycystin-1 by
immunoblotting lysates of the HPAC cell line, suggest-
ing that polycystin-1 is expressed at low levels in these
cells or that the efficiency of electroblotting this large
membrane protein is very low, or both.
E-cadherin and the catenins are coprecipitated with polycystin-
1. We precipitated HPAC cell lysates with antibody to poly-
cystin-1 and probed the precipitates with antibodies to E-
cadherin and α-, β-, and γ-catenin. We found that
E-cadherin and the catenins were coprecipitated with
polycystin-1 (Figure 3a, – peptide). Adding B145 peptide
to the immunoprecipitation reaction blocked the precip-
itation of E-cadherin and the catenins (Figure 3a, + pep-
tide), demonstrating that the coprecipitation of E-cad-
herin and the catenins was specific for the precipitation of
polycystin-1. These data show that polycystin-1 interacts
with a complex containing E-cadherin and the catenins.
To determine whether polycystin-1 was coprecipitated
with E-cadherin and β-catenin, we performed double
immunoprecipitation (Figure 3b). Antibodies to either
E-cadherin or β-catenin (2 left lanes) precipitated a com-
plex containing a high-molecular-weight polypeptide as
well as E-cadherin, α-catenin, and β-catenin. The molec-
ular weight of this polypeptide was the same as that of
precipitated polycystin-1 (fifth lane, PKD1). Reprecipi-
tation of the complex with anti–polycystin-1 antibodies
showed that the high-molecular-weight polypeptide was
polycystin-1 (center pair of lanes). Note that a small frac-
tion of the E-cadherin/catenin complex was reprecipi-
tated with antibody to polycystin-1 despite treatment of
the precipitated proteins with 1% SDS. From densito-
metric analysis, we determined that only 6.0 ± 0.5% (n =
3) of precipitable E-cadherin or β-catenin complexes
contained polycystin-1.
Polycystin-1 comigrates with
β
-catenin in sucrose density gradi-
ents. The coprecipitation of polycystin-1 with E-cadherin
and the catenins suggested that these proteins were in a
macromolecular complex. Both the colocalization data
and the coimmunoprecipitation data showed that only a
fraction of E-cadherin/catenin complexes contained poly-
cystin-1. To characterize this complex further, we subject-
ed HPAC cell lysates to sucrose density centrifugation. We
chose to use β-catenin as a marker for the
E-cadherin/catenin complex because antibodies to β-
catenin gave the strongest signal in coprecipitation exper-
iments and because β-catenin metabolism is a major deter-
minant of cell proliferation. Polycystin-1 migrated in a
fairly broad range near the center of the gradient (Figure 4,
top). The distribution of β-catenin was broader than that
of polycystin-1, extending into lighter fractions of the gra-
dient. The distribution of polycystin-1 overlapped with
that of β-catenin only in the denser parts of the β-catenin
range. As anticipated, polycystin-1 coprecipitated β-catenin
only in those fractions where polycystin-1 was present (Fig-
ure 4, bottom). The distribution of the complex contain-
ing polycystin-1 and β-catenin was not homogeneous. The
amount of β-catenin coprecipitated with polycystin-1 was
much greater in the lighter part of the polycystin-1 distri-
bution. This skewed distribution of polycystin-catenin
complexes was seen in 4 of 4 independent experiments.
These data provide additional evidence that polycystin-1 is
in a complex containing β-catenin. These results are con-
sistent with both the immunohistochemical and copre-
cipitation data, which showed that polycystin-1 and β-
catenin were not completely colocalized or coprecipitated.
The data suggest that there are 2 pools of polycystin-1, only
1 of which interacts with a complex containing β-catenin.
To discover whether the actin cytoskeleton could par-
ticipate in the formation of a unique complex containing
polycystin-1 and β-catenin, we examined the distribution
of actin within the gradient (Figure 4). Actin comigrated
1464 The Journal of Clinical Investigation | November 1999 | Volume 104 | Number 10
Figure 4
Sucrose density gradient centrifugation of HPAC cell lysates. Top: Distrib-
ution of polycystin-1, β-catenin, and actin within the gradient. Polycystin-1
was detected by immunoprecipitation and PhosphorImager analysis (Mol-
ecular Dynamics). Immunoblotting was used to detect β-catenin and actin.
Results are representative of 3 independent experiments. Bottom: Distrib-
ution of β-catenin, FAK, and complexes of β-catenin and polycystin-1 with-
in gradient. The distributions of β-catenin and FAK were determined by
immunoblotting. The distributions of complexes containing polycystin-1
and either β-catenin or FAK were determined by immunoprecipitating each
fraction of the gradient with affinity-purified antibodies to polycystin-1 and
probing immunoblots of the precipitates with antibodies to β-catenin (IP:
polycystin-1, blot: β-catenin) or FAK (IP: polycystin-1, blot: FAK). No FAK
was coprecipitated with polycystin-1. Results are representative of 3 inde-
pendent experiments.

with polycystin-1, although the distribution of actin was
broader than that of polycystin-1. The peak distribution
of polycystin-1 in the gradient matched the peak distri-
bution of actin. These results suggest that actin could be
present in polycystin-1 complexes. The distribution of
actin overlapped, but did not precisely mimic the distri-
bution of β-catenin. This result is not surprising, because
β-catenin is found both in complexes containing E-cad-
herin and in separate complexes containing APC, glyco-
gen synthase kinase, and axin.
Polycystin-1 colocalizes with
β
1integrins but not with FAK.
Although the majority of polycystin-1 was concentrated
in the apical and lateral aspects of the tubular epithelial
cells, there was consistently a small amount of peribasal
staining that did not colocalize with E-cadherin or α-, β-,
or γ-catenin. We had previously found that epithelial cells
from the cpk mouse model of autosomal recessive poly-
cystic kidney disease had increased β1-integrin–mediated
adhesion to collagen and laminin (37). We speculated that
polycystin-1 might interact with both E-cadherin and
integrin-type cell adhesion complexes. Accordingly, we
performed double-label immunofluorescence with anti-
bodies against the human β1-integrin subunit or against
FAK, the well characterized signal transduction molecule
of the focal adhesion. We found that the β1-integrin sub-
unit was not polarized in these primitive renal epithelia,
and that it colocalized with polycystin-1 (Figure 5a). FAK
was detected only in the branching tubules of the ureteric
bud, which also expressed polycystin-1. FAK was confined
to the basal plasma membrane in these epithelia, as was
paxillin, another protein of the focal adhesion (data not
shown). However, FAK did not colocalize with polycystin-
1 (Figure 5b). These data suggest that, although polycystin
could be present in a complex with a β1-integrin, it is not
present in focal adhesions containing FAK and paxillin.
Polycystin-1 does not coprecipitate or comigrate with FAK or
β
1-
integrin. To discover if a small proportion of FAK interact-
ed with polycystin-1, we probed polycystin-1 immuno-
precipitates with antibodies to β1-integrin and FAK. We
found that neither β1-integrin nor FAK were coprecipitat-
ed with polycystin-1 (Figure 3). Paxillin was precipitated
nonspecifically by agarose beads despite extensive pre-
clearing, so we could not assess coprecipitation of paxillin
with polycystin-1. In sucrose density gradient centrifuga-
tion, the distribution of FAK overlapped minimally with
that of polycystin-1 (Figure 4, bottom). FAK did not copre-
cipitate with polycystin-1 in fractions isolated by sucrose
density gradient centrifugation (Figure 4, bottom). These
data show that complexes containing FAK do not contain
polycystin-1, and that polycystin-1 is not part of the focal
adhesion. The coprecipitation data suggest that, if there
is an interaction between polycystin-1 and β1integrins, the
interaction is not strong. The observation that neither β1-
integrin nor FAK could be coprecipitated with polycystin-
1 antibodies provides additional evidence that the copre-
cipitation of E-cadherin and the catenins by polycystin-1
antibodies was specific.
Discussion
We have demonstrated that polycystin-1 is present in com-
plexes containing E-cadherin and the catenins. E-cadherin
and the catenins regulate cell proliferation, cell polarity,
and tissue morphogenesis. In ADPKD, the cysts develop
from tubules, a striking example of abnormal morpho-
genesis. The creation of this abnormal structure, the cyst,
is accompanied by perturbations in cell proliferation and
cell polarity. Our discovery that polycystin-1 interacts with
E-cadherin and α-, β-, and γ-catenin raises obvious ques-
tions about whether polycystin-1 affects the assembly of
junctional complexes or signaling from these complexes.
Because the catenins have effects on such diverse cellular
functions as the polarization of the cytoskeleton (18), reg-
ulation of gene transcription (38), and the formation of
desmosomes (39, 40), the range of possibilities is large, but
is consistent with the protean manifestations of ADPKD.
One of the first questions that arises from our work is
The Journal of Clinical Investigation | November 1999 | Volume 104 | Number 10 1465
Figure 5
Confocal images of sections of human fetal kidney stained with antibody to polycystin-1 (red) and either β1-integrin (a) or FAK (green) (b). Only the
dual-scanned images are shown. The areas where polycystin-1 colocalized with β1-integrin (a) are yellow. Polycystin-1 was expressed only in the epithe-
lial structures of the ureteric bud and the S-shaped body (arrowheads), whereas β1-integrin was ubiquitously expressed in both the epithelia (yellow
staining due to colocalization with polycystin-1) and the surrounding mesenchyme (green staining, because polycystin-1 is not expressed in the mes-
enchyme). The absence of yellow staining in (b) shows that polycystin-1 did not colocalize with FAK.

whether polycystin-1 interacts directly with E-cadherin
or with 1 of the catenins. Our data do not allow us to dis-
tinguish between a direct or an indirect interaction. Yeast
2-hybrid analysis conducted in several labs did not iden-
tify E-cadherin or any of the catenins as binding partners
for the carboxyl terminal domain of polycystin-1
(41–45), nor have preliminary biochemical experiments
identified any of these proteins in pull-down assays from
epithelial cell lysates (van Adelsberg, Huan, and Walz,
unpublished observations). Polycystin-1 is a polytopic
integral membrane protein. Current models of its struc-
ture favor 11 transmembrane domains with 5 intracel-
lular loops. Any of these loops could bind directly to E-
cadherin, to a catenin, or to an adapter molecule that
could provide a bridge between polycystin-1 and the
E-cadherin/catenin complex. β-catenin and γ-catenin
bind directly to α-catenin, giving rise to 2 distinct cad-
herin-catenin complexes (46, 47). Polycystin-1 interacts
with both of these complexes. The most parsimonious
explanation is that polycystin-1 (or the hypothetical
adapter) interacts with E-cadherin or with α-catenin,
which are common to both complexes.
We found that the interaction between polycystin-1
and the E-cadherin/catenin complex was not stoichio-
metric. The colocalization data showed separate areas of
polycystin-1 and cadherin/catenin staining, suggesting
that polycystin-1 interacts only with a discrete pool of
cadherin and catenins. Sucrose density gradient cen-
trifugation also identified a pool of polycystin-1 that did
not interact with β-catenin. These data suggest that the
interaction of polycystin-1 with the E-cadherin/catenin
complex could be regulated. Complexes containing
E-cadherin, the catenins, and polycystin-1 might have a
specialized role in signal transduction.
What is the role of an interaction between polycystin-1
and the E-cadherin/catenin complex? One possibility is
that polycystin-1 is involved in the formation of the
E-cadherin/catenin complex. This hypothesis appears
unlikely, because PKD1 null mice die during late organo-
genesis, not at the peri-implantation stage as do E-cad-
herin or β-catenin null mice (48–51). Preliminary work
(Huan and van Adelsberg, unpublished observations)
suggests that E-cadherin/catenin complexes are intact in
cyst-lining epithelial cells isolated from ADPKD kidneys.
The interaction of polycystin-1 with the E-cad-
herin/catenin complex could be involved in correct tar-
geting of polycystin-1. Mutations that affect targeting
rather than biochemical activity are well documented.
One well characterized example is the ∆F508 mutation
of the cystic fibrosis transmembrane conductance reg-
ulator (CFTR) ion channel, which produces a func-
tional channel that is not targeted to the plasma mem-
brane (52). Retention of targeted proteins in the
correct plasma membrane domain is one mechanism
for maintaining cell polarity. This mechanism has been
well characterized for Na,K-ATPase. This protein inter-
acts directly with ankyrin (27, 53), which is complexed
with nonerythrocyte spectrin (fodrin), and then,
through actin and α-catenin, to the E-cadherin/catenin
complex (24, 33, 34, 54, 55). The hypothesis that the
interaction between polycystin-1 and the E-cad-
herin/catenin complex stabilizes correctly polarized
polycystin-1 in a manner analogous to that of Na,K-
ATPase predicts several results. First, the half-life of
mutant polycystin-1 in ADPKD cyst-lining epithelial
cells should be reduced. Second, the complex should
contain cytoskeletal elements like actin and possibly
ankyrin or spectrin. We found that polycystin-1 and
actin comigrated in sucrose gradients, suggesting that
they could be part of a complex. Two of the candidate
proteins that interact with the carboxyl terminal
domain of polycystin-1 contain spectrin repeats, sug-
gesting that polycystin-1 could interact with spectrin
or fodrin. Third, polycystin-1 should be incorrectly tar-
geted in cyst-lining epithelial cells of ADPKD. A corol-
lary to the hypothesis is that only some mutations in
PKD1 would disrupt the interaction of polycystin-1
with E-cadherin and the catenins, whereas other muta-
tions might cause different effects on polycystin-1 pro-
cessing or function. Apparent mislocalization of poly-
cystin-1 has been observed in at least 1 case of ADPKD.
These data support the idea that the interaction of
E-cadherin/catenin complex with polycystin-1 could be
involved in polycystin-1 targeting.
A third possible role for the interaction of polycystin-1
with E-cadherin/catenin complexes is that of modulat-
ing signaling from these complexes, in particular by
effects on β-catenin metabolism. This is an attractive
hypothesis for a variety of reasons. First, expression of
the carboxyl terminal tail of polycystin-1 as a membrane-
targeted chimera protected cytoplasmic β-catenin from
degradation by the proteasome and increased transcrip-
tion from a reporter for Siamois, a genuine β-catenin tar-
get (56). Second, high expression of c-myc, a likely
β-catenin target, is found in both human ADPKD cyst-
lining epithelial cells and cyst-lining epithelial cells from
2 rodent models of polycystic kidney disease (57–59).
Transgenic mice that overexpress c-myc in renal epithe-
lial cells develop fatal polycystic kidney disease, demon-
strating that c-myc is in the pathway to cyst formation
(60). Third, nuclear β-catenin staining is found in the bcl-
2null mouse model of polycystic kidney disease (61).
The interaction of β-catenin with LEF transcription fac-
tors is known to target β-catenin to the nucleus (62).
These data suggest that aberrant β-catenin signaling
could be a common feature of polycystic kidney diseases.
This hypothesis predicts that the half-life of β-catenin
will be prolonged in cyst-lining epithelial cells in poly-
cystic kidney disease, and that expression of other
β-catenin target genes will be upregulated in these cells.
Our data show that polycystin-1 does not interact
directly with focal adhesions in the steady state that is
characteristic of epithelial cells in tissue or confluent cul-
tures. We had previously observed increased β1-inte-
grin–mediated adhesion to extracellular matrix in cells
isolated from a mouse model of polycystic kidney dis-
ease, suggesting that defects in cell-matrix interaction
might contribute to cyst formation (37). Our new data
suggest that this phenotype is unlikely to be caused by a
direct effect of polycystin-1 on focal adhesions, but
could be an example of cross-talk between cadherins and
integrin (63, 64). We had found that the integrin that
mediates increased matrix adhesion in cpk mouse epithe-
lial cells is α1β1-integrin (van Adelsberg and Almonte,
1466 The Journal of Clinical Investigation | November 1999 | Volume 104 | Number 10

unpublished observations). Expression of this integrin
is upregulated in autosomal recessive polycystic kidney
disease and in ADPKD (65), suggesting that upregula-
tion of α1β1-integrin–mediated adhesion is part of a
common pathway in cyst formation. This hypothesis
would place the regulation of α1β1-integrin expression
downstream of the PKD and ARPKD genes.
Our observation that β1integrins are not polarized dur-
ing renal development is not new (66–68). However, we
are the first to describe the localization of FAK and pax-
illin in developing kidney. Our data show that paxillin
and FAK are confined to the basal plasma membrane.
The β1integrins, however, are basal, lateral, and apical.
This result suggests that either these apical and lateral
integrins are not involved in signaling or that other sig-
naling complexes containing integrins but not the clas-
sic molecules of the focal adhesion are present in renal
epithelial cells. Because Madin-Darby canine kidney
(MDCK) cells express apical β1integrins and respond to
them by remodeling the axis of apicobasal polarity when
collagen is overlaid on an MDCK monolayer (69, 70), it
seems likely that the apical β1integrins are functional.
Acknowledgments
Janet van Adelsberg is an Established Investigator of the
American Heart Association. This project was funded in
part by grant 1-FY97-0513 from the March of Dimes.
Confocal microscopy was performed at the Confocal
Microscopy Facility of Columbia University, which was
established by National Institutes of Health (NIH)
Shared Instrumentation Grant 1S10-RR10406 and is
supported by NIH grant 5-P30-CA13696 as part of the
Herbert Irving Cancer Center at Columbia University.
We are grateful to Theresa Swayne of the Confocal
Microscopy Facility for her invaluable assistance in
obtaining the confocal images shown here, and to
Vivette D’Agati and Llewellyn Ward of the Department
of Pathology at Columbia University for their help in
obtaining and analyzing the kidney sections shown here.
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1468 The Journal of Clinical Investigation | November 1999 | Volume 104 | Number 10