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Interaction of Two Actin-binding Proteins, Synaptopodin
and
␣
-Actinin-4, with the Tight Junction Protein MAGI-1*
Received for publication, March 29, 2002, and in revised form, May 28, 2002
Published, JBC Papers in Press, May 31, 2002, DOI 10.1074/jbc.M203072200
Kevin M. Patrie‡§, Andrew J. Drescher‡, Ajith Welihinda‡¶, Peter Mundel储**,
and Ben Margolis‡ ‡‡§§¶¶
From the Departments of ‡Internal Medicine and ‡‡Biological Chemistry and the §§Howard Hughes Medical Institute,
University of Michigan, Ann Arbor, Michigan, 48109-0650 and the 储Departments of Medicine and of Anatomy and
Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461
In an attempt to find podocyte-expressed proteins
that may interact with the tight junction protein
MAGI-1, we screened a glomerulus-enriched cDNA li-
brary with a probe consisting of both WW domains of
MAGI-1. One of the isolated clones contained two WW
domain-binding motifs and was identified as a portion
of the actin-bundling protein synaptopodin. In vitro
binding assays confirmed this interaction between
MAGI-1 and synaptopodin and identified the second WW
domain of MAGI-1 to be responsible for the interaction.
MAGI-1 and synaptopodin can also interact in vivo,as
they can be immunoprecipitated together from HEK293
cell lysates. Another actin-bundling protein that is
found in glomerular podocytes and shown to be mutated
in an inheritable form of glomerulosclerosis is
␣
-acti-
nin-4. We show that
␣
-actinin-4 is also capable of binding
to MAGI-1 in in vitro binding assays and that this inter-
action is mediated by the fifth PDZ domain of MAGI-1
binding to the C terminus of
␣
-actinin-4. Exogenously
expressed synaptopodin and
␣
-actinin-4 were found to
colocalize along with endogenous MAGI-1 at the tight
junction of Madin-Darby canine kidney cells. The inter-
action and colocalization of MAGI-1 with two actin-bun-
dling proteins suggest that MAGI-1 may play a role in
actin cytoskeleton dynamics within polarized epithelial
cells.
The ability of the mammalian kidney to produce a protein-
free filtrate is predominantly due to specialized cells within the
renal glomerulus known as visceral epithelial cells or podo-
cytes. Mature differentiated podocytes are highly specialized
cells whose many functions, including regulating glomerular
permselectivity (1), depend on an elaborate and complex cellu-
lar morphology (2). Because of this distinct morphology, podo-
cytes can be divided into three functionally and structurally
different segments: cell body, major processes, and foot pro-
cesses. Structural differences in the segments of podocytes are
reflected in their different cytoskeletal foundation, with foot
processes exhibiting an actin-based contractile apparatus (3).
In glomerular diseases with massive proteinuria, podocytes
may undergo dramatic structural changes resulting in loss of
foot process architecture and eventual effacement. Under some
conditions, these changes are reversible, which illustrates the
morphological dynamics of foot processes. Although the regu-
lation of foot process dynamics is poorly understood at this
time, it is apparent that an alteration in the actin-based cy-
toskeleton is a fundamental aspect.
Two actin-bundling proteins have recently drawn attention
because of their expression within the podocyte. One of them,
synaptopodin, was initially identified as an antigen to a mono-
clonal antibody that showed association with the actin system
of podocyte foot processes (4). Its eventual cloning and charac-
terization showed no significant homology to any other known
proteins, and it was found in the dendritic spines of hippocam-
pal neurons in addition to renal podocytes (5). The finding that
synaptopodin associates with specialized actin-based compart-
ments of renal podocytes and neuronal dendrites suggests that
synaptopodin may play a role in the structural and/or func-
tional dynamics of these cellular extensions. The non-muscle
isoform of
␣
-actinin is another actin-associated protein shown
to be expressed in the podocyte (3, 6, 7). Of the two known
non-muscle isoforms of
␣
-actinin (
␣
-actinin-1 and
␣
-actinin-4),
␣
-actinin-4 was recently identified as the isoform that is pres-
ent in human podocytes, and mutations in the gene encoding
this protein are responsible for an autosomal dominant form of
focal and segmental glomerulosclerosis (FSGS)
1
in humans (8).
Interestingly, both
␣
-actinin-1 and
␣
-actinin-4 are found in
cultured mouse podocytes, but they exhibit differences in their
spatial distribution within these cells (9). Originally identified
as the antigen to a monoclonal antibody that exhibited a
unique immunohistochemical reactivity,
␣
-actinin-4 has also
been implicated in cell motility and carcinogenesis (10).
We recently reported on the localization of a protein,
MAGI-1, in the podocytes of rat kidneys (11). In addition, we
showed that MAGI-1 was found in the membrane fraction of
mouse glomerular preparations and that it was insensitive to
extraction with Triton X-100, which suggested to us that
MAGI-1 might be associated with the actin cytoskeleton. The
MAGI proteins consist of three members that together make up
* This work was supported in part by NIDDK Grant 2P50DK39255
from the National Institutes of Health. The costs of publication of this
article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by National Kidney Foundation Postdoctoral Research
Fellowship 5F32DK09912-02.
¶Present address: Sangstat Medical Corporation, 6300 Dumbarton
Circle, Fremont, CA 94555.
** Supported by National Institutes of Health Grant DK57683-01.
¶¶ Investigator of the Howard Hughes Medical Institute. To whom
correspondence should be addressed: Howard Hughes Medical Inst.,
University of Michigan, 4570 MSRB II, 1150 West Medical Center Dr.,
Ann Arbor, MI 48109-0650. Tel.: 734-764-3567; Fax: 734-763-9323;
E-mail: bmargoli@umich.edu.
1
The abbreviations used are: FSGS, focal and segmental glomerulo-
sclerosis; MAGI-1, membrane-associated guanylate kinase inverted-1;
MAGUK, membrane-associated guanylate kinase; PDZ, PSD-95/DLG/
ZO-1; AIP, atrophin-1-interacting protein; GST, glutathione S-transfer-
ase; HA, hemagglutinin; HEK293, human embryonic kidney 293;
MDCK, Madin-Darby canine kidney; PBS, phosphate-buffered saline.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 33, Issue of August 16, pp. 30183–30190, 2002
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org 30183
a subfamily of a larger group of proteins known as the
MAGUKs. MAGUK proteins share a common structural orga-
nization and are proposed to function as molecular scaffolds
within cells (for recent reviews, see Refs. 12 and 13). Many of
them are found at special subcellular regions such as postsyn-
aptic densities within neurons as well as the tight and adher-
ens junctions of epithelial cells and are believed to play a role
in the structure and function of these specialized complexes.
The MAGUK proteins exhibit a unique grouping of protein-
protein interaction domains that is inverted in the MAGI fam-
ily of proteins. In addition, two WW domains in the MAGI
proteins take the place of the SH3 (Src homology 3) domain
observed in the conventional MAGUKs. WW domains are small
protein interaction modules of 30 –40 amino acids in length and
are often found in association with other protein interaction
domains such as phosphotyrosine-binding and PDZ domains
(14, 15). They have been found to bind polyproline-rich peptide
sequences and can be classified into five distinct groups based
upon current understanding of their binding specificity. Group
I WW domains, like those found in the ubiquitin-protein ligase
Nedd4 and Yes-associated protein, have been extensively stud-
ied and recognize “PPXY”motifs (where P is proline, Xis any
amino acid, and Y is tyrosine; often referred to as PY motifs)
(16, 17). The binding of a number of proteins to the PDZ
domains and guanylate kinase domain of MAGI-1, MAGI-2,
and MAGI-3 has been reported; however, there is a paucity of
data on proteins that may interact with the WW domains of
MAGI proteins. Although a screen to identify interacting part-
ners for the DRPLA (dentatorubral and pallidoluysian atrophy)
gene product atrophin-1 isolated partial cDNAs containing the
WW domains of MAGI-1 (AIP-3) and MAGI-2 (AIP-1) (18), no
further data on these interactions have yet been reported.
In an initial attempt to discover potential binding partners
for MAGI-1 that are found in renal glomeruli, we screened a
cDNA expression library made from glomerulus-enriched prep-
arations of mouse kidney with the WW domains of MAGI-1.
Among the clones that were isolated was a partial cDNA coding
for a region of the actin-bundling protein synaptopodin that
contained both of its PY motifs. We provide further data
strengthening this interaction of synaptopodin with MAGI-1
using various binding assays and colocalization analysis. In
addition, we examined the potential of
␣
-actinin-4, another
actin-bundling protein whose C terminus contains a PDZ do-
main-binding motif, to bind to MAGI-1. We found that
␣
-acti-
nin-4 was also capable of binding to one of the PDZ domains of
MAGI-1. Thus, two actin-bundling proteins have been found to
bind to the MAGUK protein MAGI-1, and we discuss the po-
tential relevance of these interactions.
MATERIALS AND METHODS
Plasmid Constructs—The following MAGI-1 constructs were ob-
tained using reverse transcription-PCR on total RNA derived from
mouse glomerulus-enriched preparations: full-length MAGI-1 (amino
acids 1–1220; numbering of all MAGI-1 constructs is based on the
full-length cDNA obtained from kidney and glomerular libraries previ-
ously described (11)), which contains the shorter “A”C-terminal tail as
previously described (19); MAGI-1 WW12 (amino acids 219 –416);
MAGI-1 WW1 (amino acids 219 –334); MAGI-1 WW2 (amino acids 315–
414); MAGI-1 PDZ123 (amino acids 404 –919); MAGI-1 PDZ234 (amino
acids 607–1081); MAGI-1 PDZ45 (amino acids 905–1455), which con-
tains the “C”C-terminal tail (19); MAGI-1 PDZ4 (amino acids 905–
1081); and MAGI-1 PDZ5 (amino acids 1011–1202). The PCR products
were initially cloned into the TA cloning vector pGEM-T-Easy (Pro-
mega, Madison, WI) and then subcloned into pGSTag (20) and pRK5-
Myc (21) for production of GST fusion proteins in bacterial cells and
expression of Myc-tagged proteins in mammalian cells, respectively.
A human synaptopodin construct containing the two PPXY motifs
(amino acids 294 –350) was obtained by PCR of a human expressed
sequence tag cDNA using forward primer 5⬘-CATGGTGGAAAGGAG-
GATGATGG-3⬘and reverse primer 5⬘-ACTTGGGGTCGGAGCTGG-
GATAC-3⬘. The PCR product was first cloned into pGEM-T-Easy and
then subcloned into pGSTag to make GST-synpoPY. A full-length
mouse synaptopodin cDNA in the expression vector pRC/CMV was
subcloned into HA
3
-pcDNA3.1(⫺) (HA-synaptopodin) by PCR such that
the flanking 5⬘- and 3⬘-untranslated sequences in the original cDNA
were deleted. This HA-tagged synaptopodin construct was further sub-
cloned into pTRE2hyg for use in the Tet-Off inducible system. The T7
epitope-tagged mouse Nedd4 expression construct (T-Nedd4) was
a kind gift from Dr. D. Rotin (Hospital for Sick Children, Toronto,
Canada). The C-terminal tail of
␣
-actinin-4 was obtained by PCR of a
rat expressed sequence tag cDNA and consisted of the last 19 amino
acids including the endogenous stop codon. The
␣
-actinin-4 tail was
cloned into pGSTag to make GST-
␣
-actinin-4. A full-length mouse
␣
-actinin-4 cDNA (a kind gift from Dr. S. R. Vincent, University of
British Columbia, Vancouver, British Columbia, Canada) was cloned
into the HA
3
-pcDNA3.1(⫺) vector to make HA-
␣
-actinin-4.
Antibodies—Anti-Myc antibody 9E10 was obtained from mouse asci-
tes fluid produced at the Hybridoma Core Facility of the University of
Michigan. Anti-T7 antibody (T7䡠Tag) was from Novagen (Madison, WI).
Anti-synaptopodin polyclonal antibody NT was described elsewhere (5).
Anti-MAGI-1 polyclonal antibody UM209 was described previously (11).
An additional anti-MAGI-1 polyclonal antibody (UM223) was produced
in rabbits using a GST fusion protein containing amino acids 905–1081
(containing the “b”variant of PDZ4 (11)) of MAGI-1 and then affinity-
purified. Anti-HA antibodies 3F10 (rat monoclonal) and 12CA5 (mouse
monoclonal) were from Roche Molecular Biochemicals. Mouse anti-
␣
-
actinin-4 monoclonal antibody NCC-Lu-632 was a kind gift from Dr. T.
Yamada (National Cancer Center Research Institute, Tokyo, Japan).
Mouse anti-ZO-1 monoclonal antibody ZO1-1A12 was from Zymed Lab-
oratories Inc. (South San Francisco, CA).
Expression Cloning—Total RNA isolated from glomerulus-enriched
preparations of mouse kidneys was used to make a random-primed
cDNA library, which was then modified at its ends with EcoRI adapters.
The cDNA library was then cloned into
SCREEN phage arms contain-
ing EcoRI sites at their ends (CLONTECH, Palo Alto, CA). The library
had an initial complexity of ⬃1⫻10
6
plaque-forming units/ml prior to
one round of amplification. The GST fusion protein GST-MAGI-1 WW12
(see above) was labeled with [
␥
-
32
P]ATP and used as a probe to screen
the library (the pGSTag expression vector has a protein kinase A
phosphorylation site placed in between the GST sequence and the
downstream protein of interest). Plating and transferring of plaques to
nitrocellulose membranes were performed as described in the
SCREEN manual. The resulting membranes were blocked in Farwest-
ern buffer (20 mMHepes (pH 7.5), 1 mMKCl, 5 mMMgCl
2
,5mM
dithiothreitol, 5% nonfat dry milk, and 0.02% sodium azide) for2hat
room temperature with gentle agitation, followed by a 2-h incubation
with the
32
P-labeled probe (2 ⫻10
6
cpm/ml of Farwestern buffer) at
room temperature. The membranes were washed twice with Tris-buff-
ered saline supplemented with Triton X-100 to 0.1% and then three
times with Tris-buffered saline (5 min each wash) at room temperature.
The membranes were exposed to x-ray film at ⫺80 °C.
Cell Culture and Transfections—HEK293 cells and MDCK cells were
grown in Dulbecco’s modified Eagle’s medium (Invitrogen) supple-
mented with 10% fetal calf serum, 2 mML-glutamine, 100 units/ml
penicillin G, and 100
g/ml streptomycin. Tet-Off MDCK cells (CLON-
TECH) were grown in Dulbecco’s modified Eagle’s medium supple-
mented with 5% fetal calf serum, 4 mML-glutamine, 100 units/ml
penicillin G, 100
g/ml streptomycin, 1
g/ml puromycin, and 40 ng/ml
doxycycline.
For transient transfections, HEK293 cells at 50 –80% confluency in
10-cm dishes were transfected with epitope-tagged expression con-
structs using either a calcium phosphate precipitation method or Su-
perfect transfection reagent (QIAGEN Inc., Valencia, CA). Cells were
allowed to recover for 24 –48 h prior to harvesting for lysates. Stable cell
lines of MDCK and Tet-Off MDCK cells were obtained by transfecting
them at 50% confluency in 6-cm dishes with Superfect transfection
reagent. Twenty-four hours after transfection, the cells were trypsinized,
and one-tenth of the volume of trypsinized cells was transferred to
15-cm dishes containing complete medium supplemented with Geneti-
cin (Invitrogen) at 600
g/ml or hygromycin B at 200
g/ml for the
Tet-Off cells. After ⬃10 days in selection medium, colonies were iso-
lated and subsequently assayed for expression by immunofluorescence.
Tissue and Cell Lysates—Adult mouse brains were placed in a
Dounce homogenizer along with high salt Triton lysis buffer (50 mM
Hepes (pH 7.5), 500 mMNaCl, 1.5 mMMgCl
2
,1mMEGTA, 10% glycerol,
and 1% Triton X-100) plus protease inhibitors (Complete protease in-
hibitor mixture tablets, Roche Molecular Biochemicals) and homoge-
nized with 25–30 strokes. The lysates were centrifuged at 20,000 ⫻gfor
MAGI-1 Interacts with Actin-associated Proteins30184
30 min at 4 °C, and the resulting supernatants were transferred to
fresh tubes and stored at ⫺20 °C until used. Glomerular extracts were
prepared in radioimmune precipitation assay lysis buffer (50 mMHepes
(pH 7.5), 150 mMNaCl, 1.5 mMMgCl
2
,1mMEGTA, 10% glycerol, 1%
Triton X-100, 1% sodium deoxycholate, and 0.1% SDS) plus protease
inhibitors as described previously (11).
Transiently transfected HEK293 cells were washed once with ice-
cold phosphate-buffered saline (PBS), scraped in 400
l of Triton lysis
buffer (with 150 mMNaCl), transferred to microcentrifuge tubes, vor-
texed briefly, and incubated on ice for 10 min. The lysates were centri-
fuged at 20,000 ⫻gfor 20 min at 4 °C, and the resulting supernatants
were transferred to fresh tubes and stored at ⫺20 °C until used. Cells
transfected with synaptopodin expression constructs either alone or
with other expression constructs were scraped and lysed in radioim-
mune precipitation assay lysis buffer plus protease inhibitors.
GST Fusion Protein Precipitation (Pull-down) and Immunoprecipi-
tations—GST fusion proteins were produced in DH5
␣
bacteria cells as
previously described (21). Tissue or cell lysates were combined with
⬃10
g of GST fusion protein bound to glutathione-agarose beads and
incubated on a rocker at 4 °C overnight. The beads were then washed
twice with ice-cold HNTG buffer (20 mMHepes (pH 7.5), 150 mMNaCl,
0.1% Triton X-100, and 10% glycerol) and once with ice-cold buffer
containing 20 mMHepes (pH 7.5), 150 mMNaCl, and 0.1% Triton X-100.
Forty microliters of 1⫻SDS sample buffer was added to the beads and
then placed at 100 °C for 5 min. Proteins eluted off of the beads were
subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and
blotted with the appropriate primary and horseradish peroxidase-con-
jugated secondary antibodies or protein A-horseradish peroxidase.
Blots were then developed with chemiluminescence reagents
(PerkinElmer Life Sciences) and exposed to x-ray film.
For immunoprecipitations, lysates (200 –300
l) from transfected
HEK293 cells were brought up to a total volume of 1 ml with HNTG
buffer and rocked overnight at 4 °C with anti-Myc antibodies. The
following day, protein A-Sepharose beads were added to the samples
and rocked for an additional2hat4°C. The beads were then washed
and processed in the same manner as the GST pull-down samples
above.
Immunofluorescence—Wild-type MDCK cells or stable MDCK cell
lines were seeded onto Transwell filters (Corning, Inc., Cambridge, MA)
and allowed to reach confluency. Cells were fixed in 4% paraformalde-
hyde in PBS for 15 min at room temperature and then solubilized with
1% SDS in PBS for 5 min at room temperature. Blocking was performed
in 50% goat serum diluted in PBS for2hat30°C in a humidified
chamber. Primary antibodies were diluted in 2% goat serum in PBS
(PBS-G) and placed on cells overnight at 30 °C in a humidified chamber.
The filters were washed three times with PBS-G (5 min each wash).
Fluorophore-conjugated anti-rabbit, anti-rat, or anti-mouse secondary
antibodies diluted in PGS-G were incubated on the filters for2hat
30 °C in a humidified chamber. The filters were then washed four times
with PBS-G and mounted on glass slides with ProLong Antifade mount-
ing medium (Molecular Probes, Inc., Eugene, OR). Immunofluorescence
images were obtained with a confocal microscope.
RESULTS
In an effort to identify proteins in the kidney that interact
with the MAGUK protein MAGI-1, we screened a cDNA ex-
pression library made from a glomerulus-enriched preparation
of mouse kidneys. The library was probed with a radiolabeled
GST fusion protein containing both WW domains of mouse
MAGI-1. Screening a total of ⬃1⫻10
6
plaque-forming units
with the probe resulted in the initial isolation of 11 independ-
ent clones that survived additional rounds of selection and
purification. Sequencing of the cDNA inserts from the 11 clones
showed that five of them represented the extreme C terminus
of

-dystroglycan; four exhibited high identity to the human
cDNA KIAA0989; and the last two were single isolates of WBP2
and synaptopodin, resulting in a total of four distinct protein
fragments. Although the inserts contain partial cDNAs, all but
one of the four encoded proteins have PY motifs as expected
(the protein showing a high identity to KIAA0989 lacked a
conventional PY motif). Both synaptopodin and dystroglycan
are known to be present in podocytes and could potentially
serve as in vivo binding partners for MAGI-1. However, be-
cause we have found MAGI-1 in the Triton X-100-insoluble
fraction of glomerulus-enriched preparations (11), which sug-
gests an association with the actin cytoskeleton, we chose to
focus our attention on synaptopodin at this time. The expres-
sion clone of mouse synaptopodin (clone 11.2) contained a re-
gion of this protein encompassing amino acids 255–379 and
harboring two potential WW domain-binding PY motifs. These
two PY motifs are conserved in human synaptopodin, suggest-
ing that they may have some physiological relevance.
In parallel with the screening of the glomerular expression
library, we had PCR-amplified a region of human synaptopodin
containing its PY motifs (amino acids 294 –350) and fused it to
GST to perform analysis on proteins that could potentially bind
to the WW domains of MAGI-1. This fusion protein, GST-
synpoPY, was then used in a pull-down assay with lysates from
HEK293 cells expressing a Myc-tagged portion of MAGI-1 con-
taining only its two WW domains, Myc-MAGI-1 WW12. We
found that the GST-synpoPY fusion protein was able to pull-
down Myc-MAGI-1 WW12, whereas GST alone could not (Fig.
1A). In addition, GST-synpoPY was also capable of pulling
down a Myc-tagged full-length MAGI-1 construct expressed in
HEK293 cells (Fig. 1B) as well as endogenous MAGI-1 from
mouse brain lysates (Fig. 1C). The multiple bands observed for
MAGI-1 in mouse brain lysates are most likely due to different
forms of the protein that we (11) and others (19) have observed
previously in brain and other tissues as well. A protein whose
WW domains are very similar to those of MAGI-1 is the ubiq-
uitin-protein ligase Nedd4. We expressed a T7-tagged full-
length Nedd4 construct in HEK293 cells and tested its ability
to interact with the GST-synpoPY fusion protein. GST-syn-
poPY was unable to pull-down Nedd4 from cell lysates in this
assay (Fig. 1D), indicating that the PY motifs of synaptopodin
do not interact with any of the three WW domains of this Nedd4
construct. In reciprocal experiments, the GST-MAGI-1 WW12
fusion protein that was used as a probe to screen the glomer-
ular cDNA library was utilized in GST pull-down assays with
lysates from HEK293 cells expressing a HA-tagged full-length
mouse synaptopodin construct. As shown in Fig. 1E, GST-
MAGI-1 WW12 could pull-down HA-synaptopodin from the
lysates, whereas GST alone could not. In addition, using the
same assay on mouse brain lysates, we found that GST-
MAGI-1 WW12 could pull-down endogenous synaptopodin (Fig.
1F). We have observed that lysates from brain contain a form of
synaptopodin that is much more stable than the one found in
glomerular lysates; and therefore, brain lysates were used as
our source of synaptopodin in these binding assays. These data
suggest that a region in synaptopodin containing its two con-
served PY motifs can interact with the WW domains in
MAGI-1. Moreover, this interaction appears to be somewhat
specific, as this region of synaptopodin is unable to interact
with Nedd4, whose WW domains show a high degree of simi-
larity to those in MAGI-1.
Because MAGI-1 contains two WW domains and synaptopo-
din harbors two PY motifs that are relatively close together, it
is conceivable that both WW domains of MAGI-1 bind to the
two PY motifs of synaptopodin, thereby providing an enhanced
interaction, or alternatively, that only one WW domain of
MAGI-1 is responsible for this interaction. To address this
possibility, we performed a Farwestern assay using the WW
domains of MAGI-1 together or by themselves as
32
P-labeled
probes (Fig. 2A). As expected, the probe with both WW domains
of MAGI-1 bound to the GST-synpoPY fusion protein (Fig. 2B,
second lane). When the second WW domain of MAGI-1 was
used as a probe, nearly the same degree of binding to GST-
synpoPY was observed (Fig. 2B,sixth lane). In contrast, the
degree to which the first WW domain bound to GST-synpoPY
was significantly less and is most likely only residual in nature
MAGI-1 Interacts with Actin-associated Proteins 30185
(Fig. 2B,fourth lane). None of the three probes bound to GST
alone (Fig. 2B,first,third, and fifth lanes). Taken together,
these data indicate that MAGI-1 and synaptopodin interact
with each other in a direct manner in vitro and that the second
WW domain of MAGI-1 specifically mediates this interaction.
To investigate whether MAGI-1 and synaptopodin interact
in an in vivo situation, HEK293 cells were transfected with
full-length expression constructs of Myc-tagged MAGI-1 and
HA-tagged synaptopodin either alone or together, and the re-
sulting cell lysates were used in co-immunoprecipitation as-
says. Using anti-Myc antibodies to precipitate MAGI-1 from
the cell lysates, we found that synaptopodin was precipitated
along with MAGI-1 in cells transfected with both expression
constructs (Fig. 2C), indicating that these two proteins are able
to interact together in cells.
We have previously shown that MAGI-1 is found in Triton
X-100-insoluble fractions of mouse glomerular preparations
(11), which is suggestive of MAGI-1 association with the actin
cytoskeleton. Although we have not ascertained whether
MAGI-1 is present in lipid rafts, the above finding that MAGI-1
interacts with the actin-bundling protein synaptopodin lends
support to its actin cytoskeletal association as the reason for its
Triton X-100 insolubility. An additional actin-bundling protein
expressed in the glomerular podocytes is one of the non-muscle
isoforms of
␣
-actinin, viz.
␣
-actinin-4. Recent data provide ev-
idence that mutations in the gene for
␣
-actinin-4 cause a he-
reditary form of FSGS in humans. In addition, both non-muscle
isoforms of
␣
-actinin,
␣
-actinin-1 and
␣
-actinin-4, contain a
consensus binding motif for PDZ domains (ESDL) at their
extreme C termini. These data and observations provided the
impetus to determine whether
␣
-actinin-4 could interact with
any of the PDZ domains of MAGI-1. For our initial binding
assay, the last 19 amino acids of
␣
-actinin-4 (Fig. 3A) were
fused to GST and used in pull-down assays with lysates of
HEK293 cells expressing different Myc-tagged constructs of
MAGI-1. The GST-
␣
-actinin-4 fusion protein was able to effi-
ciently pull-down full-length MAGI-1, whereas GST alone was
not (Fig. 3B). Of the MAGI-1 deletion constructs tested, only
FIG.2. Farwestern and co-immunoprecipitation analyses for
interactions of MAGI-1 with synaptopodin. A, shown is a diagram
illustrating the GST-MAGI-1 constructs used as radiolabeled probes.
Filled boxes denote the WW domains of MAGI-1 and are indicated as
such above each. Probe 1, GST-MAGI-1 WW12; Probe 2, GST-MAGI-1
WW1; Probe 3, GST-MAGI-1 WW2. B, 100 ng of GST (first,third, and
fifth lanes) and 100 ng of GST-synpoPY (second,fourth, and sixth lanes)
were resolved on a 12% SDS-polyacrylamide gel and then transferred to
a nitrocellulose membrane. The membrane was cut into three strips
(each strip containing a lane of GST and GST-synpoPY), which were
blocked in Farwestern buffer and then incubated with separate radio-
labeled probes. After washing, each strip was exposed to x-ray film at
⫺70 °C. The probed used on each strip is indicated at the bottom of the
autoradiograph. Molecular mass markers (in kilodaltons) are indicated
on the left. C, lysates from HEK293 cells transiently transfected with
full-length (FL) HA-synaptopodin and Myc-MAGI-1 either alone or
together were incubated with anti-Myc antibodies and protein A-Sepha-
rose beads. Proteins bound to the beads were eluted off, separated on a
7% SDS-polyacrylamide gel, and blotted onto a nitrocellulose mem-
brane. The blot was developed with anti-Myc or anti-HA antibodies and
visualized with chemiluminescence reagents. Input control lysate lanes
(lower two panels) represent 5% of the amount that was used in the
immunoprecipitation (IP).
FIG.1. GST pull-down assays involving MAGI-1 and synaptopodin. Lysates of HEK293 cells expressing epitope-tagged constructs of
MAGI-1, synaptopodin, or Nedd4 and lysates of mouse brain were incubated with GST and the GST-synpoPY and GST-MAGI WW12 fusion
proteins as indicated. The resulting blots were incubated with anti-Myc, anti-MAGI-1 (UM209), anti-T7, anti-HA, or anti-synaptopodin (NT)
antibody and the appropriate secondary antibodies and then developed with chemiluminescence reagents. The following lysates were used:
HEK293 cells expressing Myc-MAGI-1 WW12 (A), Myc-tagged full-length MAGI-1 (myc-MAGI-1 FL)(B), T-Nedd4 (D), and HA-synaptopodin (E)
and mouse brain for endogenous (endo) proteins (Cand F). Lysate lanes represent 10% of that which was used in the pull-down assays. Molecular
mass markers (in kilodaltons) are indicated on the left of each panel.
MAGI-1 Interacts with Actin-associated Proteins30186
those with an intact fifth PDZ domain retained binding to
GST-
␣
-actinin-4 (Fig. 3B). We next performed the reciprocal
experiment, in which the MAGI-1 construct containing only the
fifth PDZ domain was fused to GST (GST-MAGI-1 PDZ5), and
used this fusion protein in pull-down assays with HEK293 cells
expressing a HA-tagged full-length
␣
-actinin-4 construct. GST-
MAGI-1 PDZ5 was sufficient to pull-down HA-
␣
-actinin-4,
whereas GST alone was not (Fig. 4A). In addition, we found
that GST-MAGI-1 PDZ5 was able to pull-down endogenous
␣
-actinin-4 from lysates made from mouse glomerulus-enriched
preparations (Fig. 4B). As with synaptopodin and MAGI-1, we
wished to explore whether
␣
-actinin-4 and MAGI-1 could form
a complex when expressed together exogenously in HEK293
cells. Lysates from cells expressing a Myc-tagged full-length
MAGI-1 construct and a HA-tagged full-length
␣
-actinin-4 con-
struct either alone or together were used in immunoprecipita-
tion assays with anti-Myc antibodies. As shown in Fig. 4C,
HA-
␣
-actinin-4 can be precipitated along with Myc-MAGI-1
from lysates expressing both constructs, but not from lysates
expressing either construct alone. Together, these data
strongly suggest that an additional actin-bundling protein (
␣
-
actinin-4) is able to bind to the MAGUK protein MAGI-1.
The data presented thus far using in vitro binding assays
and co-immunoprecipitation analyses indicate that MAGI-1 is
able to bind to two distinct actin-bundling proteins, synaptopo-
din and
␣
-actinin-4. To further strengthen these observations,
we stably expressed full-length HA-synaptopodin in Tet-Off
MDCK cells and HA-
␣
-actinin-4 in regular MDCK cells to
determine whether they would colocalize with endogenous
MAGI-1 in the cells. When normal MDCK cells were stained
with an antibody against MAGI-1 and ZO-1, we found that
there was complete colocalization of the two endogenous pro-
teins (Fig. 5C), confirming the previous results of others (22)
that MAGI-1 is a tight junction-associated protein in these
cells. MDCK cells stably expressing HA-
␣
-actinin-4 showed a
localization pattern that was found all along the lateral mem-
brane of most cells that extended up to and overlapped with
MAGI-1 at the tight junction (Fig. 5, Hand I). This localization
pattern mimics the pattern observed for endogenous
␣
-acti-
nin-1 in these cells.
2
Because our initial attempts to express
HA-synaptopodin in regular MDCK cells were unsuccessful, we
chose to express this construct in the Tet-Off MDCK inducible
system. When stably transfected cells of this system are main-
2
K. M. Patrie and B. Margolis, unpublished data.
FIG.3.The cytoplasmic tail of
␣
-actinin-4 binds to the fifth PDZ domain of MAGI-1. A, illustration of the Myc-tagged MAGI-1 constructs
used for the analysis of
␣
-actinin-4 binding. The different domains in MAGI-1 are indicated above the full-length (FL) construct. The amino acid
sequence of the
␣
-actinin-4 C-terminal tail that was fused to GST is shown below the MAGI-1 constructs (the potential PDZ domain-binding motif
is in boldface letters). GuK, guanylate kinase. B, lysates from HEK293 cells expressing the Myc-tagged constructs of MAGI-1 were incubated with
GST or GST-
␣
-actinin-4 (GST-
␣
-act4) as described under “Materials and Methods.”The resulting blots were incubated with anti-Myc primary and
horseradish peroxidase-conjugated sheep anti-mouse secondary antibodies and then developed with chemiluminescence reagents. Input control
lysate lanes represent 10% of the amount that was used in the pull-down assays. The Myc-tagged MAGI-1 constructs used for each pull-down assay
are indicated below the appropriate blot. Molecular mass markers (in kilodaltons) are indicated on the left of each panel.
MAGI-1 Interacts with Actin-associated Proteins 30187
tained in the presence of doxycycline, the construct of interest
is not expressed, thereby avoiding complications such as toxic-
ity or instability observed in constitutive expressing systems.
Tet-Off MDCK cells stably transfected with HA-synaptopodin
and maintained in the absence of doxycycline expressed syn-
aptopodin in a pattern very similar to that observed with
␣
-ac-
tinin-4 (Fig. 5, Eand F). Synaptopodin was seen to partially
colocalize with MAGI-1 at the tight junction of cells. The over-
lapping expression of synaptopodin and
␣
-actinin-4 with
MAGI-1 at the tight junctions of MDCK cells supports the
binding data that MAGI-1 can interact with these two actin-
bundling proteins and that these interactions may have some
biological significance.
DISCUSSION
The complex morphology observed for the renal podocyte is
crucial for its function in helping to establish the glomerular
filtration barrier. Although this morphology is well character-
ized at the light and electron microscope level, its establish-
ment and regulation at the molecular level are only slowly
being realized (23). We have recently shown that the MAGUK
protein MAGI-1 is found in the glomerular podocytes of rat
kidneys (11). MAGI-1, like most MAGUK proteins that contain
numerous protein-protein interaction domains, is envisioned as
filling a scaffolding role in cells that would facilitate the nucle-
ation of a multiprotein complex. In an effort to identify proteins
in the glomerulus that interact with MAGI-1, we screened a
cDNA expression library made from glomerulus-enriched prep-
arations of mouse kidneys with a probe containing both WW
domains of MAGI-1. One clone that was isolated in this screen
contained a region of synaptopodin harboring two PY motifs,
which are potential binding sites for Group I WW domains.
Additional in vitro and in vivo binding data using full-length
expression constructs as well as endogenous proteins con-
firmed this interaction. Therefore, two independent approaches
to investigating protein-protein interactions reveal the direct
association of synaptopodin with MAGI-1. We have provided
additional evidence that this interaction is biologically relevant
by showing that a full-length synaptopodin exogenously ex-
pressed in MDCK cells partially colocalized with endogenous
MAGI-1 at tight junctions. Of the two WW domains present in
MAGI-1, we found that the second, or C-terminal, WW domain
is responsible for mediating the interaction with synaptopodin.
Although both PY motifs of synaptopodin share the consensus
PPXY sequence at their core, we have yet to establish to which
PY motif of synaptopodin MAGI-1 preferentially binds or if
both PY motifs can serve as binding sites for MAGI-1. Because
the amino acids flanking the two core PPXY sequences of
synaptopodin are quite different, they may contribute to the
specificity of these PY motifs in binding to different WW do-
mains. Initially, synaptopodin was found only in the neurons of
the telencephalon-derived regions of the brain and glomerular
podocytes of the kidney; but more recently, it has been found in
other tissues as well (24). MAGI-1 has been found in most
tissues examined (19), making it likely that synaptopodin is a
common binding partner for MAGI-1 in those tissues express-
ing both proteins. Although this does not exclude the possibility
that other proteins may bind to the second WW domain of
MAGI-1 when synaptopodin is absent, to date, no other pro-
teins have been reported to bind this protein interaction do-
main of MAGI-1. With synaptopodin as a binding partner for
the second WW domain of MAGI-1, the first WW domain would
be available to bind to a protein yet to be identified.
The recent discovery of mutations in the gene for
␣
-actinin-4
that cause a hereditary form of autosomal dominant FSGS (8)
provides a link between the actin cytoskeleton and disease in
the kidney. Interestingly, the FSGS-associated mutations in
␣
-actinin-4 occur between the actin-binding domain and the
first rod domain of the protein and cause an increase in
␣
-ac-
tinin-4 association with F-actin in co-sedimentation assays.
These
␣
-actinin-4 mutations would not be expected to affect
MAGI-1 binding to
␣
-actinin-4, but instead would presumably
increase MAGI-1 association with the actin cytoskeleton. This
increase in MAGI-1 association with the actin cytoskeleton
could have an effect on the dynamics of actin cytoskeleton
regulation by increasing the local concentration of potential
MAGI-1-binding proteins that are known to have an effect on
actin cytoskeleton dynamics (see below). We also have shown
here that
␣
-actinin-4 exogenously expressed in MDCK cells
partially localized at the tight junction along with MAGI-1. In
addition, we have observed endogenous
␣
-actinin (
␣
-actinin-1)
in MDCK cells to be localized all along the lateral membrane
border stretching up to and overlapping with the tight junction,
as is seen with
␣
-actinin-4. Of the 19 amino acids from
␣
-acti-
nin-4 that were used as a GST fusion protein in this study, 18
are identical to the other non-muscle isoform
␣
-actinin-1, in-
cluding the PDZ domain-binding motif. It was not surprising
FIG.4. GST pull-down assays and co-immunoprecipitation of
␣
-actinin-4. A, lysates from HEK293 cells transiently transfected with
a HA-
␣
-actinin-4 construct were incubated with GST alone or GST-
MAGI-1 PDZ5 and processed as described under “Materials and Meth-
ods.”The resulting blot was incubated with an anti-HA antibody and
the appropriate horseradish peroxidase-conjugated secondary antibody.
Input control lysate lanes represent 10% of the amount that was used
in the pull-down assay. B, lysates from glomerulus-enriched prepara-
tions of mouse kidney were incubated with GST alone or GST-MAGI-1
PDZ5 and processed as described under “Materials and Methods.”The
resulting blot was incubated with an anti-
␣
-actinin-4 antibody (NCC-
Lu-632) and the appropriate horseradish peroxidase-conjugated sec-
ondary antibody. Input control lysate lanes represent 10% of the
amount that was used in the pull-down assay. Molecular mass markers
(in kilodaltons) are indicated on the left of each panel. C, lysates of
HEK293 cells transfected with full-length Myc-MAGI-1, full-length HA-
␣
-actinin-4, or both constructs were incubated with anti-Myc antibodies
and protein A-Sepharose beads. Proteins bound to the beads were
eluted off, separated on a 7% SDS-polyacrylamide gel, and blotted onto
a nitrocellulose membrane. The blot was developed with anti-Myc or
anti-HA antibodies and visualized with chemiluminescence reagents.
Input control lysate lanes (lower two panels) represent 5% of the
amount that was used in the immunoprecipitations (IP).
MAGI-1 Interacts with Actin-associated Proteins30188
therefore to find that a GST fusion protein containing only the
fifth PDZ domain of MAGI-1 was able to pull-down endogenous
␣
-actinin-1 from lysates of HEK293 cells (data not shown).
MAGI-1 appears to be capable of binding to both non-muscle
isoforms of
␣
-actinin. Our previous results showing MAGI-1 in
the Triton X-100-insoluble fraction of membranes from mouse
glomerular preparations suggests a cytoskeletal association of
MAGI-1 (11). Alternatively, this observed insolubility could
also be due to the association of MAGI-1 with detergent-resis-
tant microdomains, or lipid rafts. However, our finding of
MAGI-1 interaction with the two actin-binding proteins
␣
-ac-
tinin and synaptopodin suggests an association with the actin
cytoskeleton as the reason for its resistance to extraction with
Triton X-100.
Although the distinct subcellular localization of MAGI pro-
teins in tissues is somewhat limited at this time, MAGI-1 was
found localized at the tight junctions in intestinal epithelium
using immunoelectron microscopy (22). It was also shown that
the localization of endogenous MAGI-1 in MDCK cells overlaps
perfectly with the localization of the tight junction protein
ZO-1, a finding that we confirmed in this study. Our data
provide additional evidence that MAGI-1 provides a link from
membrane-associated protein complexes to the actin cytoskel-
eton in epithelial cells by way of its interaction with
␣
-actinin
and/or synaptopodin. This indirect association of MAGI-1 with
the actin cytoskeleton appears to be an increasingly evident
phenomenon among MAGUK proteins. For example, the hu-
man homolog of Drosophila DLG (discs large) and CASK bind
to protein 4.1, a member of the FERM protein family that binds
to actin (25, 26). Moreover, a direct association with the actin
cytoskeleton is seen with the MAGUK protein ZO-1 (27).
It is apparent that the morphological changes observed in
the foot processes of podocytes in the nephrotic syndrome are
dependent on the reorganization of the actin-based cytoskele-
ton. Understanding the regulation of the actin cytoskeleton in
podocytes is therefore crucial. The finding of MAGI-1 at mem-
brane-associated complexes in epithelial cells suggests a model
in which MAGI-1 would be localized at the membrane of foot
processes and tethered to the actin cytoskeleton by way of its
interaction with synaptopodin and
␣
-actinin-4. Although the
localization of MAGI-1 in podocytes at the ultrastructural level
is not currently available, we feel that MAGI-1 could be local-
ized in a manner that would at least partially overlap with
synaptopodin and
␣
-actinin-4. In addition to actin and
␣
-acti-
nin, the microfilament contractile apparatus of podocyte foot
processes is also composed of myosin II, talin, and vinculin,
which extends down to and is linked with the glomerular base-
ment membrane by an
␣
3

1
integrin- and
␣
/

-dystroglycan-
based electron-dense protein complex called the sole plate (3,
28, 29). Like the localization of
␣
-actinin at integrin-based
protein complexes in cultured cells,
␣
-actinin-4 is observed to
be partially localized at the sole plate (3, 30). Although
␣
-acti-
nin-4 itself can provide a link from the sole plate protein
complex to the actin cytoskeleton, its interaction with MAGI-1
FIG.5. Colocalization of exog-
enously expressed
␣
-actinin-4 and
synaptopodin with endogenous
MAGI-1 in MDCK cells. Wild-type
MDCK cells (A–C) and MDCK cells stably
expressing HA-synaptopodin (D–F)or
HA-
␣
-actinin-4 (G–I) were stained with
an anti-MAGI-1 polyclonal antibody (A,
D, and G)(green), an anti-ZO-1 mono-
clonal antibody (B)(red), or a rat anti-HA
monoclonal antibody (Eand H)(red). The
merged image of each co-staining is de-
picted in C,F, and I. Below each X-Y
panel is the X-Z plane.
MAGI-1 Interacts with Actin-associated Proteins 30189
would provide an additional link to the actin microfilament
array via synaptopodin. Alternatively, it is plausible that
MAGI-1 is providing a platform for regulators of actin dynam-
ics in the foot process instead of merely playing an additional
passive structural link between the actin cytoskeleton and the
membrane. It is well established that integrin-based focal con-
tacts in cell culture provide a signaling link from the substrate-
contacting plasma membrane to the actin cytoskeleton and are
regulated by the Rho family of small GTPases. Interestingly,
the guanine nucleotide exchange factor mouse NET1 has re-
cently been identified as a binding partner for the first PDZ
domain of MAGI-1 (31). Mouse NET1 activates RhoA and the
stress-activated protein kinase/c-Jun N-terminal kinase sig-
naling pathways (32). RhoA activation stimulates actomyosin-
based contractility, which contributes to the assembly of stress
fibers and focal contacts (33, 34). Additionally, the tumor sup-
pressor PTEN has been shown to bind to the second PDZ
domain of all three MAGI proteins (35, 36) and is implicated in
focal contact assembly by antagonizing the phosphatidylinosi-
tol 3⬘-kinase signaling pathway. However, the precise localiza-
tion of mouse NET1 and PTEN within the kidney is not known
at this time.
The precise mechanism by which MAGI-1 associates with
distinct plasma membrane subdomains or what function it may
serve there is speculative at this time. Expression of the last
two PDZ domains (PDZ4 and PDZ5) of MAGI-1 as a GFP fusion
protein in normal rat kidney cells is sufficient to target the
fusion protein to the lateral plasma membrane (37). Further-
more, when a mutant MAGI-1 construct that lacks its fifth PDZ
is expressed in MDCK cells, the mutant protein no longer is
found in the membrane fraction, as is the wild-type protein, but
is instead exclusively observed in the cytosolic fraction (38). We
have generated a point mutation in the fifth PDZ domain of
MAGI-1 that abolished binding of cognate ligands to this do-
main and expressed this in the context of the full-length
MAGI-1 protein within MDCK cells. This MAGI-1 mutant ex-
hibited neither tight junction nor plasma membrane localiza-
tion compared with the wild-type protein and was found
throughout the cell (data not shown). This confirms that the
fifth PDZ domain is required for proper localization of MAGI-1
to the plasma membrane, but it is not clear if the fifth PDZ
domain alone is sufficient to properly target MAGI-1 to the
tight junction of MDCK cells once it is at the membrane. We are
not sure at this time whether the interaction of MAGI-1 with
␣
-actinin-4 (or other proteins known to bind to the fifth PDZ
domain of MAGI-1) is solely responsible for the plasma mem-
brane localization of MAGI-1 in MDCK cells. We are currently
investigating the potential involvement of the other protein
interaction domains of MAGI-1 regarding their role in proper
membrane localization.
The identification of the proteins in renal podocytes and
other cells that bind to MAGI-1, whether they are integral
transmembrane proteins or peripheral proteins, will undoubt-
edly help reveal the function of MAGI-1. This proteomic ap-
proach combined with transgenic technology will help provide
an understanding of the role that MAGUK proteins play in the
function and regulation of podocyte dynamics.
Acknowledgments—We thank Chia-Jen (Albert) Liu for expert as-
sistance with the confocal microscopy work, Dr. Daniela Rotin for the
T-Nedd4 cDNA construct, Dr. Steven R. Vincent for the
␣
-actinin-4
cDNA, and Dr. Tesshi Yamada for the anti-
␣
-actinin-4 monoclonal
antibody NCC-Lu-632.
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MAGI-1 Interacts with Actin-associated Proteins30190
