Báo cáo khoa học: Characterization of the interactions of the nephrin intracellular domain Evidence that the scaffolding protein IQGAP1 associates with nephrin potx

Characterization of the interactions of the nephrin
intracellular domain
Evidence that the scaffolding protein IQGAP1 associates
with nephrin
Xiao Li Liu
1
, Pekka Kilpela
¨
inen
1
, Ulf Hellman
3
, Yi Sun
1
, Jorma Wartiovaara
4
, Ekaterina Morgunova
1
,
Timo Pikkarainen
1
, Kunimasa Yan
5
, Anders P. Jonsson
2
and Karl Tryggvason
1
1 Divisions of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden
2 Medical Chemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden
3 Ludwig Institute for Cancer Research, Uppsala, Sweden
4 Electron Microscopy Unit, Institute of Biotechnology, University of Helsinki, Finland

5 Department of Pediatrics, Kyorin University School of Medicine, Mitaka, Tokyo, Japan
Keywords
Fyn, IQGAP1, phosphoinositide 3-kinase,
podocyte, slit diaphragm
Correspondence
K. Tryggvason, Karolinska Institutet
Department of Medical Biochemistry and
Biophysics, Division of Matrix Biology,
Scheeles Va
¨
g 2 B1, Plan 4 SE-17177,
Stockholm, Sweden
Fax: +46 8 316165
Tel: +46 8 5248 7720
E-mail:
(Received 22 July 2004, revised 21 September
2004, accepted 22 September 2004)
doi:10.1111/j.1432-1033.2004.04408.x
Nephrin is a signalling cell–cell adhesion protein of the Ig superfamily and
the first identified component of the slit diaphragm that forms the critical
and ultimate part of the glomerular ultrafiltration barrier. The extracellular
domains of the nephrin molecules form a network of homophilic and
heterophilic interactions building the structural scaffold of the slit dia-
phragm between the podocyte foot processes. The intracellular domain of
nephrin is connected indirectly to the actin cytoskeleton, is tyrosine phos-
phorylated, and mediates signalling from the slit diaphragm into the podo-
cytes. CD2AP, podocin, Fyn kinase, and phosphoinositide 3-kinase are
reported intracellular interacting partners of nephrin, although the biologi-
cal roles of these interactions are unclarified. To characterize the structural
properties and protein–protein interactions of the nephrin intracellular

domain, we produced a series of recombinant nephrin proteins. These were
able to bind all previously identified ligands, although the interaction with
CD2AP appeared to be of extremely low stoichiometry. Fyn phosphory-
lated nephrin proteins efficiently in vitro. This phosphorylation was required
for the binding of phosphoinositide 3-kinase, and significantly enhanced
binding of Fyn itself. A protein of 190 kDa was found to associate with
the immobilized glutathione S-transferase–nephrin. Peptide mass finger-
printing and amino acid sequencing identified this protein as IQGAP1, an
effector protein of small GTPases Rac1 and Cdc42 and a putative regula-
tor of cell–cell adherens junctions. IQGAP1 is expressed in podocytes at
significant levels, and could be found at the immediate vicinity of the slit
diaphragm. However, further studies are needed to confirm the biological
significance of this interaction and its occurrence in vivo.
Abbreviations
CD2AP, CD2-associated protein; CHAPS, 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate; FITC, fluorescein-isothiocyanate;
HEK293, human epidermal kidney cells; HT1080, human fibrosarcoma; HT1080 L, mouse fibroblast; pAb 2, polyclonal antibody 2; PI 3,
phosphoinositide 3.
228 FEBS Journal 272 (2005) 228–243 ª 2004 FEBS
We have seen in recent years the rapid unravelling of
the molecular components of the slit diaphragm, a spe-
cialized structure connecting the podocyte foot proces-
ses. An important finding in this regard was the
identification of nephrin as a gene mutated in congen-
ital nephrosis NPHS1 [1,2]. NPHS1 patients show a
massive proteinuria starting in utero and their podo-
cyte foot processes are effaced. Identical symptoms can
be brought about in mice by inactivating the nephrin
gene by homologous recombination [3]. These mice die
from renal failure within 24 h after birth, which under-
lines the role of nephrin as a key component of the slit

diaphragm.
The nephrin protein has an apparent molecular mass
of 180 kDa [1]. The extracellular part of nephrin has
eight Ig-like modules, one fibronectin type III-like
module, and an unknown number of N-linked carbo-
hydrate moieties [4,5]. The intracellular domain of 156
amino acids has no homology with other known pro-
teins but contains nine tyrosine residues, some of
which are phosphorylated [6]. The domain structure
and biochemical properties of nephrin brought about
the immediate suggestion that nephrin molecules may
form dimers through homophilic interactions spanning
the slit diaphragm [1]. Very recently, our electron
tomography studies showed that the slit diaphragm is
a network of intertwined strands containing nephrin
[7]. Consistently, biochemical studies have indicated
that nephrin has an ability to form homo- and hetero-
dimers with NEPH1 [8–10], another component of
the slit diaphragm. NEPH1 belongs to the Ig super-
family, has five extracellular Ig domains, and its dele-
tion leads to heavy proteinuria and early postnatal
death [11]. P-cadherin and FAT, a large cadherin pro-
tein, have been localized at the slit diaphragm area or
close to it [12,13]. Their participation and role in the
formation of the slit diaphragm structure is not
known. Inactivation of the FAT gene in mice leads to
severe renal phenotype with the fusion of foot proces-
ses and loss of slit junctions [14], but P-cadherin-
knockout mice do not show any abnormalities in the
kidney [15,16].

The best-documented intracellular interaction part-
ner of nephrin is podocin [17,18]. It is a member of the
stomatin protein family and is predicted to form a
membrane-associated hairpin-like structure with cyto-
solic N- and C-terminal domains. Podocin has been
localized to the slit diaphragm area [19], and it is
mutated in a form of autosomal recessive familial focal
segmental glomerulosclerosis (SRN1) [20]. Podocin is
required for recruitment of nephrin into lipid rafts
[21], and podocin-knockout mice die from renal failure
within a few days of birth [22]. In addition to nephrin,
it interacts with the NEPH proteins and CD2-associ-
ated adaptor protein CD2AP [18,23]. Furthermore,
nephrin itself has been reported to coimmunoprecipi-
tate with CD2AP [24] that connects several membrane
proteins to the actin cytoskeleton [25]. The importance
of CD2AP for glomerular ultrafiltration is emphasized
by the fact that CD2AP-knockout mice develop neph-
rotic syndrome 1–2 weeks after birth that leads to
renal failure and death at 6–7 weeks of age [24]. More-
over, heterozygous CD2AP+ ⁄ –, mice are haploinsuffi-
cient and display glomerular changes at 9 month’s of
age with a histological pattern similar to that in
human focal segmental glomerulosclerosis [26].
CD2AP has been localized close to the attachment site
of the slit diaphragm [27], and its interaction with
nephrin has been examined also in vitro, although the
mapping of interacting domain for nephrin binding
resulted in controversial reports [27,28]. Studies
employing gradient centrifugation and immunofluores-

cence techniques have demonstrated that nephrin is
indeed either directly or indirectly associated with the
actin cytoskeleton [29–31].
Altered morphological characteristics of podocytes
such as foot process effacement are typical to many
experimental and human glomerulopathies [32,33]. All
these changes are reversible and recovery from these
anomalies can only be achieved by the reformation of
foot processes and slit diaphragms. Nephrin expression
levels and subcellular localization are affected in sev-
eral renal diseases and proteinuric diseases such as dia-
betes [34]. This implies that there has to be signalling
from the slit diaphragm into the podocytes, and vice
versa. Nephrin is very probaly a major player in this
signalling. Transfection of nephrin into HEK293 cells
activates protein kinase p38 and c-jun aminoterminal
kinase (JNK), thereby activating transcription factor
AP-1 [17]. Nephrin has been found to be dislocated to
the apical pole of the narrowed filtration slits and tyro-
sine phosphorylated, when the slit diaphragm is dis-
rupted by in vivo injection of antibodies recognizing a
podocyte-specific 9-O-acetylated GD3 ganglioside [6].
Fyn kinase has been shown to bind and phosphorylate
nephrin [35], and we have observed that tyrosine
phosphorylation of nephrin is induced robustly, when
nephrin is clustered on the cell surface, by using
anti-nephrin Igs [36]. In addition, a central signalling
molecule phosphoinositide 3 (PI 3)-kinase has been
reported to associate with nephrin [37].
These observations demonstrate how a complicated

network of interactions and signalling is needed to
organize and maintain the slit diaphragm. They also
indicate how important the elucidation of the protein–
protein interactions formed by nephrin are for the
X. L. Liu et al. Intracellular domain of nephrin
FEBS Journal 272 (2005) 228–243 ª 2004 FEBS 229
understanding of the function and regulation of the
glomerular ultrafiltration barrier. Here, as a step
towards this goal, we have studied the functions of the
nephrin intracellular domain. Utilizing recombinant
nephrin proteins, we analysed the requirements for the
interactions with the known intracellular partners. Fur-
thermore, we identified IQGAP1, an effector protein
of small GTPases Rac1 and Cdc42, as a potential new
interacting partner of nephrin. Nephrin and IQGAP1
were found to colocalize both in cultured cells and in
kidney sections, but the demonstration of the biologi-
cal significance requires further studies.
Results
The intracellular domain of mouse nephrin is com-
posed of 156 amino acids, of which 116 are conserved
in human and rat nephrin [38]. However, it is not
possible to show any homology to other known
proteins in databases. Nevertheless, there are several
putative docking and phosphorylation sites in neph-
rin (Table 1). Prediction of secondary structures
was carried out with several programs that were
able to recognize only two a-helical segments (http://
www.expasy.ch, />The first one, RRRLRRLAEE(1100–1110), follows
immediately the transmembrane domain, and was

detected by all programs employed, while the second,
EEDRIRNEY(1120–1128), was suggested by most
programs.
Using these in silico analyses as a background, we
proceeded to analyse the properties of the recombinant
intracellular domain. For these studies, we produced
and purified the intracellular domain without any tags,
as well as a series of glutathione S -transferase (GST)-
fusion proteins containing various parts of the intracel-
lular domain. The GST-fusion proteins migrated as
expected from their calculated molecular masses.
However, in particular, the GST–C terminus and
GST–nephrincyt were found to be partially degraded,
indicating that the C-terminal half of the intracellular
domain is sensitive to bacterial proteases (Fig. 1A).
Western blot analysis with the antibody pAb 2 gener-
ated against the intracellular domain of human neph-
rin confirmed that all polypeptides detected in Fig. 1A
originate from nephrin (results not shown). We first
tested whether these fusion proteins were able to pull-
down proteins found in previous studies to associate
with nephrin. As demonstrated in Fig. 1B, both podo-
cin and Fyn kinase readily associated with GST–C ter-
minus and GST–nephrincyt. CD2AP did not interact
with the fusion proteins in the initial experiments, and
we therefore investigated its association with nephrin
in various conditions. We were able to detect a weak,
occasional interaction in the lysis buffer containing 1%
(v ⁄ v) Chaps as a detergent, but not in 1% (v ⁄ v) digito-
nin, 1% (v⁄ v) NP-40, or 0.5% (v ⁄ v) Triton X-100.

Recently, we and others have reported that nephrin
is tyrosine phosphorylated, probably by Fyn kinase
in vivo [35,36]. Here we show direct phosphorylation
by Fyn kinase in vitro. Fyn was able to phosphory-
late the untagged full-length intracellular domain,
as well as all GST–nephrin fusion proteins indicating
that there are phosphorylation sites on both halves
of the intracellular domain (Fig. 2A). Furthermore,
several degradation fragments of GST–C terminus
Table 1. Amino acid sequence analysis of nephrin. Sequences presented are of mouse nephrin. Only sites conserved in human, mouse and
rat are mentioned.
Putative site Sequence in nephrin Consensus sequence and ⁄ or reference
Docking and phosphorylation site for Src kinase LYDEV(1207–1211) YE ⁄ D ⁄ TE ⁄ N ⁄ DI ⁄ V ⁄ M ⁄ L[42]
Docking site for Nck adaptor proteins LYDEV(1207–1211) YDEP ⁄ D ⁄ V [42]
Proline-rich sequence for SH3 domain binding PQLPP(1160–1164) PXXP [63]
a-Helical region RRRLRRLAEE(1100–1109)
EEDRIRNEY(1120–1128) />Repeated sequences GHLYDEVE(1188–1195) />GPLYDEVQ(1205–1212)
Protein kinase C phosphorylation site SEK(1112–1114) S ⁄ TXR ⁄ K
SMR(1155–1157) />Casein kinase II phosphorylation site STAE(1145–1148) S ⁄ TXD ⁄ E
SMRD(1155–1158) />TLEE(1166–1169)
Most probable tyrosine phosphorylation sites Y1153, Y1208, Y1225, Y1232 />Most probable serine or threonine
phosphorylation sites
S1119, S1142, S1145, S1155, S1160, S1171 />Intracellular domain of nephrin X. L. Liu et al.
230 FEBS Journal 272 (2005) 228–243 ª 2004 FEBS
were phosphorylated, whereas only the full-length
GST-1173 was phosphorylated at a detectable level.
Prolonged incubation or increased amount of Fyn
kinase did not significantly increase phosphorylation
suggesting that the in vitro phosphorylation was
efficient and close to quantitative (Fig. 2B). Next, we

wanted to investigate whether in vitro phosphorylation
provides new properties for the nephrin intracellular
domain. First, we examined whether the phosphoryl-
ated GST–nephrin fusion proteins were able to bind
phosphatidylinositol 3-kinase that has been reported
recently to associate with nephrin [37]. We found that
PI 3-kinase bound only to the phosphorylated GST–
nephrincyt and GST-1173 implying that one of the
SH2 domains in the p85 subunits of PI 3-kinase is
involved in the interaction (Fig. 2C). These findings
are also well in line with the prediction that the
segment YYSM(1153–1156) constitutes a docking site
for the SH2 domains of PI 3-kinase.
Having demonstrated the functionality of the phos-
phorylated GST–nephrin fusion proteins, we proceeded
to investigate how the phosphorylation effects on the
interaction of nephrin with Fyn and podocin. Interest-
ingly, we observed that the tyrosine phosphorylation
enhanced association of Fyn with nephrin, but did not
have any significant effect on the interaction with
podocin (Fig. 3).
Identification of IQGAP1 as a putative interacting
partner for nephrin
The above described findings encouraged us to utilize
GST–nephrincyt in a search for novel interactors of
nephrin. Indeed, incubation of metabolically labelled
podocyte, HEK293, HT1080, and L-cell lysates with
the fusion protein conjugated to the glutathione-Seph-
arose beads resulted in the strong and reproducible
binding of a 190 kDa protein, as demonstrated by ana-

lysing the eluates by SDS ⁄ PAGE and autoradiography
(Fig. 4A).
To identify the 190 kDa protein, it was purified in
quantities sufficient for peptide mapping by MALDI-
TOF-MS (Fig. 4B). Pooled fractions from three separ-
ate purifications were concentrated, and loaded on a
one-dimensional SDS ⁄ PAGE gel, which was then
stained by silver. The bands containing the 190 kDa
protein were excised, and the gel pieces treated as
described in Experimental procedures. After overnight
in-gel digestion with trypsin, the resulting peptide
mixture was analysed by MALDITOF-MS. Fifty-seven
peptide masses were determined, and used to search
the database. Up to 30 masses corresponded with com-
puted masses of tryptic peptides of mouse IQGAP1
(Table 2), a 189 kDa effector protein of small GTPases
Rac1 and Cdc42. These peptides covered 19% of the
mouse IQGAP1 sequence (GenBank accession number
AF240630). These results allowed an unequivocal iden-
tification of the 190 kDa protein as IQGAP1. In addi-
tion, we sequenced three of the mapped peptides and
two other peptides, which were not detected in the ori-
ginal MALDITOF-MS analysis. All obtained peptide
sequences could be positioned in the mouse IQGAP1
sequence, confirming the identification.
Fig. 1. The C-terminal half of the nephrin intracellular domain is pro-
tease-sensitive and interacts with Fyn and podocin. (A) GST fusion
proteins containing various parts of the nephrin intracellular domain
were produced and purified by standard methods in the presence
of protease inhibitors. The purified proteins were analysed by

SDS ⁄ PAGE and Coomassie Brilliant Blue-staining. (B) Cell lysates
prepared from parental HEK293 cells, or from those stably expres-
sing recombinant human podocin, were incubated with immobilized
GST fusion proteins, after which equal amounts of bound proteins
were separated on two SDS ⁄ PAGE gels. Gels were subjected
either to Western blot analysis with anti-Fyn or anti-podocin Igs, or
to Coomassie Brilliant Blue-staining to control the loading of the
GST proteins to the beads.
X. L. Liu et al. Intracellular domain of nephrin
FEBS Journal 272 (2005) 228–243 ª 2004 FEBS 231
Characterization of the IQGAP1–nephrin
interaction
IQGAP1 was purified from L-cells rather than podo-
cytes as L-cells proliferate more rapidly, and are there-
fore more convenient for a large-scale application. In
order to demonstrate that the 190 kDa protein bound
to GST–nephrincyt from the podocyte cell lysate was
IQGAP1, we carried out pull-down assays from these
lysates. The eluates were analysed by Western blotting
with anti-IQGAP1 Igs, and, as expected, a protein of
190 kDa recognized by the antibodies was found to
bind to GST–nephrincyt (Fig. 5B). We also used var-
ious deletion constructs to map the areas of the neph-
rin intracellular domain responsible for IQGAP1
binding. All constructs containing the C-terminal half
of the intracellular domain (amino acids 1167–1256)
bound IQGAP1, whereas we could not detect any
interaction between IQGAP1 and N-terminal half on
the nephrin intracellular domain, or between IQGAP1
and GST alone. The last 11 residues of the intracellu-

lar domain are fully conserved between human, mouse
and rat nephrin. However, a fusion protein lacking this
part did bind IQGAP1, indicating that the region was
not needed for IQGAP1 binding. We were not able to
convincingly demonstrate the nephrin – IQGAP1 inter-
action in NPH5 cells by the immunoprecipitation
method, as IQGAP1 tended to coimmunoprecipitate
also with the Finn-minor mutant form of nephrin lack-
ing nearly the whole intracellular domain. Tyrosine
phosphorylation of nephrin did not appear to effect
significantly on the in vitro interaction of GST–neph-
rins and IQGAP1 (Fig. 5B).
Nephrin and IQGAP1 colocalize in the HEK293
cell line expressing recombinant nephrin and
IQGAP1 can be found at the slit diaphragm
As the binding experiments described above were all
in vitro studies, and the immunoprecipitation experi-
ments did not give a definite demonstration of the
interaction in intact cells, we performed immunolocali-
zation studies to see whether the subcellular distribu-
tion of nephrin and IQGAP1 supports the possibility
that IQGAP1 is an intracellular binding partner of
nephrin. First, formaldehyde-fixed NPH5 cells were
double-labelled for nephrin and IQGAP1. IQGAP1 is
known to localize at the cortical cytoskeleton and
cell–cell adhesion sites [39,40]. The monoclonal anti-
IQGAP1 Ig was found to stain the cortical cytoskele-
ton in the NPH5 cells, and also diffusely the plasma
membrane (Fig. 6B). The strongest nephrin staining
was detected at the plasma membrane and the cortical

cytoskeleton, and due to overexpression, nephrin could
also be found in the cytoplasm (Fig. 6A). The expres-
sion patterns of IQGAP1 and nephrin were overlap-
ping (Fig. 6C).
Our pull-down and Western blotting experiments
indicated that IQGAP1 is expressed in podocytes at
significant levels (Fig. 4A; X. L. Liu & P. Kilpela
¨
inen,
unpublished observations). To examine the distribution
of IQGAP1 in podocytes in detail, we carried out
Fig. 2. The intracellular domain of nephrin is efficiently phosphorylated by Fyn kinase in vitro. Phosphorylation of a tyrosine residue within
the N-terminal half of the domain generates a binding site for phosphoinositide 3-kinase. (A) After in vitro phosphorylation of the different
nephrin proteins with a commercial Fyn kinase preparate,  0.5 lg of each protein was analysed by SDS ⁄ PAGE and Western blotting with
anti-phosphotyrosine Igs. (B) The untagged full-length intracellular domain of nephrin was in vitro phosphorylated with the Fyn kinase prepa-
rate for varying incubation times or by using different amounts of the kinase. The results demonstrate that already incubation with 1·
amount of the kinase preparate for 90 min (conditions used in the panel A experiment) results in the efficient phosphorylation of the fusion
protein. (C) Equal amounts of immobilized, nonphosphorylated and phosphorylated GST fusion proteins were incubated with HEK293 lysates,
after which bound proteins were analysed by SDS ⁄ PAGE and Western blotting with a monoclonal anti-PI 3-kinase Ig. An identical gel was
stained with Coomassie Brilliant Blue. A third gel with same samples was analysed by Western blotting with anti-phosphotyrosine Igs.
Intracellular domain of nephrin X. L. Liu et al.
232 FEBS Journal 272 (2005) 228–243 ª 2004 FEBS
immunoelectron microscopic studies of human kidney
sections (Fig. 6D–F). In immunogold labelling, IQ-
GAP1 was distributed in podocyte foot-processes
exclusively intracellularly and the cytoplasmic aspect
of the slit diaphragm or its vicinity was often notably
labelled, whereas the label was rarely found near to
other sites of the podocyte surface (Fig. 6D,E). In the
cytoplasm the label was quite often found in densities

(Fig. 6F) that could correspond to the endoplasmic reti-
culum better resolved in well-fixed nonimmunosamples.
Immunogold staining with the anti-nephrin Ig pAb 2
gave fairly abundant labelling that was located like the
label for IQGAP1 but was concentrated to the slit
diaphragm region and not found at other membrane
sites (results not shown).
Discussion
The work that resulted in the identification of the neph-
rin gene as a gene mutated in congenital nephrotic syn-
drome of Finnish type (NPHS1) [2,41], demonstrated
also the crucial role of the nephrin intracellular domain
for the structure of the slit diaphragm. A number of
mutations or deletions in the intracellular domain lead
to severe NPHS1. Analysis of the amino acid sequence
reveals that this domain is significantly conserved
between different species [38], but it does not show any
homology to other known proteins. It contains a few
very typical consensus sequences found in the most
proteins, such as two putative phosphorylation sites
for protein kinase C and three for casein kinase II.
However, a much more conspicuous and significant
feature are the six conserved tyrosines found in human,
mouse and rat [38], some of which (Tyr1191, Tyr1208
and Tyr1232, numbering according to mouse sequence)
match with the consensus sequence of Src family kin-
ase and Nck adaptor protein SH2 domain docking site
[42]. These sites correspond also to Src family kinase
phosphorylation sites. It is worth pointing out that the
amino acid sequences around these tyrosines are indeed

very similar (LYDEV, LYDEV, IYDQV), and these
could be phosphorylated by the same kinase, for exam-
ple by Fyn. Phosphorylation at multiple tyrosines pro-
vides several docking sites, which could, for example,
result in the assembly of large protein complexes
involved in the organization and maintenance of the
slit diaphragm. As the three putative docking sites are
similar, one possibility is that such complexes could
contain oligomers of same ligand that adjust or lock
nephrin at the right location. Finally, as a curiosity,
Tyr1128 is flanked by the most conserved sequence
that can be found around the tyrosines in nephrin.
However, this site is an unorthodox substrate for non-
receptor tyrosine kinases as it has a negatively charged
residue at position )1 before Tyr1128 (EYEES) [43].
On the contrary, several receptor tyrosine kinases have
this kind of substrate sequences.
As the nephrin intracellular domain did not show
any homology to other known proteins, molecular
modelling was difficult, and we could only predict the
conformation for two short sequences that potentially
form a -helixes. These were both located close to the
N-terminus of the intracellular domain. When expres-
sing and purifying the bacterial recombinant proteins,
we noticed that the intracellular domain appears to
consist of two clearly separate parts, the N-terminal
Fig. 3. Tyrosine phosphorylation of nephrin enhances association
with Fyn, but does not have an effect on the interaction with podo-
cin. Cell lysates prepared from parental HEK293 cells, or from
those stably expressing recombinant human podocin, were incuba-

ted with equal amounts of nonphosphorylated and phosphorylated
GST proteins immobilized to the glutathione-Sepharose beads.
After extensive washings, bound proteins were analysed by
SDS ⁄ PAGE and Western blotting with anti-Fyn or anti-podocin Igs.
The anti-Fyn blot was reprobed with anti-nephrin Igs, and the
podocin pull-down samples were subjected to SDS ⁄ PAGE and
Coomassie Brilliant Blue-staining to show equal loading of the
phosphorylated and nonphosphorylated fusion proteins to the
beads. The eluates were also analysed by SDS ⁄ PAGE and Western
blotting with the anti-phosphotyrosine Igs.
X. L. Liu et al. Intracellular domain of nephrin
FEBS Journal 272 (2005) 228–243 ª 2004 FEBS 233
half (amino acids 1102–1173 in mouse nephrin) and
the C-terminal half (1167–1256). The N-terminal part
was resistant to proteolysis during production, purifi-
cation and storage implying that it may form a tightly
packed domain. On the contrary, the C-terminal half
was very sensitive to proteolysis occurring already dur-
ing production in Escherichia coli BL21. It contains
most of the putative consensus sites for tyrosine phos-
phorylation and protein–protein interactions, and as
we show in this study, all known interacting partners
of the intracellular domain except PI 3-kinase, bind to
this region. The C-terminal half may form a less
tightly folded domain that has a flexible structure. This
might be needed to create several interaction sites, or
to render the area more accessible to the interacting
proteins.
Nephrin has been found to be associated and likely
phosphorylated by Src family kinase Fyn in vivo

[35,36]. In this study, we showed that Fyn kinase
directly phosphorylates nephrin in vitro (Fig. 2A,B).
Phosphorylation was efficient and apparently close to
quantitative. Both N- and C-terminal halves were
phosphorylated. Phosphorylation at the N-terminal
part never reached the same intensity as that at the
C-terminal half implying that the anti-phosphotyrosine
Ig may detect phosphotyrosines in the C-terminus
more efficiently, or that the C-terminal half may be
more accessible to kinases, or simply that the C-ter-
minal half contains more phosphorylation sites than
the N-terminal half. The last possibility is the most
likely one, as the three highly potent tyrosine phos-
phorylation sites for Src family kinases, Tyr1191
Tyr1208 and Tyr1232, are all within this region. Fur-
thermore, judging from the phosphorylation of degra-
dation fragments, the C-terminal half contains at least
two phosphorylation sites.
Progress with the investigations of the nephrin intra-
cellular domain have been relatively rapid, and have to
date resulted in the identification of several putative
interaction partners for nephrin [17,18,24,35–37]. The
biological significance and function of some interactions
is obvious, whereas those of others are more difficult to
predict. Podocin is able to interact with nephrin,
NEPH1, and CD2AP, all components of the slit dia-
phragm [17,18,23]. It may function as a scaffolding pro-
tein, and it augments nephrin signalling by facilitating
recruitment of nephrin to the slit diaphragm area
[17,21]. An adapter protein CD2AP has been reported

to interact both with podocin and nephrin, and puta-
tively connects the slit diaphragm to the actin cytoskele-
ton [18,24,25]. Src family kinase Fyn phosphorylates
nephrin, and associates with it [35,36]. This phosphory-
lation may be critical for the integrity of the glomerular
filtration barrier, as the Fyn kinase-knockout mice
display a renal phenotype having structurally distorted
or coarsened podocyte foot processes, and in some,
Fig. 4. The intracellular domain of nephrin
interacts with a 190-kDa protein. (A)
35
S-
labeled extracts from podocytes, HT1080
cells,
L-cells, and HEK293 cells were incu-
bated with immobilized GST or the GST
fusion protein containing the full-length
nephrin intracellular domain. Bound
proteins were resolved by SDS ⁄ PAGE,
and analysed by autoradiography. (B) The
190-kDa protein was purified from the
L-cell extract using GST–nephrin affinity
chromatography. An aliquot corresponding
to a 1 ⁄ 50 portion of the purified protein is
analysed here by SDS ⁄ PAGE and silver
staining. Three similar large-scale
preparations were performed to obtain
enough protein for MALDITOF-MS.
Intracellular domain of nephrin X. L. Liu et al.
234 FEBS Journal 272 (2005) 228–243 ª 2004 FEBS

podocytes foot processes are completely effaced [35,44].
PI 3-kinase has been found to be able to bind to
nephrin via the p85 subunit, and in a cell culture model
this binding is related to activation of antiapoptotic
PI 3-kinase ⁄ AKT pathway [37]. However, further char-
acterization of nephrin–ligand interactions is required,
and especially their regulation is nearly completely
unknown. In this study, we could detect binding of
podocin, Fyn, and PI 3-kinase to GST–nephrin. We
tested binding of CD2AP to GST–nephrin using four
different detergent conditions and two cell lines in pull-
down assays. Two different antibodies were employed
to visualize CD2AP in Western blot analysis. Nonethe-
less, we were able to detect CD2AP-binding only occa-
sionally, and only in a lysis buffer containing 1%
Chaps. Neither did in vitro phosphorylation of GST–
nephrin induce CD2AP-binding. It is likely that neph-
rin–CD2AP interaction is of very low stoichiometry also
in vivo. It has not been detected in all previous studies
[17], and there are two contradictory reports on which
domains of CD2AP are involved in the nephrin-binding
[27,28]. Consequently, it is possible that the biologically
more critical interaction occurs between podocin and
CD2AP, and it may be mainly the lack of this inter-
action that contributes to the phenotype found in the
CD2AP-knockout mice [24].
Surprisingly, more Fyn kinase bound to the phos-
phorylated GST–nephrin than to the unphosphory-
lated fusion protein. This may imply that nephrin is
phosphorylated processively by Fyn. In this process,

Fyn at first would phosphorylate a site in nephrin that
becomes a high affinity binding site for the SH2
domain. Interaction between this site and the SH2
domain of Fyn facilitates phosphorylation of subse-
quent tyrosines in nephrin – alternatively Fyn could
phosphorylate other substrates that are forming a pro-
tein complex with the intracellular domain of nephrin.
The processive phosphorylation by Src family kinases
has been demonstrated with several multiphosphory-
lated substrates [45,46].
Interestingly, another important signalling molecule,
PI 3-kinase bound only to the N-terminal half of the
intracellular domain. The classical motif for the binding
of PI 3-kinase SH2 domains is YXXM [42]. The match-
ing sequence YYSM is conserved in mouse and rat
(Tyr1153 in mouse), but is replaced in human nephrin
by the sequence YYRSL. This segment is the most
probable candidate for the PI 3-kinase binding site
also as the phosphorylated tyrosine in GST-1173 is
obviously located very close to the C-terminal end of
the fusion protein. The PI 3-kinase binding site is
apparently needed mainly for signalling purposes, and
this same tyrosine may not be involved in the forma-
tion of an intracellular protein complex around nephrin
or in the regulation of contacts with the cytoskeleton.
Tyrosine phosphorylation in the C-terminal half of
nephrin has more likely this kind of functions.
Having confirmed the functionality of GST–nephri-
ncyt, we employed GST–nephrincyt affinity chromato-
Table 2. Peptide masses obtained by MALDITOF-MS analysis after

in-gel tryptic digestion of the 190-kDa protein identify IQGAP1 as
an interacting partner for nephrin. Measured masses are obtained
by MALDITOF-MS analysis after in-gel tryptic digestion of the 190-
kDa protein. Data base searches with this set of peptide masses
assigned a total of 30 peptides to mouse IQGAP1 corresponding to
a sequence coverage of 19%. The peptide mass error was less
than 100 p.p.m. with all except one peptide. The identification was
confirmed by sequencing three peptides shown in bold and two
other peptides not detected in the original MALDITOF-MS analysis.
Their sequences, TLQALQIPAAK and LFQTALQEEIK, correspond,
respectively, to residues 557–567 and 1025–1035 of the mouse IQ-
GAP1 sequence.
Measured
mass
Computed
mass Sequence
Residues in
IQGAP1
Start To
725.453 725.386 LQYFR 829 833
735.453 735.373 IPYGMR 1156 1161
746.487 746.444 IQAFIR 842 847
769.434 769.401 MHQARK 818 823
800.439 800.429 ATGLHFR 105 111
800.439 800.425 DIRNQR 1488 1493
826.521 826.527 LIVDVIR 1391 1397
851.431 851.432 MVVSFNR 1054 1060
908.486 908.475 LGNFFSPK 81 88
934.464 934.512 EEYLLLR 1018 1024
934.464 934.454 QDKMTNAK 267 274

1040.576 1040.634 ALQSLALGLR 327 336
1117.543 1117.595 LIFQMPQNK 989 997
1117.543 1117.566 LDNSIRNMR 1131 1139
1123.602 1123.638 IIGNLLYYR 1186 1194
1140.508 1140.563 YGIQMPAFSK 192 201
1148.563 1148.585 TCLDNLASKGK 1533 1543
1258.676 1258.706 MREEVITLIR 892 901
1274.681 1274.701 MREEVITLIR 892 901
1318.665 1318.713 LFQTALQEEIK 1025 1035
1408.769 1408.851 RLAAVAAINAAIQK 388 401
1414.729 1414.781 LGLAPQIQDLYGK 162 174
1482.670 1482.666 ATFYGEQVDYYK 1517 1528
1531.698 1531.719 IFYPETTDIYDR 131 142
1564.613 1564.646 FDVPGDENAEMDAR 1369 1382
1566.757 1566.843 TLINAEDPPMIVVR 857 870
1715.841 1715.908 GVLLEIEDLQANQFK 1572 1586
1723.791 1723.837 EEIQSSISGVTAAYNR 723 738
1752.870 1752.911 IELEKYGIQMPAFSK 187 201
1752.870 1752.961 VDQIQEIVTGNPTVIK 1038 1053
1889.909 1889.948 FALGISAINEAVDSGDVGR 623 641
1953.882 1953.967 LPYDVTPEQALSHEEVK 1112 1128
2020.976 2020.998 NVIFEIGPTEEVGDFEVK 1587 1604
2054.108 2054.079 LEGVLAEVAQHYQDTLIR 568 585
X. L. Liu et al. Intracellular domain of nephrin
FEBS Journal 272 (2005) 228–243 ª 2004 FEBS 235
graphy and peptide mass fingerprinting to search for
new intracellular interaction partners for nephrin. This
work resulted in the identification of IQGAP1, an
effector protein of small GTPases Rac1 and Cdc42
and a putative regulator of cell–cell adheren junctions

[39,40]. According to the GST pull-down assays, the
interaction took place strictly and specifically between
the C-terminal half of the nephrin intracellular domain
and IQGAP1. The binding was not lost even after rel-
atively harsh washing (four times in the lysis buffer
containing 0.5 m NaCl), and IQGAP1 could be detec-
ted in the pull-down samples also by silver staining of
SDS ⁄ PAGE gels (Fig. 5C). Compared to the inter-
actions with the previously identified ligands, that
between nephrin and IQGAP1 appeared to be clearly
the strongest and most specific one. However, despite
the fact that these two proteins are according to our
immunoelectron microscopic and immunocytochemical
findings physically located very close to each other,
immunoprecipitation could not demonstrate direct
association between nephrin and IQGAP1 in HEK293
cells. This could imply that the nephrin–IQGAP1
interaction is transient, taking place only during
certain situations, such as during the formation of
the slit structure, and possibly even before nephrin is
associated with other intracellular interactors.
Although it is not clear whether the nephrin–
IQGAP1 interaction is a biologically relevant one,
study of the IQGAP1 literature strongly supports the
notion that this could be the case. Rac1 and Cdc42
belong to the Rho subfamily of small GTPases that
has been implicated in the regulation of a wide range
of biological processes, including cell motility and
adhesion, cytokinesis, cell morphology and polariza-
tion, and cell growth [47]. They affect these processes

mostly by controlling the reorganization of the actin
cytoskeleton, but they also regulate signal transduction
pathways affecting gene transcription in the nucleus.
Rac proteins stimulate the formation of lamellipodia
and membrane ruffles and are involved also in actin
polymerization, cell–cell adhesion and cell motility,
whereas Cdc42-activation triggers the formation of
filopodia and affects cell–cell adhesion. IQGAP1 accu-
mulates at the polarized leading edge and areas of
Fig. 5. Characterization of the nephrin–IQGAP1 interaction. (A) Mouse podocyte lysates were incubated for 4 h with various immobilized
GST proteins, after which bound proteins were analysed by SDS ⁄ PAGE and Western blotting with monoclonal anti-IQGAP1 Ig. The results
show that the binding site for IQGAP1 resides within the C-terminal half of the nephrin intracellular domain. (B) L-cell lysates were incubated
with equal amounts of nonphosphorylated and phosphorylated GST proteins immobilized to glutathione-Sepharose beads. The bound
proteins were analysed, as in panel A, with monoclonal anti-IQGAP1 Ig. An identical SDS ⁄ PAGE gel was run to show equal loading of the
phosphorylated and nonphosphorylated fusion proteins to the beads. The eluates were also analysed by SDS ⁄ PAGE and Western blotting
with the anti-phosphotyrosine Igs. The results demonstrate that IQGAP1-binding to nephrin is not affected by the nephrin tyrosine phos-
phorylation. (C) GST–nephrin immobilized to glutathione-Sepharose was incubated with the
L-cell lysate, after which the beads were washed
four times with the lysis buffer containing indicated concentrations of NaCl. Bound proteins were fractionated by SDS ⁄ PAGE and visualized
by silver staining. A significant fraction of bound IQGAP1 remains associated even after washing with 0.5
M NaCl.
Intracellular domain of nephrin X. L. Liu et al.
236 FEBS Journal 272 (2005) 228–243 ª 2004 FEBS
membrane ruffling, as well as at the cell–cell adheren
junctions [39,40,48]. It can bind to the activated
GTP-bound Rac1 and Cdc42 maintaining them in an
activated state, but not to the inactive GDP-bound
forms [39,48]. Calmodulin is another signalling mole-
cule IQGAP1 binds to [48], and it has also a
well-documented actin-binding activity [49,50]. Thus

IQGAP1 is a scaffolding protein connecting Ca
2+
⁄
calmodulin and Rac1 ⁄ Cdc42-mediated signalling and
cytoskeleton [51].
Very interesting is the recent observation that
IQGAP1 interacts with CLIP-170, a member of a
protein family that specifically accumulates at the
plus ends of growing microtubules [52]. The CLIP-
170–IQGAP1 complex appears to function as a linker
between the plus ends of microtubules and cortical
actin meshwork. It may recruit the microtubules at
special cortical sites leading to cell polarization. Acti-
vated Rac1 or Cdc42, both known as key regulators
of cell polarization [53], is present as a third member
in the CLIP-170–IQGAP1 complex. Furthermore,
IQGAP1 was reported recently to interact with
S100B [54] that colocalizes with IQGAP1 at the
polarized leading edge and areas of membrane ruf-
fling in glioma cells. The strongest S100B immuno-
reactivity was found in cells that are characterized
by long processes. In these cells, a colocalization of
S100B and IQGAP1 was evident at plasma mem-
brane and in the growing processes. However, maybe
the most notable issue is that IQGAP1 is involved
in the formation and maintenance of cell–cell adheren
junctions. It has been suggested to associate both
with E-cadherin–catenin and nectin–afadin systems,
and to regulate E-cadherin-mediated cell–cell adhe-
sion [40,55]. IQGAP1 may, together with cell adhe-

sion proteins, organize F-actin to form specific
structures and morphology at the cell–cell adhesion
sites in epithelial cells. It is possible that the neph-
rin–IQGAP1 interaction plays a similar role in the
slit diaphragm, which, in fact, has been suggested to
be a modified adherens junctions, as a-, b- and c-
catenins as well as P-cadherin have been found in
Fig. 6. Nephrin and IQGAP1 colocalize in cultured cells. IQGAP1 is found at the slit diaphragm in human kidneys. HEK293 cells stably
expressing human nephrin were double-stained for nephrin (A) and IQGAP1 (B). Panel C shows the merged picture, indicating that nephrin
and IQGAP1 colocalize at the sites of cell–cell contacts. Some of the cells had already lost nephrin expression. (D–F) IQGAP1-immunogold
label in podocyte foot-processes. FP, foot processes; SD, slit diaphragm; GBM, glomerular basement membrane; arrowheads, gold label. In
D, shorter and longer stretches of slit diaphragm are seen in oblique tangential view in filtration slit between foot processes of human kidney
podocytes. Note intracellular gold-label for IQGAP1 (arrowheads) mostly along SD. In higher magnification (E) gold label for IQGAP1 is seen
close to cell surface both at the SD area and elsewhere. In F, IQGAP1-label deeper in the podocyte cytoplasm is associated with cytoplas-
mic density at the level of cross-cut SD.
X. L. Liu et al. Intracellular domain of nephrin
FEBS Journal 272 (2005) 228–243 ª 2004 FEBS 237
podocytes close to the slit diaphragm [12]. While
revising this manuscript, nephrin was reported to
form a multiprotein complex with cadherins and
p120 in kidney glomeruli [56] which further supports
a role for IQGAP1 as a biological ligand of nephrin.
It is apparent that the slit-diaphragm is connected to
the actin cytoskeleton that has a well-documented and
critical role in the formation of the slit diaphragm and
foot processes, as well as in the recovery after disrup-
tion of the filtration barrier [32,33]. One possibility is
that the nephrin–IQGAP1 interaction plays a role
mainly during the formation of the slit diaphragm and
the specific actin structures, or in such situations as

recovery processes after injury. Therefore, it would be
interesting to challenge the IQGAP1-knockout mice
[57] that do not have any obvious defects in kidney
functions with protamine sulfate or anti-nephrin Igs.
The slit diaphragm is a highly specialized structure,
the formation of which requires strictly targeted pro-
tein transport and localization. These processes and
the functional activity of nephrin are regulated via a
plethora of protein–protein interactions. The function
and regulation of nephrin can not be fully understood
before the protein complex associated with its intracel-
lular domain is described and characterized. There are
a number of questions waiting to be addressed. The
most important question is how this complex responds
to different physiological or pathological states. For
example, do changes occur in the molecular composi-
tion of the complex? Or how are the regulation of the
nephrin function and the rearrangement of the actin
cytoskeleton connected? If IQGAP1 is a biologically
significant ligand for nephrin, it is possible that the cell
adhesive properties of nephrin and ⁄ or its connections
to the cytoskeleton are regulated in concert with cyto-
skeletal rearrangements.
Experimental procedures
Antibodies
Rabbit antisera pAb 2 against the intracellular domain of
human nephrin has been described previously [1,58]. Affin-
ity-purified polyclonal rabbit anti-podocin is also described
elsewhere [59]. Affinity-purified polyclonal rabbit anti-Fyn
and anti-CD2AP Igs were obtained from Santa Cruz Bio-

technology, Inc (Santa Cruz, CA, USA). Affinity-purified
hamster anti-CD2AP was kindly provided by A. Shaw
(Washington University School of Medicine, St. Louis,
MO, USA). Mouse monoclonal anti-PI 3-kinase (clone
UB93-3) was supplied by Upstate Biotechnology (Lake Pla-
cid, NY, USA). Anti-phosphotyrosine Ig RC20:HRPO was
obtained from Transduction Laboratories (Lexington, KY,
USA). Mouse monoclonal anti-IQGAP1 was from Zymed
Laboratories Inc (San Francisco, CA, USA). Secondary
antibodies for immunocytochemistry were purchased from
Dako A ⁄ S (Glostrup, Denmark). Colloidal gold goat anti-
rabbit F(ab)
2
for immunoelectron microscopy was from
British BioCell International (Cardiff, UK).
DNA constructs
The expression construct for the untagged full-length
human nephrin intracellular domain (amino acids 1088–
1241) was generated by PCR using the human nephrin
cDNA [2] as a template. The upstream and downstream
primers contained cutting sites for NdeI and XhoI, respect-
ively. The amplified fragment was ligated to the corres-
ponding sites in the pET24a(+) vector (Novagen Inc.,
Madison, WI, USA). The Nde I recognition site provided
the translation initiation codon ATG, thus adding one
extra residue, a methionine residue, to the N-terminus of
the recombinant protein. To generate the constructs for
GST–nephrin fusion proteins, the cDNA encoding the full-
length cytoplasmic domain of mouse nephrin (amino acids
1102–1256) with BamHI and EcoRI sites at the ends was

produced by PCR using the mouse nephrin cDNA [38] as a
template, and cloned into the pGEX-2TK vector (Amer-
sham Pharmacia Biotech AB, Uppsala, Sweden). Using this
GST–nephrincyt vector as a template in PCR, the nephrin
cDNAs containing amino acids 1102–1122 (GST-1122),
1102–1173 (GST-1173), 1102–1245 (1245), and 1167–1256
(GST–Cterminus) were created, and subsequently cloned
into BamHI and EcoRI sites of the pGEX-2TK vector.
The authenticity of all constructs was confirmed by sequen-
cing with BigDye Terminator Cycle Sequencing Ready
Reaction kit (PE Biosystems, Foster City, CA, USA). The
expression construct encoding the full-length human podo-
cin in the pcDNA3.1 vector has been described elsewhere
[59].
Production and purification of recombinant
bacterial proteins
The GST fusion proteins and the untagged nephrin intra-
cellular domain were expressed in Escherichia coli BL21
strain. The GST fusion proteins were purified from bac-
terial lysates with Glutathione Sepharose
TM
4B beads
according to the manufacturer’s instructions (Amersham
Pharmacia Biotech AB). The untagged intracellular
domain was purified with a Q Sepharose XL anion-
exchange column, Source 15 PHE hydrophobic interac-
tion column and a Superdex 200 gel filtration column
was utilised as a polishing step. All chromatographic pro-
cedures were performed with HPLC system of Waters Co
(Milford, MA, USA). Chromatographic media were sup-

plied by Amersham Pharmacia Biotech AB.
Intracellular domain of nephrin X. L. Liu et al.
238 FEBS Journal 272 (2005) 228–243 ª 2004 FEBS
Cell culture
HEK293, HT1080, NPH5, HeLa and L-cells were grown in
Dulbecco’s modified Eagle’s medium supplemented with
10% (v ⁄ v) fetal bovine serum, 100 UÆmL
)1
penicillin, and
100 lgÆmL
)1
streptomycin. NPH5 cells are derived from
HEK293 cells, and stably express human nephrin [60]. For
maintaining the human nephrin expression, the NPH5 cells
were grown under G418 selection (1 mg mL
)1
).
Conditionally immortalized mouse podocytes were
obtained from P. Mundel (Albert Einstein College of
Medicine, Bronx, New York), and cultivated as previously
described [61] at 33 °C in RPMI 1640 medium supple-
mented with 10% (v ⁄ v) fetal bovine serum, 100 UÆmL
)1
penicillin, 100 lgÆmL
)1
streptomycin, and 10 UÆmL
)1
mouse recombinant c-interferon (Sigma, St. Louis, MO,
USA).
Cell extracts

Cells washed three times in ice-cold NaCl ⁄ P
i
were lysed in
a buffer containing 25 mm Hepes (pH 7.4), 150 mm NaCl,
5mm MgCl
2
, 10% (v ⁄ v) glycerol, 1% (v ⁄ v) Chaps (or in
some cases 1% (w ⁄ v) digitonin, 1% (v ⁄ v) NP-40, or 0.5%
(v ⁄ v) Triton X-100), 1 mm phenylmethanesulfonyl fluoride,
and Complete
TM
EDTA-free protease inhibitor cocktail
(Boehringer Mannheim, Mannheim, Germany). Extracts
were cleared by centrifugation either at 15 000 g for 5 min
at 4 °C, or at 12 000 g for 30 min at 4 °C, depending on
the volume of an extract.
Metabolic labelling
Near confluent cultures were washed with NaCl ⁄ P
i
, and fed
with methionine- and cysteine-free Dulbecco’s modified
Eagle’s medium containing 10% (v ⁄ v) NaCl ⁄ P
i
-dialyzed
fetal bovine serum and l-[
35
S]methionine ⁄ cysteine mixture
(100 lCiÆmL
)1
, NEN

TM
Life Science Products, Inc., Bos-
ton, MA, USA). After 12 h incubation, cell extracts were
prepared as described above. They were precleared by 1-h
end-to-end incubation with the glutathione-Sepharose
TM
4B
beads (Amersham Pharmacia Biotech AB) before use in
GST pull-down assays.
In vitro phosphorylation by Fyn kinase
Proteins to be phosphorylated were mixed with an equal
volume of the Src Kinase Reaction Buffer [100 mm
Tris ⁄ HCl (pH 7.2), 125 mm MgCl
2
,25mm MnCl
2
,2mm
EGTA, 0.25 sodium orthovanadate and 2 mm dithiothrei-
tol] and 0.25 milliunitsÆlL
)1
Fyn kinase (Upstate Biotech-
nology, Lake Placid, NY, USA), and incubated for 90 min
or indicated times at 30 °C. Before use in pull-down assays,
phosphorylated GST proteins immobilized to the glutathi-
one Sepharose beads were extensively washed with the lysis
buffer containing 1% (v ⁄ v) Triton X-100 and 1.0 m NaCl.
Pull-down assay
GST–nephrin fusion proteins or GST alone bound to the
glutathione-Sepharose beads (20–30 lL, depending on
experiment) were mixed with 500–1000 lL of cell extracts,

and incubated for 4 h with end-to-end mixing at 4 °C.
After washing with the lysis buffer, bound proteins were
eluted by boiling in the SDS-sample buffer. The samples
were separated by SDS ⁄ PAGE under reducing conditions,
and subjected to Western blot analysis. When analyzing
bound proteins from cells labelled with l-[
35
S]methio-
nine ⁄ cysteine, the gel was soaked after the electrophoresis
in Amplify reagent (Amersham Pharmacia Biotech, Buck-
inghamshire, UK), dried and exposed for autoradiography.
Large-scale affinity chromatography with the
GST–nephrincyt fusion protein
Cell extracts were precleared by a 4-h end-to-end incuba-
tion with the GST–glutathione-Sepharose beads 4 °C, and
then applied to a GST–nephrincyt-coupled glutathione-
Sepharose column. The column was washed with 25 mm
Hepes (pH 7.4), 150 mm NaCl, 1 mm dithiothreitol, and
the bound proteins were eluted with 50 mm Tris ⁄ Cl
(pH 8.0), 10 mm glutathione. Collected fractions were ana-
lysed with SDS ⁄ PAGE and silver staining as described
below. Fractions containing a protein of interest were
pooled and concentrated on UltrafreeÒ centrifugal concen-
trators equipped with a 100 kDa or 50 kDa cut-off mem-
brane (Millipore Corporation, Bedford, MA, USA). The
concentrate was electrophoresed on a 7% SDS ⁄ polyacryl-
amide gel under reducing conditions. After the electrophor-
esis, proteins were visualized by silver staining. For
staining, the gel was fixed with 30% methanol ⁄ 10% acetic
acid, (v ⁄ v ⁄ v), and then sensitized in 2% (w ⁄ v) potassium

hexacyanoferrate before incubating for 30 min in 0.1%
(w ⁄ v) AgN0
3
. After rinsing with four changes of distilled
water, the silver stain was developed in 6% Na
2
C0
3
, 0.11%
formaldehyde, 0.008% sodium thiosulfate (w ⁄ v ⁄ w ⁄ v). The
bands containing protein of interest were excised and used
for mass spectrometry analysis.
Peptide mass fingerprinting
The excised silverstained gel pieces containing the protein
sample were destained and treated for in-gel digestion
according to Gharahdaghi et al. [62]. Incubation with por-
cine trypsin (modified, sequence grade from Promega,
Madison, WI, USA) was done overnight at 30 °C. Incuba-
tion was stopped by acidification with trifluoroacetic acid,
and peptides were extracted and concentrated by vacuum
X. L. Liu et al. Intracellular domain of nephrin
FEBS Journal 272 (2005) 228–243 ª 2004 FEBS 239
centrifugation. The peptide mixture was desalted and fur-
ther concentrated by revers-phase chromatography on
Poros R2 20 packed in a hand-made micro column in a
GelLoader tip. The peptide mixture was analysed by
MALDITOF-MS on a Bruker Biflex III (Bruker Daltonics,
Bremen, Germany), using alfa cyano 4-hydroxy cinnamic
acid in a dried droplet sample preparation. The resulting
spectrum was calibrated internally using two autodigestion

peptides from the protease (842.51 and 2211.10 Da,
MH +, monoisotopic). The peptide mass table was used in
ProFound to scan the nonredundant sequence database
(NCBInr) for matching proteins.
Amino acid sequencing
For obtaining amino acid sequence, Nano-ES mass spectra
were recorded using a Q-TOF (Micromass, Manchester,
UK). The instrument was equipped with a Z-spray nano-
ES interface (Micromass). Metal-coated borosilicate capil-
laries (Protana, Odense, Denmark) were used and opened
manually on the stage of a light microscope to give a spray-
ing orifice of  5 lm. The needle voltage was optimized in
the range 900–1700 V, with the cone voltage set at 40 V.
For the acquisition of collision-induced dissociation (CID)
spectra, the collision energy was varied between 20 and
50 V depending on the mass and charge state of the pep-
tides. Argon was used as the collision gas.
Immunocytochemistry
The NPH5 cells grown for 2 days on glass coverslips were
fixed with 4% (v⁄ v) paraformaldehyde for 20 min at room
temperature. Free aldehyde groups were quenched by incu-
bating the cells in NaCl ⁄ P
i
containing 50 mm NH
4
Cl for
5 min, after which the cells were permeabilized by incuba-
ting with 0.1% (v ⁄ v) Triton X-100 in NaCl ⁄ P
i
for 2 min.

Non-specific binding was blocked by incubating cells with
2% BSA ⁄ 2% fetal bovine serum in NaCl ⁄ P
i
(v ⁄ v ⁄ v) for at
least 45 min. pAb 2 antisera was used at a dilution of
1 : 200 and the monoclonal antibody anti-IQGAP1 at a
1 : 400 dilution. The primary antibodies were incubated
for 1–2 h at room temperature. Bound antibodies were
visualized by incubating the cells with either tetramethyl-
rhodamine–isothiocyanate-conjugated swine anti-(rabbit
IgG) for 1–2 h at room temperature or with biotinylated
goat anti-(mouse IgG) for a similar period followed by a
15-min incubation with fluorescein-isothiocyanate (FITC)-
conjugated streptavidin (both secondary antibodies and
FITC-streptavidin were from Dako A ⁄ S, Denmark). The
coverslips were mounted by inverting them into NaCl ⁄ P
i
containing 50% (v ⁄ v) glycerol and 1 mgÆmL
)1
p-phenylene-
diamine. For double-labelling for nephrin and IQGAP1,
the cells were at first stained for nephrin as described
above, fixed again in the paraformaldehyde solution, and
stained for IQGAP1.
Immunoelectron microscopy
Processing of tissue for immunoelectron microscopy was
performed as described earlier [1]. In short, kidney tissue
from two brain-dead intended donors (a 49-year-old female
unsuitable as donor due to HbsAg positivity and a 30-year-
old female that was found to have variation in the kidney

vasculature) were used. The kidneys were stored 12 and
15 h in ViaSpanÒ solution (DuPont Merck Pharmaceutical
Ltd, Herts, England) until processing. The samples were
fixed in 3.5% (v ⁄ v) paraformaldehyde (Sigma, St Louis,
Missouri, USA) alone or combined to 0.01%, 0.05% or
0.1% (v ⁄ v) glutaraldehyde (Electron Microscopy Sciences,
Fort Washington, PA, USA) in 0.1 m phosphate buffer
(pH 7.3), at room temperature for 1 or 1.5 h. Fixed sam-
ples were washed in phosphate buffer, dehydrated in graded
ethanol and embedded in LR White (London Resin Com-
pany Ltd, Reading, Berkshire, UK) resin.
Thin sections were cut on Pioloform (Agar Scientific Ltd,
Stansted, Essex, UK) and carbon coated nickel grids. For
indirect immunostaining, the grids were incubated in the first
antibody diluted in 3% BSA ⁄ NaCl ⁄ P
i
(to 10–50 lgÆmL
)1
for
the protein A affinity purified rabbit anti-(nephrin IgG) (pAb
2) and 1 : 50 for monoclonal anti-IQGAP1 Ig), for 60 min.
Grids were then treated with the second antibody [5 nm or
10 nm colloidal gold goat anti-rabbit F(ab)
2
] or with 5 nm or
10 nm protein A-gold (Department of Cell Biology, Utrecht
School of Medicine, Utrecht, the Netherlands), diluted in 3%
BSA ⁄ NaCl ⁄ P
i
(1 : 50 or 1 : 65, v ⁄ v), for 30 min at room

temperature. The sections were poststained with uranyl acet-
ate and examined under a Jeol 1200 EX electron microscope
at 60 kV accelerating voltage.
Acknowledgements
We thank Dr A. Shaw (Washington University School
of Medicine, St. Louis, MO, USA) for anti-CD2AP Ig,
and Dr Vesa Ruotsalainen (University of Oulu, Oulu,
Finland) for anti-nephrin Ig. Mouse podocyte cell line
was very kindly provided by Dr Peter Mundel (Albert
Einstein College of Medicine, Bronx, New York). We
are grateful to Protein Analysis Center (Karolinska
Institutet, Stockholm, Sweden) for their help in amino
acid sequencing, and to Ms Olga Beltcheva for her
help in transfection experiments. This work was sup-
ported in part by NIH grant no. DK54724 as well as
grants from Novo Nordisk Foundation and the Sigrid
Juse
´
lius Foundation.
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