PIST regulates the intracellular trafficking and plasma membrane expression of cadherin 23.

Abstract

The atypical cadherin protein cadherin 23 (CDH23) is crucial for proper function of retinal photoreceptors and inner ear hair cells. As we obtain more and more information about the specific roles of cadherin 23 in photoreceptors and hair cells, the regulatory mechanisms responsible for the transport of this protein to the plasma membrane are largely unknown.
PIST, a Golgi-associated, PDZ domain-containing protein, interacted with cadherin 23 via the PDZ domain of PIST and the C-terminal PDZ domain-binding interface (PBI) of cadherin 23. By binding to cadherin 23, PIST retained cadherin 23 in the trans-Golgi network of cultured cells. The retention was released when either of the two known cadherin 23-binding proteins MAGI-1 and harmonin was co-expressed. Similar to MAGI-1 and harmonin, PIST was detected in mouse inner ear sensory hair cells.
PIST binds cadherin 23 via its PDZ domain and retains cadherin 23 in trans-Golgi network. MAGI-1 and harmonin can compete with PIST for binding cadherin 23 and release cadherin 23 from PIST's retention. Our finding suggests that PIST, MAGI-1 and harmonin collaborate in intracellular trafficking of cadherin 23 and regulate the plasma membrane expression of cadherin 23.

Full text

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RESEARCH ARTICLEOpen Access
PIST regulates the intracellular trafficking and
plasma membrane expression of Cadherin 23
Zhigang Xu1,2*, Kazuo Oshima1, Stefan Heller1
Abstract
Background: The atypical cadherin protein cadherin 23 (CDH23) is crucial for proper function of retinal
photoreceptors and inner ear hair cells. As we obtain more and more information about the specific roles of
cadherin 23 in photoreceptors and hair cells, the regulatory mechanisms responsible for the transport of this
protein to the plasma membrane are largely unknown.
Results: PIST, a Golgi-associated, PDZ domain-containing protein, interacted with cadherin 23 via the PDZ domain
of PIST and the C-terminal PDZ domain-binding interface (PBI) of cadherin 23. By binding to cadherin 23, PIST
retained cadherin 23 in the trans-Golgi network of cultured cells. The retention was released when either of the
two known cadherin 23-binding proteins MAGI-1 and harmonin was co-expressed. Similar to MAGI-1 and
harmonin, PIST was detected in mouse inner ear sensory hair cells.
Conclusions: PIST binds cadherin 23 via its PDZ domain and retains cadherin 23 in trans-Golgi network. MAGI-1
and harmonin can compete with PIST for binding cadherin 23 and release cadherin 23 from PIST’s retention. Our
finding suggests that PIST, MAGI-1 and harmonin collaborate in intracellular trafficking of cadherin 23 and regulate
the plasma membrane expression of cadherin 23.
Background
Cadherins are calcium-dependent transmembrane pro-
teins. They play important roles in cell adhesion, which
is crucial for establishing and maintaining tissue archi-
tecture and function [1]. Around 80 cadherin proteins
have been identified, which can be divided into different
subgroups, including classic cadherins, desmogleins,
desmocollins, protocadherins, CNRs, Fats, seven-pass
transmembrane cadherins, and Ret tyrosine kinase [2].
All cadherins have extracellular cadherin (EC) repeats,
the extracellular Ca2+-binding domains that mediate
cell-cell adhesion, but their cytoplasmic domains are
diverse. Classic cadherins (E- and N-cadherins) have a
b-catenin-binding motif in their cytoplasmic domain,
which is important for the cell adhesion function [2].
The atypical cadherin protein cadherin 23 (CDH23) is
closely related to the Fat subgroup, which is character-
ized by a large number of EC repeats (27 for cadherin
23 and Dachsous, 34 for Fat), a transmembrane domain,
and a short cytoplasmic domain. Although it bears no
homology with classical cadherins except for the EC
repeats, cadherin 23 can mediate cell-cell adhesion
when over-expressed in L cells [3]. The cytoplasmic
domain of cadherin 23 lacks the b-catenin-binding
motif, suggesting that it may not be able to interact with
b-catenin directly. However, recently cadherin 23 was
shown to bind to the PDZ4 domain of a scaffolding pro-
tein, MAGI-1 [4], which in turn can bind to b-catenin
via its PDZ5 domain [5], suggesting that MAGI-1 may
act as a bridge between cadherin 23 and b-catenin.
The CDH23 gene gives rise to different transcripts
through two mechanisms. The first mechanism utilizes
different promoters, giving rise to proteins with different
numbers of EC repeats [6]. The second type involves
the alternative splicing of exon 68, which encodes part
of the cytoplasmic domain of the cadherin 23(+68) iso-
form that is preferentially expressed in the inner ear
[3,4]. It has been shown that the longest cadherin 23
variant with 27 EC repeats is a part of the tip-links in
hair cell stereocilia [3,7], which are the mechanical links
that are essential for gating of the mechanoelectrical
* Correspondence: xuzg@sdu.edu.cn
1Departments of Otolaryngology - Head & Neck Surgery and Molecular &
Cellular Physiology, Stanford University School of Medicine, Stanford, CA
94305, USA
Full list of author information is available at the end of the article
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© 2010 Xu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
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transduction channels. Mutations of CDH23 gene have
been identified to associate with blindness and hearing
loss [8,9].
Several cadherin 23-binding proteins have been
reported, including harmonin [10,11], myosin 1c [3],
and protocadherin 15 [7], all of which have been shown
to be involved in hearing transduction and/or retinal
function. As we learn more and more about the func-
tion of cadherin 23, our knowledge about its genesis is
limited. Understanding how cadherin 23 is being
shuttled to the apical hair cells membrane for example,
and ultimately, how tip links are being assembled is cru-
cial for shedding light on the molecular mechanisms of
hair cell mechanosensation. Recently, EHD4, a EH
domain-containing protein involved in endocytic recy-
cling was identified as a novel cadherin 23-binding part-
ner, and was suggested to play a role in regulating the
membrane localization of cadherin 23 [12]. Nevertheless,
the regulatory mechanism responsible for the transport
of cadherin 23 to the plasma membrane remains
unclear.
We have conducted yeast two-hybrid screens of a
cochlear cDNA library using the cadherin 23(+68) intra-
cellular domain as a bait, and identified MAGI-1, a
MAGUK protein containing multiple PDZ domains, as a
novel cadherin 23-interaction partner [4]. Here we
report another PDZ domain-containing protein identi-
fied from the screen, PIST (also known as GOPC, or
CAL). PIST was first reported as a putative binding pro-
tein of the Rho protein TC10, which is reflected in its
name PIST: PDZ domain protein interacting specifically
with TC10 [13]. As a Golgi-associated protein, PIST has
been shown to interact with some transmembrane pro-
teins and regulate the intracellular sorting and plasma
membrane expression of these proteins [14-19].
Here we showed that PIST and cadherin 23 interact
via the PDZ domain of PIST and the cytoplasmic PDZ-
binding interface (PBI) of cadherin 23, respectively.
When co-expressed in cultured cells, PIST retained cad-
herin 23 in trans-golgi networks (TGN). MAGI-1 and
harmonin, two known cadherin 23-binding proteins that
are expressed in hair cells were able to compete with
PIST and to release cadherin 23 from its retention.
Immunostaining showed that PIST is expressed in inner
ear hair cells. Our results suggest that PIST, MAGI-1
and harmonin regulate the intracellular sorting of cad-
herin 23, thereby affecting the membrane localization of
cadherin 23.
Methods
Yeast Two-hybrid Screen
The yeast two-hybrid screen was performed as described
before [4]. Briefly, a chicken basilar papilla cDNA library
[20] was screened using the carboxyl-terminal 265
amino acids (aa) of chicken cadherin 23(+68) as a bait.
5 × 106total transformants were selectively screened
using HIS3 (at the presence of 2.5 mM of 3-amino-
1,2,4-triazole) as the primary reporter gene, then two
more reporter genes ADE2 and lacZ were used to verify
the positive colonies. The prey vectors in triple-positive
yeast colonies were recovered and cDNA inserts were
sequenced.
Expression Vectors
Mouse CDH23 cDNA is a gift from K. Noben-Trauth
(National Institute on Deafness and Other Communica-
tion Disorders, Rockville, MD), which consists of
CDH23 cDNA encoding the first three extracellular cad-
herin (EC) repeats (1-348 aa) fused to cDNA encoding
the protein’s carboxyl-terminus (2975-3354aa in cad-
herin 23(+68), 2975-3319aa in cadherin 23(-68)) includ-
ingthe transmembrane
manipulation, we added His and c-Myc tags between
the signal peptide and the first EC repeat. The cDNA
encoding cadherin 23 lacking the last 4 aa (ITEL) at the
carboxyl-terminus was PCR amplified and cloned into
pcDNA3.1(+) to generate expression vectors for Myc-
cadherin 23 (-ITEL). Human HA-PIST cDNA is a gift
from Dr. W. B. Guggino (Johns Hopkins Medical Insti-
tute, Baltimore, MD). PIST cDNA was PCR amplified
and cloned into pEGFP-C2 to express full length PIST,
PIST CC2-plus domain (146-274aa), and PIST PDZ
domain (276-366aa) as EGFP fusion proteins. Mouse
MAGI-1c cDNA is a gift from K. M. Patrie (University
of Michigan, Ann Arbor, MI), and was PCR-amplified
and cloned into pEGFP-C2 for the expression of EGFP-
MAGI-1c protein. Mouse harmonin cDNA was PCR-
amplified from mouse organ of Corti cDNA and cloned
into pEGFP-C2 for the expression of EGFP-harmonin
protein.
domain.For protein
Co-immunoprecipitation
HEK293 cells were transfected with the expression vec-
tors using GeneJammer transfection reagent (Stratagene,
La Jolla, CA). Transfected cells were washed with PBS
24-48 hours after transfection and lysed in ice-cold lysis
buffer consisting of 150 mM NaCl, 50 mM Tris at pH
7.5, 1% (vol/vol) Triton X-100, 1 mM PMSF, and 1 ×
protease inhibitor cocktail (Sigma-Aldrich, Saint Louis,
MO). For immunoprecipitation, we used immobilized
monoclonal anti-c-Myc agarose beads (Sigma-Aldrich)
and performed the experiments according to the manu-
facturer’s recommendation. Following 2 hours of incu-
bation at 4°C, immunoprecipitated proteins were
washed five times with washing buffer (a modified lysis
buffer containing 500 mM NaCl instead of 150 mM),
separated by polyacrylamide gel electrophoresis, then
transferred to nitrocellulose membrane. The proteins
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were probed with corresponding antibodies and detected
with an Odyssey Infrared Imaging System (LI-COR
Biosciences, Lincoln, NE).
Immunocytochemistry
All steps were performed at room temperature unless
otherwise indicated. Transfected cells (HEK293, CHO,
or COS-7) growing on Gelatin-coated glass cover slips
were fixed with 4% paraformaldehyde (PFA) in phos-
phate-buffered saline (PBS) for 15 minutes, then per-
meabilized and blocked with PBT1 (0.1% Triton X-100,
1% BSA, 5% heat-inactivated goat serum in PBS, pH 7.3)
for 30 minutes, followed by incubation with mouse
anti-myc antibody 9E10 (The Developmental Studies
Hybridoma Bank, Iowa City, IA) and/or rabbit anti-PIST
antibody (Affinity purified, a gift from Dr. K. Nagata
(Institute for Developmental Research, Aichi Human
Service Center, Kasugai, Japan)), 1:500 diluted in PBT1,
over night at 4°C. In other experiments, polyclonal rab-
bit antibody against cadherin 23 (a gift from Dr. Ulrich
Muller (Scripps Research Institute, La Jolla, CA)) and
monoclonal mouse antibody against golgin-97 (A-21270,
Invitrogen, Carlsbad, CA), 1:200 diluted in PBT1, were
used to label cadherin 23 and trans-golgi network mar-
ker golgin-97. After washing twice with PBT1 for 10
minutes and twice with PBT2 (0.1% Triton X-100, 0.1%
BSA in PBS) for 5 minutes, cells were incubated with
7.5 μg/ml fluorescence-conjugated secondary antibody
(Jackson ImmunoResearch Inc., West Grove, PA) in
PBT2 for 1 hour, followed by two 5 minutes PBT2
washes and two 5 minutes PBS washes. For nuclei stain-
ing, cells were then incubated with TOTO-3 (Molecular
Probes, Eugene, OR), 1:2,000 diluted in PBS for 1 hour,
followed by three 10 minutes PBS washes, then
mounted in Glycerol/PBS (1:1). Immunostaining was
imaged with a confocal microscope (LSM Pascal, Zeiss,
Germany).
Tissue section staining
All animal procedures followed guidelines set forth by
the National Institutes of Health. The cochleae were dis-
sected from P5 and adult (4-week old) C57 mice, imme-
diately fixed in 4% PFA for 3 hours, then immersed in
30% sucrose solution overnight and embedded in O.C.T.
compound (Tissue-Tek, Sakura Finetek, Japan). Blocks
were then frozen at -20°C and sectioned at 14 μm thick-
ness onto slide glasses with Cryostat (Leica CM3050S,
Germany). Staining was performed as described above.
Briefly, sectioned tissues were incubated with rabbit
anti-PIST antibody (1:100) and guinea pig anti-myosin
VIIa antibody (1:200) [21], then 7.5 μg/ml fluorescence-
conjugated secondary antibody (Jackson ImmunoRe-
search Inc.). After the final wash with PBS, tissues were
stained with TRITC-conjugated phalloidin (Sigma-
Aldrich) to visualize F-actin in the hair bundles of hair
cells and DAPI (Molecular Probes) to visualize nuclei.
The slides were analyzed by fluorescence microscopy
and digital image acquisition (Zeiss Axioimager and
AxioCam).
Results
PIST’s interaction with cadherin 23 is PDZ mediated
To identify proteins that interact with cadherin 23, we
performed yeast two-hybrid screens of a chicken basilar
papilla cDNA library using the intracellular part of
chicken cadherin 23(+68) protein as a bait (Figure 1A).
Proteins identified through the yeast two-hybrid screen
include scaffolding proteins (for example, MAGI-1 [4])
and chaperones (such as PIST, see below), most but not
all of which are PDZ domain-containing proteins.
Twenty-nine clones, which account for more than half
of all the isolated positive clones, encode the chicken
PDZ-containing, Golgi-associated chaperone protein
PIST. The isolated 29 PIST clones fall into 10 groups of
unique cDNAs, covering different lengths of PIST’s
amino acid sequence (Figure 1B). The longest clone
encodes nearly the full-length chicken PIST, only miss-
ing the N-terminal 85 amino acids comparing to the
predicted chicken PIST sequence in NCBI database, and
the shortest contains the second coiled-coil domain and
the PDZ domain.
To verify the interaction biochemically, we performed
co-immunoprecipitation (co-IP) experiments. Both pro-
teins are highly conserved among different species.
Mammalian cadherin 23 and PIST share about 90%
homology with their chicken counterparts. Comparing
to the predicted chicken PIST protein, mammalian PIST
misses about 150 aa at the N-terminus (Figure 2A).
There are no predicable domains in this region. Mam-
malian hair cells are becoming more and more the main
focus of research, which prompted us to focus the rest
of the investigation on mammalian proteins. The full-
length CDH23 cDNA is more than 10 kb long and diffi-
cult to manipulate, so instead of using the full-length
cDNA, we made a construct that expresses a Myc-
tagged cadherin 23 protein missing EC4-27 (Figure 3,
left panel). When over-expressed in HEK293 cells,
EGFP-tagged human PIST was co-immunoprecipitated
with Myc-tagged mouse cadherin 23, and this interac-
tion was not affected by the splicing of exon 68 (Figure
1C). Co-IP experiments with EGFP-tagged PIST coiled-
coil domain 2 (CC2) plus the amino acids between CC2
and the PDZ domain (CC2-plus, 146-274aa) or the PDZ
domain (276-366aa) revealed that both domains were
co-IPed with Myc-cadherin 23 (Figure 2B,C). The inter-
action between PIST’s PDZ domain and cadherin 23
was not affected by the splicing of exon 68, while the
interaction between PIST CC2-plus and cadherin 23
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was isoform-dependent, with the cadherin 23(-68) inter-
action being much stronger than cadherin 23(+68). The
interaction between PIST CC2-plus and cadherin 23
(+68) was barely detectable.
The last 4 amino acids (ITEL) at cadherin 23’s car-
boxyl-terminus constitute a class-I PDZ domain binding
interface (PBI), which is important for the interaction of
cadherin 23 with two other PDZ domain containing
proteins, harmonin and MAGI-1 [4,11]. We then tested
whether these 4 amino acids are also important for the
association of cadherin 23 with PIST. We found that
cadherin 23 lacking the last 4 amino acids (cadherin 23
(-ITEL)) displayed decreased binding ability to PIST
when compared to intact cadherin 23 (Figure 1C).
Although the removal of binding could be the conse-
quence of misfolding of the truncated protein, our data
Figure 1 Interaction between PIST and cadherin 23. A. Schematic diagram of chicken cadherin 23 domain structure and the intracellular
fragment used as the bait for yeast two-hybrid screens. B. Schematic diagram of chicken PIST domain structure and PIST protein fragments
encoded by the clones isolated from yeast two-hybrid screens. C. Western blots showing co-immunoprecipitation (co-IP) of EGFP-tagged human
PIST protein with Myc-tagged mouse cadherin 23(+68) and cadherin 23(-68). The co-IP is almost undetectable when the last 4 amino acids (ITEL)
of cadherin 23 are deleted. IP indicates antibody used for immunoprecipitation and WB indicates antibody used for detection.
Figure 2 PIST binds to cadherin 23 via its PDZ domain, as well as the CC2-plus region. A. Schematic diagram of chicken and human PIST
domain structures, and the domains of human PIST used in co-IP experiments. B. EGFP-tagged PIST CC2-plus region is co-IPed with Myc-tagged
cadherin 23(-68), but not cadherin 23(+68). C. EGFP-tagged PIST PDZ domain is co-IPed with Myc-tagged cadherin 23(+68) and cadherin 23(-68)
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as well as other published results suggested that the
principal interaction between the two proteins utilizes
the PDZ binding interface.
PIST retains cadherin 23 in the trans-Golgi network
It has been shown that PIST can regulate the subcellular
localization of its interacting partners. To test whether
this also applies to cadherin 23, we analyzed the subcel-
lular distribution of PIST and cadherin 23 in transfected
HEK293 cells by immunocytochemistry and confocal
microscopy. Myc-cadherin 23 was found in the cyto-
plasm as well as associated with the plasma membrane
(Figure 3A). The association with the plasma membrane
was clearly visible when we stained for the extracellular
Myc epitope of Myc-cadherin 23 without permeabilizing
the cells (Figure 4A). The Myc epitope is placed in the
extracellular part of cadherin 23, so when a non-permeable
protocol is used, only the plasma membrane cadherin 23
can be visualized. When co-expressed with EGFP-PIST,
however, Myc-cadherin 23 was found to co-localize with
EGFP-PIST (Figure 3B), which exhibited a TGN-like
staining pattern, and the plasma membrane expression of
cadherin 23 was dramatically reduced to an undetectable
level (Figure 3B and 4B). To make sure that the reduced
membrane expression of Myc-cadherin 23 in Figure 4B is
not caused by unsuccessful transfection, we used a con-
struct that expresses Myc-cadherin 23 and mCherry at the
same time (Myc-cadherin 23-IRES-mCherry), so when
mCherry is detected in a cell, Myc-cadherin 23 is
expressed in the same cell. When we disrupted the interac-
tion between these two proteins by removing the last 4
amino acids of cadherin 23, the subcellular localization of
cadherin 23(-ITEL) was not affected by EGFP-PIST any
more (Figure 3C and 3D).
The retention of cadherin 23 by PIST was also
observed in transfected CHO and COS-7 cells (data not
shown). PIST has been shown to associate with trans-
golgi network (TGN) in mammalian cells [14]. To
explore whether PIST retains cadherin 23 in TGN, we
stained transfected cells with an antibody against a TGN
maker, glogin-97. When cotransfected in COS-7 cells,
Myc-cadherin 23 co-localized with EGFP-PIST, as well
Figure 3 Co-localization of cadherin 23 with PIST in transfected HEK293 cells. A. Myc-tagged cadherin 23 localizes in the cytoplasm. B. In
the presence of EGFP-tagged PIST, Myc-tagged cadherin 23 co-localizes with EGFP-PIST at TGN-like structures. C. Myc-tagged cadherin 23
without the last 4 amino acids (ITEL) localizes in the cytoplasm. D. In the presence of EGFP-PIST, Myc-tagged cadherin 23 without the last 4
amino acids (ITEL) remains in the cytoplasm, albeit EGFP-PIST localizes at TGN-like structures. Myc-cadherin 23 was stained with mouse anti-Myc
antibody and then TRITC-conjugated goat anti-mouse secondary antibody. Nuclei were stained with TOTO-3. Scale bars, 10 μm.
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as golgin-97 (Figure 5). Our data suggests that by bind-
ing to cadherin 23, PIST retains cadherin 23 in TGN,
hence reduces its membrane expression.
MAGI-1 and harmonin compete with PIST, and release
cadherin 23 from trans-Golgi network retention
MAGI-1, harmonin, and PIST can independently inter-
act with cadherin 23 via a PDZ domain-mediated
mechanism [4,10,11]. Given the fact that they all bind to
the same binding site at the carboxyl-terminus of cad-
herin 23 (harmonin can also bind to an internal peptide
of cadherin 23(-68) [22]), a single cadherin 23 protein
might only be able to bind to either PIST, or MAGI-1,
or harmonin. We consequently explored whether
MAGI-1 or harmonin compete with PIST for binding
cadherin 23, and furthermore, whether they are able to
release cadherin 23 from the PIST-mediated trans-Golgi
network retention. EGFP-MAGI-1 exists as protein
aggregates in the cytoplasm when overexpressed in
HEK293 cells (Figure 6B). When we co-expressed Myc-
cadherin 23 and HA-PIST in presence of EGFP-MAGI-1,
Myc-cadherin 23 (both +68 and -68 isoforms) co-localized
with EGFP-MAGI-1 in the cytoplasm, and was no longer
associated with HA-PIST in the trans-golgi network
(Figure 6A,B). This suggests that MAGI-1 is able to com-
petitively displace PIST from binding to cadherin 23, and
that this competition releases the retention of cadherin 23
in the trans-golgi network.
The interaction between harmonin and cadherin 23 is
more complex, involving multiple binding sites. The
PDZ2 domain of harmonin binds weakly to cadherin
23’s carboxyl-terminus. A second interaction happens
between the region immediately upstream of harmonin’s
PDZ1 domain and an internal peptide of cadherin 23’s
intracellular domain [22]. The amino acids encoded by
Figure 4 PIST reduced the membrane expression of cadherin 23 in transfected HEK293 cells. A. Myc-cadherin 23 immunoreactivity using
a non-permeable protocol shows clear plasma membrane localization. B. In the presence of EGFP-PIST, Myc-cadherin 23’s plasma membrane
immunoreactivity is undetectable. Staining was performed as normal except Triton X-100 was excluded. Myc-cadherin 23 was stained with
mouse anti-Myc antibody and then Cy5-conjugated goat anti-mouse secondary antibody. mCherry was used as a control indicating Myc-
cadherin 23-IRES-mCherry cassette was expressed successfully. Scale bar, 10 μm.
Figure 5 Retention of cadherin 23 by PIST in trans-golgi
network in transfected COS-7 cells. A. Myc-tagged cadherin 23
localizes in the cytoplasm. B. In the presence of EGFP-tagged PIST,
Myc-tagged cadherin 23 co-localizes with EGFP-PIST and a trans-
golgi network marker golgin-97. Myc-cadherin 23 was stained with
rabbit anti-cadherin 23 antibody and golgin-97 was stained with
mouse anti-golgin-97 antibody. Scale bar, 20 μm.
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exon 68 are adjacent to this binding site in cadherin 23
and may affect the binding to harmonin, since it has
been shown that harmonin binds to cadherin 23(-68)
much more robustly than to cadherin 23(+68) [4,11].
This preferential splice variant-dependent binding was
functionally confirmed in our subcellular localization
assays. When Myc-cadherin 23(-68) and HA-PIST were
co-expressed in HEK293 cells in presence of EGFP-
harmonin, Myc-cadherin 23(-68) co-localized with
EGFP-harmonin, which was associated with filamentous
structures in the cytoplasm as described before [10], but
not in trans-golgi networks where HA-PIST localizes
(Figure 6C). Conversely, when Myc-cadherin 23(+68)
and HA-PIST were co-expressed in HEK293 cells in
Figure 6 MAGI-1 and harmonin release cadherin 23 from PIST’s retention in transfected HEK293 cells. A. Myc-tagged cadherin 23 co-
localizes with HA-tagged PIST at TGN-like structures. B. In the presence of EGFP-MAGI-1c, Myc-cadherin 23 colocalizes in the cytoplasm with
EGFP-MAGI-1c, instead of co-localizing with HA-PIST at TGN-like structures. C. In the presence of EGFP-harmonin, Myc-cadherin 23(-68) co-
localizes with EGFP-harmonin, instead of co-localizing with HA-PIST at TGN-like structures. D. In the presence of EGFP-harmonin, Myc-cadherin 23
(+68) co-localizes with EGFP-harmonin, as well as with HA-PIST at TGN-like structures. Myc-cadherin 23 was stained with mouse anti-Myc
antibody then TRITC-conjugated goat anti-mouse secondary antibody. HA-PIST was stained with rabbit anti-PIST antibody then Cy5-conjugated
goat anti-rabbit secondary antibody. Scale bar, 10 μm.
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presence of EGFP-harmonin, Myc-cadherin 23(+68) dis-
played a mixed localization, partially colocalized with
EGFP-harmonin in the cytoplasm, as well as partially
colocalized with HA-PIST in the trans-golgi network,
which is consistent with a weaker binding ability of cad-
herin 23(+68) to harmonin (Figure 6D).
PIST protein is expressed by all types of hair cells
Cadherin 23 expression is restricted to a few cell types;
cadherin 23(+68) especially has only been detected in
inner ear sensory hair cells so far [3,4]. If PIST function-
ally interacts with cadherin 23, we would expect it to be
expressed in these cell types. We used affinity-purified
antibodies to examine the expression pattern of PIST in
the mouse inner ear. In the cochlea of postnatal day 5
(P5) mice, PIST immunoreactivity was detectable in
inner and outer hair cells as well as supporting cells,
including pillar cells and Deiter’s cells (Figure 7A,B).
Also in the five vestibular organs (utricle, saccule, and
the three ampullae of the semicircular canals), PIST
immunoreactivity was confined to the sensory epithelia,
where it labeled hair cells and probably also supporting
cells (Figure 7C-E). In the adult mouse cochlea, PIST
shows a similar expression pattern (data not shown).
Discussion
Our findings support the hypothesis that the Golgi-
associated, PDZ domain-containing protein PIST, in con-
junction with two other PDZ domain-containing proteins
MAGI-1 and harmonin, plays roles in trafficking cad-
herin 23 to different subcellular locations. It has been
shown that PIST interacts with some membrane proteins,
and regulates the intracellular trafficking and localization
of these membrane proteins: over-expression of PIST
leads to a dramatic decrease in the plasma membrane
expression of CFTR [15], ClC-3 chloride channels [14],
the b1 adrenergic receptor [23], and the somatostatin
receptor subtype 5 [18]. Here we show that over-expres-
sion of PIST retains cadherin 23 in the trans-golgi net-
work and decreases the plasma membrane expression of
cadherin 23, suggesting that it is able to regulate the
intracellular localization and/or sorting of cadherin 23.
This regulation requires the C-terminal PDZ domain-
binding interface (PBI) of cadherin 23.
Interestingly, we found that besides the PDZ domain,
the CC2 domain and its downstream amino acids (PIST
CC2-plus) also interacts with cadherin 23 in an isoform-
dependent way. Nevertheless, this binding site does not
appear to mediate the principal interaction between
these two proteins because interference of the PDZ/PBI-
mediated interaction between PIST and cadherin 23
abolishes the interaction of these two proteins nearly
completely.
We also found that the other two cadherin 23-binding,
PDZ domain-containing proteins, MAGI-1 and harmo-
nin, can compete with PIST in cellular assays, resulting
in release of cadherin 23 from trans-golgi networks.
Consistent with its equal binding strength to cadherin
23(+68) and cadherin 23(-68), MAGI-1 can release both
cadherin 23 isoforms from PIST’s retention. On the
other hand, harmonin has a lower binding ability to cad-
herin 23(+68) when compared to cadherin 23(-68),
hence although it releases cadherin 23(-68) efficiently, it
only partially releases cadherin 23(+68) from PIST’s
retention.
We used HEK293 cells as a model to study the com-
petitions among these PDZ domain-containing proteins
for binding cadherin 23. In polarized cells such as hair
cells, both MAGI-1 and harmonin have been shown to
associate with the plasma membrane and hair cell
stereocilia [4,10,24]. In our study, over-expressed
MAGI-1 and harmonin are not targeted to the mem-
brane; instead, they showed cytoplasmic localization and
aggregation. This mislocalization is likely the result of
overexpression in a cell line. In addition, other unknown
factors that are potentially present in hair cells might
contribute to the membrane localization of MAGI-1 and
harmonin. Nevertheless, our data provide the first clues
on posttranslational targeting of cadherin 23 through
the trans-golgi network to other places inside the cell.
We hypothesize that PIST in hair cells can transiently
interact with cadherin 23 and retain cadherin 23 in the
trans-golgi networks, and that this retention is released
by either MAGI-1 or harmonin. We speculate that
MAGI-1/cadherin 23 or harmonin/cadherin 23 are tar-
geted together to the apical hair cell plasma membrane.
Cadherin 23 has a limited expression profile, only
detected in some cell types. Cadherin 23(+68), which is
suggested to be the hair cell tip-link component, is only
reported in hair cells so far [3]. In mouse inner ear,
PIST expression was restricted to hair cells and support-
ing cells, both in the auditory and vestibular systems.
This places all four proteins, cadherin 23, PIST, harmo-
nin, and MAGI-1 into hair cells. It has been shown that
in hair cells, cadherin 23 is detected on the stereocilia,
and not detectable in the trans-golgi network, but it is
very clear the native cadherin 23 needs to pass through
the TGN (and very likely interacts with other proteins
in the TGN) on its way to the apical hair cell plasma
membrane. Our data suggests that PIST may play an
important role in regulating the intracellular sorting/
localization of cadherin 23. PIST, harmonin, and MAGI-
1, the three cadherin 23-binding, PDZ domain-contain-
ing proteins, may work together, perhaps sequentially,
to regulate cadherin 23’s transport to the plasma
membrane.
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Figure 7 PIST is expressed in P5 mouse inner ear. (A) Mouse organ of Corti immunolabeled with rabbit antibodies to PIST (detected with
FITC-conjugated secondary antibodies, green) and with guinea pig antibodies to myosin VIIa (Myo7a, detected with Cy5-conjugated secondary
antibodies, red). (B) Mouse organ of Corti at higher magnification reveals PIST immunoreactivity in organ of Corti supporting cells, outer hair cells
(OHC), and albeit weaker, in inner hair cells (IHC). The extracellular tectorial membrane (TM) shows unspecific labeling with the PIST antibody. (C)
Mouse utricle and saccule staining. (D) Mouse utricle staining at higher magnification. (E) Mouse ampulla staining. DAPI (blue) was used to label
cell nuclei. TRITC-conjugated Phallodin (white) was used to label stereocilia. Scale bars, 50 μm.
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Conclusions
Our data suggests a possible regulatory mechanism
responsible for the transport of cadherin 23 to the
plasma membrane. We show that cadherin 23 interacts
with PIST, a Golgi-associated, PDZ domain-containing
protein, which retains cadherin 23 in the trans-golgi
network, and reduces the membrane expression of cad-
herin 23. In this way PIST plays a negative role in tar-
geting cadherin 23 to the plasma membrane. We also
show that MAGI-1 and harmonin can compete with
PIST for binding cadherin 23 and release cadherin 23
from PIST’s retention. Taken together, PIST, MAGI-1
and harmonin may collaborate in intracellular trafficking
of cadherin 23 and regulate the plasma membrane
expression of cadherin 23.
List of abbreviations
CDH23: cadherin 23; EC: extracellular cadherin; PBI: PDZ binding interface;
TGN: trans-golgi network; HEK: human embryonic kidney; Co-IP: co-
immunoprecipitation; PBS: phosphate buffered saline; PFA:
paraformaldehyde; CC2: coiled-coil domain 2; TM: tectorial membrane.
Authors’ contributions
ZX performed the yeast two-hybrid experiment, the co-immunoprecipitation
experiment, and the immunochemistry experiment. KO performed the tissue
section staining experiment. ZX and SH designed the experiments, analyzed
the results and wrote the manuscript. All authors read and approved the
final manuscript.
Acknowledgements
This work was supported (in part) by National Institute on Deafness and
Other Communication Disorders Grants R01 DC4563 and P30 DC010363 (to
S.H.) and Independent Innovation Foundation of Shandong University
(IIFSDU) (to Z.X.). We thank Dr. K. Nagata (Institute for Developmental
Research, Aichi Human Service Center, Kasugai, Japan) for the anti-PIST
antibody; Dr. U. Muller (Scripps Research Institute, La Jolla, CA) for the anti-
cadherin 23 antibody; Dr. K. Noben-Trauth (National Institute on Deafness
and Other Communication Disorders, Rockville, MD) for the CDH23 cDNA;
Dr. K.M. Patrie (University of Michigan, Ann Arbor, MI) for the MAGI-1 cDNA;
Dr. W.B. Guggino (Johns Hopkins Medical Institute, Baltimore, MD) for the
PIST cDNA. We thank Dr. A.W. Peng and Y. Yu for critically reading this
manuscript.
Author details
1Departments of Otolaryngology - Head & Neck Surgery and Molecular &
Cellular Physiology, Stanford University School of Medicine, Stanford, CA
94305, USA.2Key Laboratory for Experimental Teratology of the Ministry of
Education, Institute of Developmental Biology, School of Life Sciences,
Shandong University, Jinan, Shandong 250100, China.
Received: 12 March 2010 Accepted: 19 October 2010
Published: 19 October 2010
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doi:10.1186/1471-2121-11-80
Cite this article as: Xu et al.: PIST regulates the intracellular trafficking
and plasma membrane expression of Cadherin 23. BMC Cell Biology 2010
11:80.
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