Molecular Biology of the Cell
Vol. 18, 1744–1755, May 2007
The MAGUK Protein MPP7 Binds to the Polarity Protein
hDlg1 and Facilitates Epithelial Tight Junction Formation□
Volker M. Stucke,* Evy Timmerman,†Joel Vandekerckhove,†Kris Gevaert,†
and Alan Hall*‡
*Medical Research Council Laboratory for Molecular Cell Biology, Cancer Research UK Oncogene and Signal
Transduction Group, University College London, London WC1E 6BT, England; and†Department of Medical
Protein Research, Flanders Interuniversity Institute for Biotechnology and Department of Biochemistry, Ghent
University, B-9000 Ghent, Belgium
Submitted November 1, 2006; Revised January 16, 2007; Accepted February 15, 2007
Monitoring Editor: Ben Margolis
Three groups of evolutionarily conserved proteins have been implicated in the establishment of epithelial cell polarity:
the apically-localized proteins of the Par (Par3-Par6-aPKC-Cdc42) and Crumbs groups (Crb3-PALS1-PATJ) and the
basolaterally localized proteins of the Dlg group (Dlg1-Scribble-Lgl). During epithelial morphogenesis, these proteins
participate in a complex network of interdependent interactions that define the position and functional organization of
adherens junctions and tight junctions. However, the biochemical pathways through which they control polarity are
poorly understood. In this study, we identify an interaction between endogenous hDlg1 and MPP7, a previously
uncharacterized MAGUK-p55 subfamily member. We find that MPP7 targets to the lateral surface of epithelial cells via
its L27N domain, through an interaction with hDlg1. Loss of either hDlg1 or MPP7 from epithelial Caco-2 cells results in
a significant defect in the assembly and maintenance of functional tight junctions. We conclude that the formation of a
complex between hDlg1 and MPP7 promotes epithelial cell polarity and tight junction formation.
The establishment of cell polarity and the formation of mul-
ticellular tissues and organs are fundamentally important in
the development of higher eukaryotes (Schock and Perrimon,
2002). In epithelial sheets, cells are polarized with apical and
basolateral membrane domains separated by tight junctions,
specialized sites of intimate cell–cell contact that act as a
barrier to the free diffusion of lipid and proteins (Yeaman et
al., 1999). Numerous proteins and signal transduction path-
ways have been implicated in the establishment of cell po-
larity, but genetic analyses in Drosophila and Caenorhabditis
elegans have identified three groups of highly conserved
proteins that play a critical role in this process (Macara,
2004). These are: 1) Par3, Par6, and aPKC (Joberty et al., 2000;
Lin et al., 2000; Wodarz et al., 2000; Petronczki and Knoblich,
2001; Tepass et al., 2001); 2) Crb3, PALS1 (MPP5), and PATJ,
which are both located predominantly apically (Bachmann
et al., 2001; Hong et al., 2001; Tepass et al., 2001; Medina et al.,
2002); and 3) Dlg1, Scribble, and Lgl, located mostly on the
basolateral surfaces (Bilder et al., 2000; Bilder and Perrimon,
2000; Bossinger et al., 2001; Tepass et al., 2001). With the
exception of aPKC, members of these three groups of pro-
teins lack catalytic activity. Instead they contain multiple
protein–protein interaction domains, in particular PDZ,
SH3, and guanylate kinase domains often referred to as
membrane-associated guanylate kinase or MAGUK proteins
(Funke et al., 2005), indicating a complex network of protein
interactions controlling cell polarity.
Par3, Par6, and the activity of aPKC regulate the assembly
of functional tight junctions in mammalian cells (Yamanaka
et al., 2001; Suzuki et al., 2002). These proteins physically
interact with each other. Par3, which contains three PDZ
domains, for example, binds to the PDZ domain of Par6
(Joberty et al., 2000; Lin et al., 2000) and is able to associate
with cell adhesion molecules such as Jam1 (Ebnet et al., 2001)
or nectin (Takekuni et al., 2003). Both Par3 and Par6 can bind
to aPKC, whereas the GTP-bound form of Cdc42 interacts
with Par6 via a Cdc42/Rac-interactive binding (CRIB)-like
motif to regulate aPKC activity (Izumi et al., 1998; Tabuse et
al., 1998; Joberty et al., 2000; Lin et al., 2000; Qiu et al., 2000;
Suzuki et al., 2001). The transmembrane protein Crb3 and its
binding partners PALS1 (MPP5) and PATJ are also required
for tight junction integrity (Roh et al., 2003; Straight et al.,
2004; Shin et al., 2005). Crb3 binds through a carboxy-termi-
nal motif to the PDZ domain of PALS1 (MPP5; Makarova et
al., 2003; Roh et al., 2003) and in turn, PALS1 binds to PATJ
through L27 domain interactions (Lemmers et al., 2002; Roh
et al., 2002b). The multi-PDZ domain protein PATJ is able to
associate with tight junction–associated proteins such as
ZO-3 and claudin-1 (Roh et al., 2002a), providing a physical
link to tight junctions. The third group of polarity proteins,
the tumor suppressor proteins Dlg1, Scribble and Lgl have
not been shown to physically associate with each other.
However, mutations in any one of these genes disrupt api-
This article was published online ahead of print in MBC in Press
on March 1, 2007.
DThe online version of this article contains supplemental material
at MBC Online (http://www.molbiolcell.org).
‡Present address: Memorial Sloan-Kettering Cancer Center, 1275
York Avenue, Box 572, New York, NY 10021.
Address correspondence to: Alan Hall (firstname.lastname@example.org).
Abbreviations used: PDZ, PSD-95, ZO-1, and Discs-large; MPP,
1744© 2007 by The American Society for Cell Biology
protein (amino acid numbers are given above) as indicated. (B) Colloidal blue-stained gel of an hDlg1 immunoprecipitate from MCF7 cell
extracts (lane 1). A control immunoprecipitation was performed with mouse monoclonal ?-myc antibody (mIgG; lane 2) or with anti-hDlg1
mAb alone without incubation in cell extracts (lane 3). Note the precipitated ?140-kDa hDlg1 isoforms, the ?110-kDa CASK protein, and the
65-kDa MPP7 protein identified by tryptic peptide fingerprinting. Molecular weight (MW) markers are indicated on the right. (C) Cell lysates
of different epithelial cell lines were immunoprecipitated with hDlg1-antibodies. Proteins were detected by immunoblotting with antibodies
against hDlg1, MPP7, and CASK. Note the very low MPP7 expression in MCF10A mammary breast epithelial cells and 16HBE lung epithelial
cells. (D) Lysates made from polarized MCF7 cell monolayers were fractionated on a Superose-6 gel filtration column and fractions analyzed
by Western blots for hDlg1, MPP7, and CASK. The elution volume and the native molecular mass standards are indicated. (E) Immuno-
precipitations were performed from extracts of HEK 293T cells expressing myc-MPP7 (lanes 1–5), VSVG-CASK (lanes 6–9) or untransfected
cells (lanes 10–13) with antibodies as indicated. Expression of the transfected constructs was monitored by Western blotting with anti-myc
Identification and characterization of CASK and MPP7 as binding partners for hDlg1. (A) Schematic representation of the hDlg1
hDlg1 Protein Complexes and Cell Polarity
Vol. 18, May 2007 1745
We find that the N-terminal L27 domain in MPP7 interacts
with hDlg1. Through its SH3-HOOK domain, MPP7 also
interacts with PALS1 (MPP5), a member of the Crb3/PALS1
(MPP5)/PATJ group of apically localized polarity proteins,
and in turn PALS1 (MPP5) interacts with Crb3 (Bachmann et
al., 2001; Hong et al., 2001; Roh et al., 2002b, 2003). In Caco-2
cells, we find that both hDlg1 and MPP7 show partial colo-
calization with Occludin and ZO-1, suggesting that the in-
teraction of these polarity complexes might occur at the level
of cell junctions. However, it remains unclear whether all
these proteins are simultaneously present in one complex.
We also provide evidence that MPP7 can interact with
Crb3 likely through its interaction with MPP5 (PALS1). A
recent report on the identification of new polarity complexes
showed that MPP7 can be coprecipitated with PALS1
(MPP5) and PATJ (Wells et al., 2006), thus validating our
data. This raises the possibility that MPP7 might act as a
bridge between the Dlg and Crb groups of polarity proteins.
However, we have been unable to detect any Crb3 or PALS1
(MPP5) in hDlg1 immunoprecipitates (data not shown) and
so this remains speculative. It is possible that the interaction
between MPP7 and the Crb3/PALS1 (MPP5) complex is
transient and that MPP7 is recruited to the plasma mem-
brane through an interaction between its SH3-HOOK do-
main and PALS1 (MPP5) and is then retained at the cell
membrane through binding to hDlg1. This would account
for our observation that hDlg1 is required, but not sufficient,
for the localization of MPP7 to the plasma membrane. Such
a two-step mechanism for plasma membrane recruitment is
already known for several polarity proteins, e.g., Dlg1
(Thomas et al., 2000) and Scribble (Albertson et al., 2004;
Zeitler et al., 2004).
Finally, we provide evidence that the interaction of hDlg1
and MPP7 is functionally important in tight junction forma-
tion. Retroviral-shRNA mediated depletion of either MPP7
or hDlg1 compromised functional tight junctions assembly
in Caco-2 cells after a calcium switch, as revealed by quan-
titative TER. It is clear, however, that although the loss of
hDlg1 or MPP7 in Caco-2 cells leads to a defect in tight
junction functionality, it does not lead to significant mislo-
calization of tight junction markers (occludin, ZO-1), or ad-
herens junction proteins (E-cadherin, ?-catenin). A similar
conclusion has been reached in hScrib-depleted cells (Qin et
al., 2005). This may suggest that MPP7 and the Dlg/Scrib
group of polarity proteins are not required for cell junction
assembly, but rather for some functional activity perhaps
related to its dynamic stability. Alternatively, possible re-
dundancy in the activities and/or protein–protein interac-
tions associated with the numerous polarity proteins, cou-
pled with the relatively unsophisticated nature of these 2D
tissue culture assays may account for the lack of dramatic
We are grateful to Zenta Walther (Yale) for the VSVG-CASK construct, James
Staddon (EISAI Research Laboratories, London, United Kingdom) for help
with gel filtration chromatography and members of the laboratory for valu-
able discussions. V.M.S. is supported by an European Union Marie-Curie
fellowship, and A.H. is generously supported by Cancer Research UK. K.G. is
a Postdoctoral Fellow of the Fund for Scientific Research, Flanders, Belgium
(F.W.O. Vlaanderen). The project was supported by research grants from the
Fund for Scientific Research, Flanders, Belgium (project number G.0008.03)
and the GBOU-research initiative (project number 20204) of the Flanders
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hDlg1 Protein Complexes and Cell Polarity
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