1 The Netherlands Cancer Institute, Division of Cell Biology, Plesmanlaan 121,
1066 CX Amsterdam, The Netherlands
2 University Hospital Geneva, Department of Dermatology, CH-1211 Geneva,
Switzerland
* Present address: Department of Human Genetics, Academic Medical Center,
University of Amsterdam, Meibergdreef 9, 1105 AZ, Amsterdam
Author for correspondence (e-mail:
a.sonnenberg{at}nki.nl)
Accepted 28 October 2002
![]() |
Summary |
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Key words: Hemidesmosome, Integrin, Bullous pemphigoid, Plakin, Keratinocyte
![]() |
Introduction |
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BP230 and plectin are cytoplasmic proteins that belong to the plakin
protein family, which also includes desmoplakin, envoplakin and periplakin.
These proteins are crucially involved in the organization of the cytoskeleton
(Ruhrberg and Watt, 1997;
Leung et al., 2001
). They are
composed of domains that have considerable sequence homology. Their N-terminus
consists of a plakin domain containing a number of subdomains of high
-helical content, designated NN, Z, Y, X, W and V, whereas the central
coiled-coil rod domain is composed of heptad repeats thought to be involved in
the dimerization of the plakin (Green et
al., 1992
). Finally, their C-terminal and exhibits one or more
homologous repeat sequences designated A, B or C. In plectin as well as in
neuronal and muscular isoforms of BP230 (BPAG1-a and BPAG1-b, respectively), a
calponin-type actin-binding domain (ABD) precedes the plakin domain
(Brown et al., 1995
;
McLean et al., 1996
;
Leung et al., 2001
). The
C-terminal end of plakins has intermediate filament binding properties
(Meng et al., 1997
;
Wiche et al., 1993
;
Yang et al., 1996
;
Leung et al., 1999
), whereas
the N-terminal end harbors specific sequences that target the proteins to
distinct membrane sites, such as HDs or desmosomes, cell-cell adhesion
complexes in a variety of epithelia
(Kowalczyk et al., 1997
;
Rezniczek et al., 1998
;
Geerts et al., 1999
;
Hopkinson and Jones,
2000
).
The 6ß4 integrin plays a central role in the assembly of HDs.
Loss of
6ß4 due to mutations in the genes for either the
6
or ß4 subunit causes a distinct form of pyloric atresia associated with
junctional epidermolysis bullosa (PA-JEB), and is characterized by fragility
and extensive blistering of the skin. In affected patients HDs are rudimentary
or completely absent (Vidal et al.,
1995
; Niessen et al.,
1996
; Ruzzi et al.,
1997
). A similar phenotype is observed in
6 or ß4 null
mutant mice (van der Neut et al.,
1996
; Georges-Labouesse et al.,
1996
; Dowling et al.,
1996
). The large cytoplasmic domain of the integrin ß4
subunit is essential for the formation of HDs
(Nievers et al., 1998
;
Murgia et al., 1998
). It is
over 1000 amino acids long and harbors two pairs of fibronectin type III
(FNIII) repeats, separated by a connecting segment (CS)
(Hogervorst et al., 1990
;
Suzuki and Naitoh, 1990
). The
second FNIII repeat and the first 35 amino acid residues of the CS of the
ß4 integrin are required for the recruitment of plectin into HDs
(Niessen et al., 1997a
;
Nievers et al., 2000
;
Niessen et al., 1997b
). The CS
and the third FNIII repeat have been implicated in the binding to BP180
(Borradori et al., 1997
;
Aho and Uitto, 1998
;
Schaapveld et al., 1998
).
Furthermore, the cytoplasmic domain of ß4 has been reported to interact
with BP230 (Hopkinson and Jones,
2000
).
BP180 is a type II transmembrane protein with a 466-amino acid cytoplasmic
domain and a large collagenous extracellular domain
(Giudice et al., 1992). There
is evidence that, like
6ß4, BP180 binds laminin-5
(Reddy et al., 1998
). In
cultured epithelial cell lines, the localization of BP180 in HDs is dependent
on its interaction with the cytoplasmic domain of ß4
(Borradori et al., 1997
;
Schaapveld et al., 1998
).
Furthermore, the extracellular domain of BP180 is also able to interact with
the
6 integrin subunit (Hopkinson
et al., 1995
; Hopkinson et
al., 1998
). Finally, BP180 is involved in the recruitment of BP230
into HDs (Borradori et al.,
1998
; Hopkinson and Jones,
2000
).
The aim of our study was: (1) to further assess the potential of the
6ß4, BP180, BP230 and plectin to associate with each other; (2) to
map the involved binding sites and, finally, (3) to define the importance of
these interactions for the recruitment of these proteins into HD in combined
yeast two-hybrid assays and cell-transfection studies. Our findings show that
interactions between these hemidesmosomal components are more complex than
previously anticipated, uncover the potential of BP180 to interact with
plectin and reveal that the recruitment of these proteins into HDs is
regulated by a hierarchy of interactions that each appear to have a different
impact on HD assembly.
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Materials and Methods |
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Mouse monoclonal antibody (mAb) 233 against BP180
(Nishizawa et al., 1993) and
mouse mAb 121 against plectin/HD1 (Hieda
et al., 1992
) were kind gifts from K. Owaribe (University of
Nagoya, Nagoya, Japan). Rat mAb 439-9B recognizes an extracellular epitope on
the integrin ß4 subunit and mouse mAb 450-11A directed against the
cytoplasmic domain of ß4 were purchased from Pharmingen (San Diego, CA).
The rabbit polyclonal antiserum against BP230
(Tanaka et al., 1990
) was a
kind gift from J. R. Stanley (University of Pennsylvania, Philadelphia, PA).
The human mAbs 5E and 10D against BP230 were generously provided by T.
Hashimoto (Keio University, Tokyo, Japan)
(Hashimoto et al., 1993
).
Rabbit polyclonal antibody against laminin-5 was kindly provided by P.
Rouselle (Lyon, France). The rabbit polyclonal antibodies against the
extracellular domain of ß4 (sc-9090) and against hemagglutinin (HA)
epitope tag (sc-805) were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). Secondary antibodies were purchased from Rockland (Gilbertsville, PA)
(FITC-conjugated goat anti-mouse immunoglobulin (Ig) G) and Molecular Probes
(Eugene, OR) (Alexa-488 conjugated goat anti-human IgG and
Texas-Red-conjugated goat anti-rat and anti-rabbit IgG).
DNA transfections
PA-JEB or GABEB cells were grown to 40% confluence in 12-well
tissue-culture plates (Falcon; Becton Dickinson, Lincoln Park, NJ). Transient
transfections were performed with 0.8 µg cDNA using Lipofectin, according
to the manufacturer's instructions (Gibco-BRL). Transfection mixtures were
replaced by SFM medium after 6 hours and incubated in this medium for 12
hours. Subsequently, the SFM medium was replaced by Nutrient Mixture Ham's
F12/Dulbecco's MEM (1:3) for an additional 24 hours after which the cells were
assayed for gene expression.
Immunofluorescence microscopy
PA-JEB and GABEB cells grown on glass coverslips were fixed with 1%
paraformaldehyde in PBS for 10 minutes and permeabilized with 0.5% Triton
X-100 in PBS for 5 minutes at room temperature. After rinsing in PBS and
blocking with 2% BSA in PBS for 30 minutes at room temperature, the cells were
incubated with primary antibodies for 60 minutes at room temperature and then
washed three times with PBS. Cells were subsequently incubated with Alexa-488,
FITC- and Texas Red-conjugated secondary antibodies directed against mouse,
rat, rabbit or human IgG for 45 minutes at room temperature. Coverslips were
washed three times, mounted in Mowiol/DABCO and viewed under a Leica confocal
scanning laser microscope.
Yeast two-hybrid interaction assay
The yeast strain Saccharomyces cerevisiae PJ69-4A (a kind gift
from P. James, University of Wisconsin, Madison, WI), which contains the
following genetic markers: trp1-901, leu2-3, his3-200, gal4,
gal80
, LYS2::GAL1-HIS3 and GAL2-ADE2
(James et al., 1996
) was used
as host for the two-hybrid assays. This strain contains two tightly regulated
selectable Gal4-driven reporter genes, his and ade, allowing sensitive
detection of protein-protein interactions between Gal4 fusion proteins. The
Gal4 activation domain (AD)- and Gal4 binding domain (BD)-fusion plasmids were
co-transformed into PJ69-4A, as described previously
(Schaapveld et al., 1998
), and
equal aliquots of transformed cells were spread out on plates containing yeast
synthetic complete medium lacking leu and trp (vector markers) or leu, trp,
his and ade (vector and interaction markers). Plates were incubated at
30°C and growth of colonies was scored after 6 and 10 days. The plating
efficiencies on -leu,-trp,-his,-ade (SC-LTHA) plates, as compared with the
plating efficiency on -leu,-trp (SC-LT) was used as a measure for the strength
of the two-hybrid protein interaction. Autonomous activation of the reporter
genes was suppressed by the addition of 2 mM 3-amino-1,2,4-triazole (a His
antagonist) (A8506; Sigma Chemical Co.). Expression of the Gal4-fusion
proteins was analyzed by immunoblotting with antibodies against the
Gal-activation or -DNA binding domain (sc-1663 and sc-510, respectively; Santa
Cruz).
Constructs for the yeast two-hybrid studies were generated using standard cloning techniques. All nucleotide and amino acid positions are numbered with the ATG initiation codon at position one. The cDNA sequences used for alignment and designation of primers are available from GenBank under accession number m77830 (desmoplakin); mn_000494 (BP180); m69225 (BP230); u53204 (plectin) and x53587 (ß4). Plasmid inserts were generated by restriction digestion or PCR using the proofreading Pwo DNA polymerase (Boehringer Mannheim) and gene-specific sense and antisense primers containing restriction site tags. Numbers in superscript correspond to the amino acid residues of subclones encoded within the Gal4 (AD) or -(BD) fusion proteins. Vectors used were the yeast Gal4 AD or BD expression vectors pACT2 or pAS2.1 (Clontech).
ß-Galactosidase assay
For the quantitative analysis of ß-galactosidase activity, five yeast
colonies were combined and grown to an OD660 of approximately 1.0
in selective medium lacking Leu and Trp. ß-Galactosidase activity was
determined at 37°C using the Pierce yeast ß-galactosidase assay kit
(75768) with O-nitrophenyl ß-D-galactopyranoside as substrate.
The A405 was measured in an ELISA-reader and the time at which the
reaction reached a value of 0.2 was taken to calculate the
ß-galactosidase activity using the equation:
1,000xA405/(cell volume (ml) x time of reaction (min)
x OD660). Samples that did not reach this value within 4
hours were left overnight and measured the next morning. The final values are
the results from three independent determinations.
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Results |
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In ß4-deficient PA-JEB cells, the transient expression of the ß4
subunit was shown to restore the cells' ability to form HD-like structures
(Schaapveld et al., 1998). In
extension to these studies, we found that, in many cells of a PA-JEB
keratinocyte cell line that stably express ß4 (PA-JEB/ß4 cells), the
subcellular localization of laminin-5 and plectin was largely identical to
that of
6ß4 (Fig.
1A,B). Furthermore, the distribution of BP180 and BP230 was the
same, i.e. in structures appearing as dots and patches, which are typical for
HD-like structures or stable anchoring contacts
(Fig. 1C). By contrast,
co-localization of either ß4 with BP180 or of plectin with BP230 was
mostly only partial (Fig.
1D-I). These results indicate that certain, but not all, HD-like
structures contain, in addition to
6ß4 and plectin, BP180 and
BP230 as shown by immunofluorescence microscopy studies, i.e. they represent
both type I and type II HDs (Uematsu et
al., 1994
).
|
When BP180-deficient keratinocytes obtained from a patient with GABEB were
analyzed by immunofluoresence microscopy, the distribution of ß4 appeared
to be normal; it was concentrated in patches at sites of cell-substrate
contacts (Fig. 2A-C). However,
although in nearly all PA-JEB/ß4 cells the staining of ß4 and
plectin largely overlapped, in many of the BP180-deficient GABEB cells there
were several patches containing ß4 but not plectin (compare
Fig. 2A with
Fig. 1B). Furthermore,
consistent with previous observations
(Borradori et al., 1998), BP230
was not present in these HD-like structures
(Fig. 2C). Nevertheless, on
transient transfection with cDNA for BP180, we found that BP230 was recruited
into HD-like structures together with BP180
(Fig. 2D-I). Together, these
findings obtained in PA-JEB and GABEB keratinocytes indicate that the
recruitment of BP230 into HD-like structures is regulated by distinct
either direct or indirect interactions, involving mainly ß4 and
BP180. Furthermore, for the localization of plectin in HD-like structures to
be efficient, it seems that BP180 is also required.
|
The cytoplasmic domain of BP180 contains two distinct binding sites
for ß4
Previous studies have shown that the recruitment of BP180 into HDs depends
on a direct association of BP180 with ß4
(Borradori et al., 1997). To
identify the ß4 binding region on the cytoplasmic domain of BP180,
several deletion mutants were generated and expressed in yeast cells together
with a ß4 construct containing all four FNIII repeats of the ß4
cytoplasmic domain, ß41115-1666
(Fig. 3). Interactions were
detected by growth of yeast cells on plates lacking histidine and adenine (see
Materials and Methods). Although constructs BP1801-231 and
BP180145-401 bound to ß41115-1666, there was no
interaction with either BP1801-147 or BP180145-230.
Notably, the latter contains the stretch of amino acids 145-230 shared by the
above two BP180 constructs, which had binding activity. Because proper
expression of the BP180145-230 mutant was ascertained by
immunoblotting of yeast cell lysates, the lack of binding could not be due to
defective protein expression (data not shown). Furthermore, deletion of the
stretch of amino acids 145-230 from the cytoplasmic domain of BP180,
BP1801-401,
145-230 had no impact on its interaction with
ß41115-1666. Together, these results strongly suggest that
there are at least two distinct binding sites for ß4 on BP180: one
located in the first 230-amino-acid stretch of BP180 and a second in a more
C-terminally located region encompassing amino acids 231-401.
|
A stretch of 85 amino acids in the cytoplasmic domain of BP180 is
crucial for its binding to BP230
BP180 has recently been shown to associate with BP230 via a region of 280
amino acids (residues 180-460) spanning half the cytoplasmic domain
(Hopkinson and Jones, 2000).
To define the region on BP180 that binds to BP230, we carried out additional
yeast two-hybrid assays. As shown in Fig.
3, the BP180 mutants that contain the region of 85 amino acids
145-230 bound to BP2301-555 and BP2301-1156, whereas
those lacking it did not. Furthermore, the BP180145-230 mutant that
only contains this stretch of amino acids was also able to bind, albeit less
efficiently. Thus, although additional sequences might further strengthen
their interaction, this 85-amino-acid stretch is necessary and sufficient for
the binding of BP180 to BP230. Finally, two mutants carrying a deletion of the
first 37 N-terminal amino acids (BP18038-422 and
BP18038-422,
229-324), a region previously identified as
being crucial for recruiting BP230 into HDs in transfection studies
(Hopkinson et al., 1995
;
Borradori et al., 1998
), were
both able to interact with BP230. These results suggest that BP180 interacts
with BP230 through a region of 85 amino acids (BP180145-230) and
that the first 37 N-terminal amino acids are dispensable for binding in
yeast.
Deletion of a stretch of 85 amino acids from the cytoplasmic domain
of BP180 reduces the recruitment of BP230 in HDs in transfected cells
Next, we analyzed in transfection studies the importance of the stretch of
85 amino acids of BP180, which contains a binding site for BP230, for the
recruitment of the latter into HDs. When introduced in BP180-deficient
keratinocytes, BP180145-230 is correctly recruited into HDs
and, like wild-type BP180, is co-localized with ß4
(Fig. 4A). The same was
observed in cells expressing BP180
1-36
(Fig. 4C). However, at variance
with cells expressing either wild-type BP180
(Fig. 2I) or the
BP180
1-36 mutant (Fig.
4D), in cells expressing BP180
145-230, BP230 was
found only rarely in HD-like structures together with the BP180 mutant
(Fig. 4B). Thus, consistent
with the results in yeast two-hybrid interaction assays, in which a region
encompassing amino acids 145-230 of BP180 is required and sufficient for its
interaction with BP230, the deletion of this sequence from BP180 dramatically
reduced recruitment of BP230 into HDs. Deletion of the 36 most N-terminal
amino acid residues of BP180 had no effect on the recruitment of either BP230
or ß4 into HD-like structures.
|
BP230 and plectin interact with BP180 via their Y domain
We next investigated which region of BP230 is involved in binding to BP180.
For this purpose, a series of mutant forms of BP230 were generated and tested
together with BP1801-401 in yeast two-hybrid assays. The results
show that BP230 interacted with BP180 by the Z-Y subdomains
(Fig. 5A,B) and, although the Y
domain alone was able to bind to BP1801-401, albeit slightly less
efficiently (Fig. 5B), no
binding of the Z domain alone could be detected. In turn,
BP180145-230, which interacted weakly with BP2301-555
(Fig. 3), did not bind to the
isolated Z-Y or Y subdomains of BP230 (data not shown), possibly because
interaction of this short stretch of BP180 with BP230 requires additional
sequences flanking the Z-Y subdomains of BP230. Indeed, when two larger
fragments of BP230 containing the Z-Y subdomains (BP2301-555 and
BP2301-887) were tested for binding to BP1801-401 using
a ß-galactosidase assay, they did bind more strongly than the isolated
Z-Y or Y subdomains (Fig.
5C).
|
The observations that in BP180-deficient keratinocytes the localization of plectin in HD-like structures appeared to be somewhat impaired and that the Y domain of plectin is homologous to that of BP230 (Fig. 5E) prompted us to investigate whether plectin can directly bind to BP180. We therefore generated two constructs encoding the Z-Y or Y domains of plectin and tested their binding ability in yeast. As shown in Fig. 5B, both constructs interacted with BP1801-401, although less strongly than the corresponding constructs of BP230 as confirmed by a quantitative ß-galactosidase assay (Fig. 5C). Hence, BP180 is capable of binding to the Y domain of not only BP230 but also of plectin. Surprisingly, we found that the Z-Y and Y domains of desmoplakin, which is a component of desmosomes, also interacted with BP180. As in the case of BP230, the isolated Z-Y and Y subdomains of plectin or desmoplakin did not interact with BP180145-230 (data not shown).
The Z-Y domains of BP230 contain sequences important for its
recruitment into HDs in transfected cells
To assess whether an interaction of BP230 with BP180 is required for the
localization of BP230 in HDs, we generated various HA-tagged BP230 mutants in
which the Z-Y domains had been deleted. Because human keratinocytes lacking
BP230 expression have not yet been identified, we expressed these mutants in
PA-JEB/ß4 cells, whereas immunofluorescence microscopy analyses were
carried out with polyclonal antibodies against the HA-epitope tag to
distinguish the localization of the ectopically expressed BP230 mutant
proteins from that of endogenous BP230. Although, in most cells, a BP230
construct containing the first 1-887 N-terminal amino acids
(BP2301-887) was found diffusely distributed over the cytoplasm
(Fig. 6A), in very few of the
transfected cells it was concentrated in HD-like structures, together with
BP180 (Fig. 6B). By contrast, a
mutant form of BP230, BP2301-2161, which encompasses the rod
domain, was found not only in HD-like structures in a larger proportion of the
cells, but also in aggregates in the cytoplasm, particularly in transfected
cells that strongly expressed the cDNA
(Fig. 6C). Other than
BP2301-2161, BP2301-2161,ZY from which the Z-Y
domains had been deleted was not colocalized with BP180 in HD-like structures,
but was found in small aggregates (Fig.
6D).
|
Because both BP2301-2161 and BP2301-2161,ZY
contain the central rod domain of BP230 and thus have the potential to form
dimers with endogenous BP230, transfected cells were also stained with an
anti-BP230 antiserum raised against a C-terminal fragment of BP230 (amino
acids 1722-2203). Because a large portion of this fragment is part of the two
BP230 mutants, the anti-BP230 antiserum will stain not only endogenous BP230,
but also the two BP230 mutants. Consistent with the localization of
transfected BP2301-2161 in HDs, the staining pattern with the
anti-BP230 and anti-HA antibodies overlapped in transfected cells and was
comparable to that seen with anti-BP230 in untransfected cells, i.e. in HDs
(Fig. 6E). By contrast,
BP2301-2161,
ZY was again found in small aggregates and it
was not obviously co-localized with endogenous BP230 in HDs
(Fig. 6F). Thus, even if
dimerization of BP2301-2161,
ZY mutants with endogenous BP230
occurs, this did not result in the recruitment of the mutated molecule into
HDs.
Collectively, these results suggest that: (1) the Z-Y domain contains
sequences important for the localization of BP230 into HDs, most likely via
binding to BP180, and (2) the presence of the rod domain of BP230 thought to
be implicated in the formation of dimers
(Ruhrberg and Watt, 1997;
Leung et al., 2001
) increases
the correct targeting of the protein into HDs.
BP230 interacts with the third and fourth FNIII repeat of
ß4
In BP180-deficient keratinocytes, the recruitment of BP230 into HD-like
structures is crucially dependent on the re-expression of BP180
(Borradori et al., 1998).
However, in PA-JEB keratinocytes, i.e. in the absence of
6ß4, no
HDs are formed, despite the fact that these cells express BP180 and BP230. We
therefore wondered whether the ß4 subunit might directly bind to BP230
and thus also affects its localization. We tested the binding activity of
several ß4 constructs, in which one or more FNIII repeats were deleted or
in which the CS was progressively shortened, with a BP230 construct containing
the first 1156 amino acids of the N-terminus in yeast two-hybrid assays. As
depicted in Fig. 7, removal of
part of the fourth FNIII repeat resulted in loss of interaction. The deletion
of the first pair of FNIII repeats from the cytoplasmic domain of ß4 had
no effect on binding, but the removal of also the CS abolished the
interaction. Specifically, an interaction between BP230 and ß4 still
occurred with progressive deletions up to amino acid 1436, but not beyond.
These data show that the extreme C-terminal portion of the CS in combination
with the third and the fourth FNIII repeats of ß4 are required for its
binding to BP230.
|
Finally, as a control, the various ß4 constructs were also tested
against the cytoplasmic domain of BP180, BP1801-401. In extension
to previous studies (Geerts et al.,
1999), we found that construct ß41457-1552
encompassing the third FNIII contains the minimal sequences necessary for the
interaction with BP180.
The ß4 subunit interacts with N-terminal sequences specific for
the epidermal isoform of BP230
We next investigated which sequences within BP230 are involved in its
binding to ß4 in yeast. Various BP230 constructs were tested with a
ß4 construct containing the first FNIII repeat up to the fourth repeat,
ß41115-1666 (Fig.
5A). The results indicate that the first stretch of 92 amino acids
at the N-terminus of BP230 is sufficient for its interaction with ß4.
To ascertain that a binding site for ß4 was not accidentally introduced by fusing the Gal4 domain to the N-terminal extremity of BP230, we generated additional mutants as depicted in Fig. 5D. All these constructs were able to interact with ß4, providing evidence that the interaction was indeed dependent on BP230-specific sequences and not on the Gal4 moiety. Furthermore, the results show that the first three amino acids of BP230 are dispensable for this interaction.
Except for the first 56 amino acids, the N-terminal region of BP230 is
almost identical to that of two isoforms, dystonin-1 and -2, encoded by the
BPAG1 gene that also encodes BP230. These two isoforms, previously
thought to be specifically expressed in the nervous system
(Yang et al., 1996), differ
from the BP230 isoform by the presence of an ABD at their extreme N-terminus
that is absent from BP230. To determine whether or not the interaction between
BP230 and ß4 is mediated by the stretch of amino acids 56-92 common to
BP230 and the two dystonin isoforms, we generated constructs comprising amino
acids 1-92, 1-56 or 56-190 of BP230. When these constructs were assayed for an
interaction with ß41115-1666, it was found that amino acids
1-56 of BP230 interacted, although less efficiently than amino acids 1-92,
with ß4, whereas amino acids 56-190 did not
(Fig. 5D). These data indicate
that the interaction of BP230 with ß4 is mediated by sequences specific
for the epidermal isoform, although obviously the common part also influences
binding.
BP230 cannot replace plectin in supporting the formation of HDs
The finding that BP230 can interact with both ß4 and BP180 prompted us
to investigate whether BP230 can replace plectin in supporting the formation
of HDs. For this, we made use of a mutant ß4 subunit
(ß4R1281W) that is unable to interact with plectin, but can
bind to BP180 and BP230 (Geerts et al.,
1999; Koster et al.,
2001
). Stable expression of ß4R1281W in PA-JEB
cells by using retroviral transduction revealed that this mutant can support
the formation of HD-like structures containing BP180 and BP230, consistent
with previous observations using transient transfection protocols
(Geerts et al., 1999
). However,
these HD-like structures appear to be less conspicuous and dense than those
formed in the presence of wild-type ß4 (compare
Fig. 1 and
Fig. 8). Furthermore, in the
vast majority of cells, the ß4 mutant was not co-localized with BP180 or
BP230. From these results, we conclude that: (1) when plectin is not bound to
ß4, ß4 binding to BP180 is strongly reduced, and (2) BP230 cannot
replace plectin in supporting proper localization of BP180 into HDs.
|
![]() |
Discussion |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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The different interactions between the various hemidesmosomal components are depicted in Fig. 9, along with a model that shows how these interactions are likely to contribute to the formation of stable type I HDs.
|
Interaction of BP180 with plectin contributes to their localization
in HDs
The localization of BP180 in HDs containing 6ß4 and plectin has
previously been shown to depend on an interaction of BP180 with the
cytoplasmic domain of ß4 (Borradori et
al., 1997
; Schaapveld et al.,
1998
). Interactions between the
6 subunit and the
extracellular NC16a domain of BP180 are probably also implicated
(Hopkinson et al., 1998
).
Here, we identified a third interaction partner of BP180, i.e. plectin.
The interaction between BP180 and plectin is not sufficiently strong to
induce the formation of HDs in the absence 6ß4, because in
ß4-deficient PA-JEB cells, which contain BP180 and plectin, no HDs are
formed. However, it may strengthen the interaction between
6ß4,
BP180 and plectin in a three-molecular complex, thereby ensuring their proper
incorporation in HDs. Consistent with this idea, we found less plectin in the
ß4-positive adhesion structures of the BP180-deficient GABEB
keratinocytes than in those of PA-JEB/ß4 keratinocytes, at least as
assessed by immunofluorescence. Moreover, studies with PA-JEB keratinocytes
stably expressing a mutant ß4 subunit (ß4R1281W) that is
unable to interact with plectin (Geerts et
al., 1999
; Koster et al.,
2001
) revealed that the incorporation of BP180 into cell-substrate
structures is severely compromised. Only few cells formed HD-like adhesion
structures containing
6ß4, BP180 and BP230. Thus, although
6ß4 can bind to BP180, proper incorporation of this protein into
HDs only occurs when plectin is also available.
The results with the PA-JEB/ß4R1281W cells also suggest
that the role of plectin in supporting efficient localization of BP180 in HDs
cannot be replaced by BP230. This is surprising considering the fact that
BP230 can interact with both ß4 and BP180. Both BP230 and plectin
probably bind via sequences contained in their Y subdomain to the same region
(amino acids 145-230) on BP180 (see below). Thus, it is possible that, when
plectin is not bound to 6ß4, it competes with BP230 for binding to
BP180. However, given the fact that the binding activity of BP180 for BP230 is
greater than that for plectin, this is not very likely. An alternative
possibility, which we favor, is that binding of plectin to ß4 is required
for activating the ß4 cytoplasmic domain, by unfolding it and rendering
it accessible for interaction with BP230 (see also
Fig. 9). Evidence for such an
intramolecular folding of the ß4 cytoplasmic domain has been presented in
both biochemical and yeast two-hybrid assays
(Rezniczek et al., 1998
;
Geerts et al., 1999
).
Regardless of what the mechanism might be, it is clear that plectin and BP230
are not exchangeable in their ability to support the formation of proper
HDs.
In view of our finding that binding to BP180 is mediated by the Y domain of
plectin, it is interesting to note that Pulkkinen et al.
(Pulkkinen et al., 1996) have
described a deletion of three amino acids, QEA, in this region in a patient
with epidermolysis bullosa simplex associated with muscular dystrophy
(EBS-MD). A pathological consequence of this deletion, therefore, could be
that plectin is unable to bind to BP180, and that the ternary complex of
6ß4, BP180 and plectin, which serves as a platform for the
incorporation of BP230 into HDs (see below), cannot be (properly) formed,
leading to deficient assembly of HDs. Future experiments, however, should
reveal whether indeed the deletion of these three amino acids in the Y domain
prevents the binding of plectin to BP180.
In previous studies (Hopkinson et al.,
1995; Borradori et al.,
1997
), it was found that the 36 most N-terminal amino acids of the
cytoplasmic domain of BP180 were implicated in the recruitment of BP180 into
HDs. However, in this study, no evidence was found for the involvement of this
region in the localization of BP180 in HDs. An important difference between
our study and the study by Borradori et al.
(Borradori et al., 1997
) is
that the latter used a chimeric protein, which consisted of the membrane
targeting sequence of K-ras fused to the cytoplasmic region of BP180, whereas
in the present study full-length BP180 has been used. Full-length BP180 forms
trimers (Hirako et al., 1996
)
and, therefore, it is predicted that it interacts more strongly with ß4
than the chimeric monomeric molecule lacking the collageneous extracellular
domain. In that case, binding of full-length BP180 to ß4 may have become
less dependent on additional sites of interaction that reside on the
cytoplasmic domain of BP180. However, it remains unexplained why we have not
been able to detect an interaction between ß4 and a BP180 mutant
containing the first 36 amino acids (BP1801-147) in yeast
two-hybrid interaction assays. Perhaps the fusion of the N-terminus of BP180
to the Gal4 (BD) has destroyed the ß4-binding site in the N-terminus of
BP180. Although the importance of the first 36 amino acids of BP180 for the
binding to ß4 could not be confirmed in the yeast two-hybrid system, the
finding that both BP1801-231 and BP180145-401, but not
the amino acids 145-230 that are shared by these two constructs, interact with
ß4 suggests that there are two ß4 binding sites on BP180. One of
them is located in the first 230 N-terminal amino acids of the BP180
cytoplasmic domain and the other is located C-terminally to this region.
Although the ß4-binding site in the first 230 N-terminal amino acids of
BP180 may overlap with that for BP230 (see below), the two sites are clearly
not identical. Binding of ß4 still occurs when the region of amino acids
145-230, which is essential and sufficient for the interaction with BP230, has
been deleted from the cytoplasmic domain of BP180
(BP1801-401,
145-230). In line with the results obtained in
yeast, a full-length BP180 molecule carrying this deletion became co-aligned
with ß4 in HD-like structures.
Expression of BP180 is required for proper localization of BP230 in
HDs
The observation that in BP180-deficient keratinocytes, BP230 was not
co-localized with 6ß4 and plectin in HD-like structures but
diffusely distributed over the cytoplasm indicates that ß4 and plectin
are not sufficient for the proper localization of BP230 into HDs. Efficient
recruitment of BP230 only occurs when BP180 is also expressed in these cells
(Schaapveld et al., 1998
).
However, it is likely that the additional binding site for BP230 that is
provided on ß4 also contributes to the recruitment of BP230 in HDs. Thus,
a model emerges in which
6ß4 interacts with plectin, after which
they bind BP180 in a ternary complex. Ultimately, BP230 binds to two
components in the complex, i.e.
6ß4 and BP180, and stable type I
HDs are then formed (Fig.
9).
Our results show that sequences in the C-terminal domain of ß4 (the
second pair of FNIII repeats) interact with residues 1-56 of BP230, which are
contained within the N-terminal domain of BP230 (residues 1-980) that has been
shown to be involved in the interaction with ß4
(Hopkinson and Jones, 2000).
In the same study, a second ß4 binding site on BP230 has been identified
in its C-terminal domain, residues 1812-2649. By contrast, we found that a
fragment of BP230 encompassing residues 2077-2649 did not associate with
ß4, but bound to intermediate filaments in yeast and was able to decorate
intermediate filaments in transfected cells (J. Koster, unpublished
observations). It is possible that the binding site for ß4 is located in
the region of amino acids 1812-2077 of BP230 and that this region contributes
to the recruitment of BP230 into HDs. In support of this, in transfection
experiments with PA-JEB/ß4 cells, BP2301-887 was localized in
only very few of the HDs, whereas a BP230 construct, which also includes the
rod domain and the region 1812-2077 (BP2301-2161), was detected in
a higher percentage of the HDs. The rod domain, which is an integral part of
all members of the plakin family and is responsible for the dimerization of
these molecules, may also contribute to the localization of BP230 in HDs by
its ability to dimerize the binding sites in the N- and C-terminal
domains.
BP230 interacts with BP180 via its Y domain
In a recent study (Hopkinson and Jones,
2000) it was shown that the first 170 amino acids, as well as
amino acids 555-700 of an N-terminal 700 amino acid fragment of BP230, are
required for its binding to BP1801-517. Our finding that
BP2301-555 interacted with BP1801-401 shows that the
amino acids 555-700 of BP230 are not essential. Furthermore, no binding was
detected when a fragment containing the first 172 amino acids of BP230 was
used, whereas this fragment did bind to ß4. Binding of BP180 to BP230
appeared to be mediated by the Y subdomain of BP230. The importance of the Y
subdomain for binding is further supported by the fact that in transient
transfection experiments using PA-JEB/ß4 cells, BP2301-2161,
but not the construct from which the Z-Y domain had been deleted
(BP2301-2161,
ZY) was recruited into HDs. However, the latter
construct had a tendency, when expressed in transfected cells, to form
aggregates in the cytoplasm, which might prevent its recruitment into HDs. The
fact that Hopkinson and Jones (Hopkinson
and Jones, 2000
) found that sequences additional to the Z-Y
subdomains of BP230 are required for interaction with BP1801-517,
in contrast to our results with BP1801-401, may be explained by
their use of a less-sensitive assay system. This is supported by the finding
that BP1801-401 binds more strongly to BP2301-555 and
BP2301-887 than to the isolated Z-Y and Y subdomains of BP230 and,
furthermore, that BP1801-401, but not BP180145-230, can
bind to the Z-Y and Y subdomains of BP230. Thus, it seems that additional
sequences stabilize the interaction of the Y subdomain with BP230.
We found, unexpectedly, that the Y subdomain of desmoplakin was also able
to interact with BP180. In fact, desmoplakin is not found in HDs
(Green and Jones, 1996), but
in desmosomes, in which BP180 is not present. It is possible that desmoplakin
does not recruit BP180 into desmosomes (despite its ability to directly
interact with BP180) because
6ß4 is absent from these adhesion
complexes. This assumption is indirectly supported by the observation that
6ß4 is necessary for the efficient recruitment of BP230 and
plectin into HDs, despite the ability of these two molecules to bind to BP180
(Borradori et al., 1997
;
Schaapveld et al., 1998
).
Alternatively, the interaction between plakins and cell-surface receptors
might be subject to regulation. Clearly, these results further stress the
important role of
6ß4 in the assembly of HDs.
Comparison of HD formation in vivo and in cultured keratinocytes in
vitro
The crucial role of plectin in the formation of HDs as observed in cultured
keratinocytes does not seem to be supported by findings in vivo. Indeed, it
has been shown that HDs can be formed in plectin-deficient mice, although the
HDs appear to be rudimentary and their number is reduced
(Andrä et al., 1997). We
think that this discrepancy is partly due to a different role of BP180 in
vitro and in vivo. As we have shown in vitro, the clustering and polarization
of BP180 at the cell basis is entirely dependent on the presence of
6ß4 and plectin, probably because the specific ligand for BP180 is
not produced by keratinocytes in culture. No polarized expression of BP180 was
observed in ß4-deficient PA-JEB keratinocytes
(Schaapveld et al., 1998
). By
contrast, in vivo, where the ligand for BP180 is almost certainly present and
deposited into the basement membrane (it may be produced by the mesenchymal
cells), clustering of BP180 is probably less dependent on plectin and
6ß4, and thus can also occur in the absence of plectin. BP230 may
then subsequently interact with the BP180-
6ß4 complexes, resulting
in the formation of rudimentary HDs. In support of this, we have found that
when
6ß4 is absent, as in patients with PA-JEB, some HDs can be
observed, the formation of which may have been initiated by interaction of
BP180 with an unidentified ligand in the epidermal basement membrane
(Niessen et al., 1996
).
In conclusion, we have characterized interactions between several molecules that are crucially involved in the assembly of HDs. We show that the different hemidesmosomal components can interact with multiple other components and that often interactions with more than one component are required for their efficient recruitment into HDs. Our findings that the cytoplasmic domain of ß4 bears a scaffold of FNIII repeats responsible for interactions with the three major hemidesmosomal components plectin, BP180 and BP230, underscores its essential role in the formation of HDs.
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