1 Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121,
1066 CX Amsterdam, The Netherlands
2 Division of Pathology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066
CX Amsterdam, The Netherlands
3 Division of Pathology, Academic Medical Center, Meibergdreef 9, 1105 AZ
Amsterdam, The Netherlands
Author of correspondence (e-mail: asonn{at}nki.nl )
Accepted 21 December 2001
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Summary |
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The anti-CD151 antibody TS151R detects an epitope at a site at which CD151
interacts with integrins, and therefore it cannot react with CD151 when it is
bound to an integrin. Comparison of the straining patterns produced by TS151R
with that by of an anti-CD151 antibody recognizing an epitope outside the
binding site (P48) revealed that most tissues expressing one or more
laminin-binding integrins reacted with P48 but not with TS151R. However,
smooth muscle cells that express 7ß1 and renal tubular epithelial
cells that express
6ß1 were stained equally well by TS151R and
P48. These results suggest that the interactions between CD151 and
laminin-binding integrins are subject to cell-type-specific regulation.
Key words: Tetraspanin, Laminin receptor, Integrin, Dystroglycan, Lutheran blood group antigens
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Introduction |
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On the basis of the stability of the formed complexes, interactions of
tetraspanins can be classified into three categories
(Class et al., 2001).
CD151-
3ß1 complexes represent the exceptionally strong, highly
stable and selective level 1 association. Most interactions between
tetraspanins and integrins, for example, the association of CD151 with
6ß4, are less strong and considered to be level 2; they are
numerous, fairly stable and selective. CD151-
6ß1 interactions are
either level 1 or level 2, probably depending on the cell type in which these
molecules are expressed. Level 3 interactions, which occur mostly between
tetraspanins and molecules other than tetraspanins or integrins, are indirect,
numerous and are the least stable of the interactions. The level 1 interaction
of CD151 with
3 occurs between the large extracellular loop of CD151
and the extracellular membrane proximal region of the
3 subunit
(Yauch et al., 2000
). A CD151
epitope recognized by the mAb TS151R is located at this site of interaction
(Serru et al., 1999
;
Yauch et al., 2000
). Recently,
it was shown that the binding site on CD151 for tetraspanins is different from
the one involved in the binding to integrins
(Berditchevski et al.,
2001
).
Integrins are heterodimeric cell surface receptors consisting of a
non-covalently associated and ß subunit, which link the
extracellular matrix to the cytoskeleton
(Hynes, 1992
;
Schwartz et al., 1995
;
van der Flier and Sonnenberg,
2001
). The integrins
3ß1,
6ß1 and
7ß1 bind preferentially to laminin and contain sequential and
structural homologies. During maturation, the
3,
6 and
7
integrin subunits are post-translationally cleaved into a heavy and a light
chain. Furthermore, the mRNAs for these integrin subunits can be alternatively
spliced, leading to extracellular and cytoplasmic variants (Collo and
Quaranta, 1993; Hogervorst et al.,
1993
,Hogervorst et al.,
1993
; de Melker et al.,
1997
). The ligand binding and signaling function of these integrin
variants is potentially different, which during development may lead to
changes in function when variants are switched
(Ziober et al., 1993
;
Thorsteindóttir et al.,
1995
; Schober et al.,
2000
).
While much is known about the function and biochemical properties of
integrins, the exact function of the associated tetraspanins is still elusive
but is gradually becoming unraveled. Previously, we have shown that CD151 is a
component of hemidesmosomes, specialized structures in the basal epidermal
cell layer that link intermediate filaments to the extracellular matrix
(Sterk et al., 2000). Others
have shown the involvement of CD151 in cell adhesion
(Hasegawa et al., 1998
;
Fitter et al., 1999
), (tumor)
cell migration and metastasizing
(Yáñez-Mó et al.,
1998
; Yauch et al.,
1998
; Sincock et al.,
1999
; Sugiura and
Berditchevski, 1999
;
Berditchevski and Odintsova,
1999
; Testa et al.,
1999
), vesicular transport of integrins
(Sincock et al., 1999
),
signaling (Berditchevski et al.,
1997
; Yauch et al.,
1998
; Sugiura and
Berditchevski, 1999
;
Berditchevski and Odintsova,
1999
; Zhang et al.,
2001
), cell polarization
(Yáñez-Mó et al.,
2001
), wound healing
(Peñas et al., 2000
)
and neurite outgrowth (Stipp and Hemler,
2000
).
In vivo, CD151 is expressed by a large variety of cells of epithelial and
mesenchymal origin. The tissue distribution of CD151 shows a striking overlap
with that of the combined patterns of expression of 3ß1,
6ß1,
6ß4 and
7ß1 - all laminin-binding
integrins (Sincock et al.,
1997
). This suggests that CD151 preferentially interacts with the
laminin-binding integrins.
Laminin is a basement membrane component that, by interaction with its cell
surface receptors, influences cell shape, movement and differentiation
(Colognato and Yurchenko, 2000). Besides representing a ligand for integrins,
laminin also interacts with the recently discovered Lutheran (LU) molecule
expressed on erythrocytes (El Nemer et
al., 1998; Parsons et al.,
2001
; Udani et al.,
1998
) and with dystroglycan (DG), an integral component of the
dystrophin glycoprotein complex (DGC) originally detected in skeletal muscle
and subsequently in epithelia (Ibraghimov-Breskovnaya et al., 1992;
Durbeej and Campbell,
1999
).
In the present study we investigated the expression of various tetraspanins
and laminin receptors in tissues that strongly express one or more of the
laminin-binding integrins, such as kidney (3ß1 in glomerular
cells;
6ß1 in tubular cells), skin (
3ß1 and
6ß4) and muscle (
7ß1). At sites of contacts between
cells and the matrix, CD151 is the only one of the tetraspanins tested that is
always colocalized with the laminin-binding integrins. The intimate relation
between CD151 and the laminin-binding integrins is also stressed by their high
stoichiometric interaction and the increase of CD151 cell surface expression
in K562 cells transfected with one of these integrins. By immunoprecipitation
it was demonstrated for the first time that CD151 is associated with
7ß1, and that their firm association is comparable to that of the
association between
3ß1 and CD151. Cytoplasmic or extracellular
splice variants or mutations preventing cleavage of the integrin
subunit were found to have no influence on the formation of integrin-CD151
complexes. Although there is a considerable overlap in expression between the
laminin receptors from the three different receptor families and CD151, no
interaction between CD151 and either LU or DG could be identified in vitro.
Finally, we provide evidence that the same site on CD151 interacts with all
the different laminin-binding integrins. Furthermore, the exposure of a CD151
epitope involved in this binding is cell type specific and may vary depending
on the (patho) physiological status of the tissues involved.
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Materials and Methods |
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The other mouse mAbs against CD151 were all gifts: 5C11 from M. E. Hemler
(Dana-Farber Cancer Institute, Boston, MA), 8C3 from K. Sekiguchi (Osaka
University, Osaka, Japan), Sfa-1 from H. Hasegawa
(Hasegawa et al., 1996) (Ehime
University, Ehime, Japan), LIA1/1 from F. Sánchez-Madrid (Hospital de
la Princesa, Madrid, Spain), IIG5 from M. Humphries (The University of
Manchester, Manchester, UK) and TS151 and TS151R
(Yauch et al., 2000
;
Serru et al., 1999
) from E.
Rubenstein (Hôpital Paul Brousse, Villejuif Cedex, France). The mouse
mAbs MEM62 (CD9;
(Ho
ej
í and
Vl
ek, 1991
)), MEM53 (CD53
(Angelisová et al.,
1994
)), M38 (CD81 (Imai and
Yoshie, 1993
)) and C33 (CD82
(Imai and Yoshie, 1993
)), were
generously provided by V. Ho
ej
í (Institute of Molecular
Genetics, Prague, Czech Republic). F. Berditchevsky (The University of
Birmingham, Birmingham, UK) kindly supplied the mouse mAb 6H1 (CD63
(Berditchevsky et al., 1995)). The sheep anti-mouse and donkey anti-rabbit
horseradish-peroxidase-coupled secondary antibodies were purchased from
Amersham (Harlington Heights, IL). FITC-or Texas Red-conjugated goat
anti-mouse, goat anti-rat or goat anti-rabbit secondary antibodies were
obtained from Rockland (Gilbertsville, PA). The FITC- and TRITC-conjugated
goat anti-mouse isotype specific antibodies were obtained from Nordic
Immunochemical Laboratory (Tilburg, The Netherlands). Phycoerythrin-conjugated
goat anti-mouse or anti-rat antibodies were obtained from Jackson laboratories
(Westgrove, PA).
Generation of retroviral expression constructs and stable cellular
transduction
The full-length c-DNA encoding the X1 and X2 splice variants of the
extracellular domains of human 7B were a kind gift from D. Mielenz and
K. von dem Mark (University of Erlangen, Erlangen, Germany).
7BX1 and
-X2 c-DNA was released from pUC-18 or pcDNA3 respectively, by digesting with
EcoRI. The resulting fragments were ligated into the retroviral
LZRS-IRES-zeo expression vector, a modified LZRS retroviral vector conferring
resistance to zeocin (Kinsella and Nolan,
1996
; van Leeuwen et al.,
1997
), to give an LZRS-IRES-zeo-
7BX1 or -
7BX2
vector. These constructs were then introduced into the Phoenix packaging cells
(Kinsella and Nolan, 1996
) by
the calcium phosphate precipitation method and virus containing supernatant
was collected. K562 cells were infected with the recombinant virus by the
DOTAP method (Boehringer Mannheim Corp.). After incubation for 8 hours at
37°C, infected cells were grown in medium as described below for the
wild-type K562 cells. Cells expressing
7 at their surface were analyzed
and sorted using FACScan® flow cytometer (Becton Dickinson, Mountain View,
CA).
Cell lines
The human erythroleukemic cell line K562 was maintained in RPMI-1640
supplemented with 10% heat-inactivated fetal calf serum (Life Technologies,
Paisly, UK), 100 U/ml penicillin and 100 µg/ml streptomycin (both from Life
Technologies). K562 cells stably expressing 3ß1,
6ß1
and
6ß4 were established as described previously
(Delwel et al., 1993
;
Niessen et al., 1994
). An
established cell line of human glomerular visceral epithelial cells
(Krishnamurti et al., 1996
)
was cultured in medium made up of 1 volume of each Dulbecco's modified Eagle's
medium (DMEM) (ICN, Costa Mesa, CA) and Hams F-10 (Life Technologies),
supplemented with 5% Nu serum (Becton Dickinson, Bedford, MA), 25 ng/ml
prostaglandin E1, 0.5 nM tri-iodothyronine, 10 nM sodium selenite,
5 µg/ml transferrin, 50 nM hydrocortisone, 5 µg/ml insulin (all hormones
from Sigma, St. Louis, MO), 100 U/ml penicillin and 100 µg/ml streptomycin.
Human proximal tubular epithelial cells (PTEC) also known as HK-2 cells are
HPV 16-immortalized cells purchased from the American Type Culture Collection.
Cells were grown in conditioned medium consisting of 1 volume of DMEM and 1
volume Ham F-12, supplemented with 5% heat-inactivated FCS, 100 U/ml
penicillin and 100 µg/ml streptomycin, 2 mM L-glutamine, 5 µg/ml
insulin, 5 µg/ml transferrin, 5 ng/ml sodium selenite, 20 ng/ml
tri-iodothyronine, 5 ng/ml hydrocortisone, 5 ng/ml prostaglandin E1
and 5 ng/ml epidermal growth factor (all from Sigma, St. Louis, MO). Cells
were grown at 37°C in a humidified, 5% CO2 atmosphere.
Immunoprecipitation of 125I-labeled cells
Transfected K562 cells, stably expressing 3A,
3B,
6A,
6B or
6ARGGD mutant, were surface labeled with
125I by the lactoperoxidase method as previously described
(Sonnenberg et al., 1987
).
Cells were solubilized in lysis buffer containing 1% (w/v) CHAPS (Sigma), 5 mM
MgCl2, 25 mM Hepes, pH 7.5, 150 mM NaCl and proteinase inhibitors
(1 mM phenylmethanesulphonyl fluoride, 10 µg/ml soybean trypsin inhibitor
and 10 µg/ml leupeptin). Lysates were clarified at 20,000 g
and precipitated with protein A-sepharose beads CL-4B (Amersham Pharmacia
Biotech Inc., Uppsala, Sweden), which had previously been sequentially
incubated with rabbit anti-mouse IgG and the precipitating antibody. After
incubation for 1.5 hour at 4°C, the beads carrying the immune complexes
were washed and treated with sodium dodecyl sulfate (SDS) sample buffer.
Precipitated proteins were analyzed by SDS-polyacrylamide gel
electrophoresis.
Immunoblotting
Wild-type and transfected K562 cells stably expressing vß1,
3ß1,
6ß1,
6ß4,
7X1ß1 or
7X2ß1, and podocytes and PTEC were lysed in 1% (w/v) CHAPS buffer
with proteinase inhibitors. Alternatively, cells were lysed in 1% (v/v)
Nonidet P-40, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl and 1 mM EDTA. Lysates were
incubated for 1 hour at 4°C with Gamma Bind-G Sepharose beads or protein
A-Sepharose beads (Amersham Pharmacia Biotech, Uppsala, Sweden), previously
incubated overnight at 4°C with the precipitating antibodies. The beads
carrying the immune complexes were washed three times with lysis buffer and
two times with PBS at 4°C. The immune complexes were eluted by the
addition of sample buffer, heated at 95°C or 65°C and separated by
SDS-PAGE under reducing (2% ß-mercaptoethanol) or non-reducing
conditions. After electrophoresis, gels were electrophoretically transferred
to a PVDF membrane (Immobilon-P, Bedford). Blots were stained with Coomassie
blue to indicate the markers, destained in 45% methanol, 5% acetic acid in
demineralized water and blocked for 30 minutes with 5% dry milk in TBST-buffer
(10 mM Tris, pH 7.5, 150 mM NaCl, 0.3% Tween-20). Subsequently blots were
incubated for 1 hour with primary antibodies in 0.5% dry milk in TBST. Primary
antibodies were mAbs 29A3 against
3 (neat supernatant) or 8C3 against
CD151 (1:1,000), or polyclonal antibodies against
3 (1:100),
7B
(1:100),
v (1:100) or ß-dystroglycan (1:100). After washing three
times with TBST/0.5% dry milk, blots were incubated for an additional hour
with secondary sheep anti-mouse, donkey anti-rabbit or goat anti-rat
Ig-horseradish-peroxidase-coupled, diluted 1:5,000 or 1:1,000 respectively, in
0.5% dry milk in TBST. After washing three times with TBST, proteins were
visualized by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech.),
as described by the manufacturer.
Analyses of integrin and CD151 surface expression
The surface expression of integrin subunits and CD151 on wild-type and
transfected K562 cells was assessed by flow cytometry. 5x105
cells were washed three times with PBS containing 2% FCS (PBS/FCS), followed
by incubation with the primary antibody or antibodies for 1hour. The cells
were washed three times with PBS/FCS and then incubated with
phycoerythrin-conjugated goat anti-mouse or anti-rat antibodies for another
hour. After another three washes with PBS/FCS, cells were resuspended in PBSA
and analyzed with a FACScan® flow cytometer.
Immunohistochemistry and immunofluorescence of tissue sections
Cryostat sections (3 µm thick) from human kidney, skin, smooth, striated
or heart muscle were fixed in acetone for 10 minutes at 4°C. For
immunoperoxidase staining, endogenous peroxidase was blocked by 0.1% NaN3 and
0.3% H2O2 in PBS for 10 minutes. For blockage, PBS
containing 2% BSA (PBSA) was used for 10 minutes, followed by overnight
incubation with the primary antibody at 4°C. Anti-rabbit or anti-mouse
horseradish-peroxidase-conjugated secondary antibodies (PowerVision,
Technologies Co., Daly City, CA) were added for 30 minutes and visualized by
hydrogen peroxide and 3',3'-amino-9-ethyl carbazole (Sigma
Chemical Co.). Sections were counterstained with hematoxylin and mounted in
Depex (Britisch Drug House Chemicals, Poole, UK). In between steps, sections
were washed three times with PBS. For double-labeled immunofluorescence
analyses, sections were blocked in PBSA for 30 minutes and incubated with the
primary antibody diluted in PBSA overnight at 4°C. The sections were
washed three times with PBSA and then, depending on the primary antibodies
used, incubated with different combinations of goat anti-mouse, goat anti-rat
or goat anti-rabbit FITC- or Texas-Red-conjugated antibodies for 45 minutes.
After washing twice with PBS, sections were mounted in Vectashield (Vector
Laboratories Inc., Burlingame, CA) and viewed under a Leica TCS NT confocal
laser-scanning microscope equipped with an Ar/Kr laser (Leica Microystems,
Heidelberg, Germany). Unless otherwise mentioned, both procedures were
performed at room temperature.
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Results |
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Analysis of kidney tissues by immunoperoxidase staining, using mAb P48,
revealed strong expression of CD151 in glomeruli, at the basal surface of
tubular epithelial cells and in vascular smooth muscle cells
(Fig. 1A). Double
immunofluorescence analysis revealed a striking colocalization with
3ß1 (Fig. 2A) and
DG (Fig. 2B) within glomeruli
in a predominant, continuous GBM-bound pattern. Along the GBM, DG was only
partially co-distributed with
3ß1
(Fig. 2C), whereas LU was
absent from the glomerulus but strongly expressed in the subendothelial matrix
and along the basal side of the tubules, colocalized with
6ß1
(Fig. 2D). Like CD9 and CD63,
the LU molecule is also focally distributed throughout the cytoplasm of
tubular epithelial cells (not shown). Transport of LU to the apical or
basolateral side of polarized cells has been described before
(El Nemer et al., 1999
). This,
together with the presence of LU at the site of cell-cell contacts between
keratinocytes (see below), suggests that LU is not merely a laminin receptor
but has other functions as well. The tetraspanins CD9 (not shown), CD63
(Fig. 2E) and CD81
(Fig. 2F) are present in
glomerular cells but are not colocalized with
3ß1 along the GBM.
Glomerular cells do not express CD53 or CD82, which however are present on a
subset of the distal tubules together with CD9 and CD81, the latter being
weakly expressed by all tubular cells (not shown).
|
|
Immunolocalization of CD151 in skin revealed strong reactivity at the
basolateral surface of basal keratinocytes
(Fig. 1B), where the integrins
3ß1 and
6ß4 are also concentrated
(Sterk et al., 2000
). Only
very small amounts of DG are present at the dermo-epidermal junction, whereas
LU is focally expressed on all basal keratinocyte surfaces (not shown). The
tetraspanins CD9 and CD81 are not only present in the basal cell layer but
also in the more superficial epidermal cell layers. They are absent from the
dermo-epidermal junction but are concentrated at inter-keratinocytic contacts
(not shown). CD63 is only expressed by melanocytes present between the basal
keratinocytes (not shown).
Skeletal, cardiac and smooth muscle cells all express CD151 at their plasma
membrane (Fig. 1C,
Fig. 9A-C). Additionally, CD151
is present at the costamers in skeletal and cardiac muscle and the
intercalated discs in the heart. In these cells, it is colocalized with
7Bß1 (Fig. 3A,C)
and DG (not shown). Besides CD151, smooth muscle cells express the
tetraspanins CD9 and CD81 and some CD53 and CD82 (not shown). Small amounts of
CD63 and CD81 are present at the sarcolemma of skeletal and heart muscle and
at the intercalated discs. LU is not expressed in myocytes (not shown). In
peripheral nerves, also present in the smooth muscle sections,
6ß4,
7ß1, LU, CD9, CD63, CD81 and CD151 are all
localized at the endo- and perineurium (not shown). In summary, CD151 is
extensively colocalized with various laminin receptors, both integrins and
non-integrins, at sites of cell-matrix interactions.
|
|
3 and
6 are coprecipitated with CD151
To determine whether CD151 is also able to associate with the integrin
3 and
6 subunits, K562 cells stably transfected with
3 or
6 were 125I-surface-labeled and lysed in 1% CHAPS. As shown
in Fig. 4, antibodies against
3 precipitated
3ß1 and antibodies against
6,
6ß1 from lysates of
3- and
6 transfected K562 cells,
respectively. The transfected integrins were also present in precipitates
prepared with antibodies against CD63, CD81 and CD151. However, although large
amounts of
3ß1 and
6ß1 were coprecipitated with CD151,
only small amounts were coprecipitated with CD63 or CD81. This difference is
not due to differences in the level of expression of the proteins, as all
three tetraspanins proved to be present at substantial levels in K562 cells,
as shown by FACS analysis (Yauch et al.,
1998
) (L.M.T.H. and A.S., unpublished). Rather, it is the result
of the high stoichiometric binding of CD151 to the laminin-binding integrins,
which indicates the unique and specific role of CD151 in the formation of
these stable complexes. Furthermore, immunoprecipitates of CD63 and CD151 from
the two transfected K562 cell lines, contained a protein of the size of CD81
(23 kDa). Because it is known that tetraspanins can interact with other
tetraspanins, we suspect that CD81 is coprecipitated because it is associated
with CD63 and CD151, and not, as in the case of CD151, with integrins. CD63,
which migrates as a smear between 45-60 kDa on gels, could not be detected in
the various immunoprecipitations, probably because it is labeled only poorly
with 125I. As anticipated, similar amounts of the
3 and
6 subunits were coprecipitated with CD151 from lysates of K562 cells
transfected with the cytoplasmic A or B variants of
3 or
6 (not
shown). Thus, the formation of CD151-
3ß1 or CD151-
6ß1
complexes is not influenced by the nature of the splice variants in the
integrin molecules.
|
CD151 stably associates with the two extracellular variants (X1 and
X2) of 7B
Association of CD151 with either splice variant of the extracellular domain
X1 or X2 of the laminin receptor 7ß1 was investigated in
coimmunoprecipitation experiments; the strength of the interaction was also
tested by using different lysis buffers. CHAPS is considered to be a mild
detergent in which weak interactions between different molecules are not
disrupted, whereas Nonidet P-40 is more stringent leaving only stable
complexes intact. As shown in Fig.
5 (upper panel),
7 is coprecipitated with CD151 from K562
cells stably expressing either the X1 or X2 splice variant, and vice versa
CD151 is co-precipitated with
7 (lower panel). The
7 subunit and
CD151 were precipitated in comparable amounts irrespective of the lysis buffer
used. Thus, CD151 not only associates with the
3ß1 and
6ß1 integrins but also with
7ß1, the strength of this
interaction being comparable with that with
3ß1.
|
No influence of extracellular cleavage from the integrin
subunit on CD151 binding
The extracellular domain of the integrin 3,
6 and
7
subunits is cleaved during biosynthesis into a heavy and a light chain. This
has been shown to be required for proper insideout signaling by
6ß1 in K562 cells but has no influence on ligand binding
(Delwel et al., 1996
). When
the RKKR sequence (amino acids 876-879) is mutated to RGGR, cleavage into a
heavy and a light chain cannot occur. For the formation of stable complexes of
tetraspanins and integrins, their extracellular domains are essential
(Yauch et al., 2000
). To
determine whether cleavage influences the binding of integrins to CD151, K562
cells were transfected with the
6ARGGR-mutant (uncleaved).
Cells were 125I-surface-labeled, lysed in 1% CHAPS and the lysates
precipitated with antibodies against
5,
6, ß1, CD81 or
CD151. As shown in Fig. 6,
6ß1 could be precipitated by antibodies against
6 or
ß1 as well as by antibodies against CD81 or CD151. Consistent with
previous results (Fig. 4), the
amount of
6 precipitated by anti-CD81 is much smaller than that
precipitated by anti-CD151. The
5ß1 integrin that is endogenously
present on K562 cells is only precipitated by antibodies against
5 or
ß1, not by antibodies against either of the two tetraspanins, CD81 or
CD151.
|
Availability of the CD151 epitope recognized by antibody TS151R in
vitro and in vivo
TS151R is a monoclonal antibody directed against an epitope on CD151 that
is located at the binding site for 3, whereas P48 recognizes an epitope
outside this binding site (Serru et al.,
1999
; Yauch et al.,
2000
). To investigate whether the CD151 epitope recognized by
TS151R is also involved in the binding to
7ß1, lysates of K562
cells stably expressing the integrin
7ß1 were subjected to
immunoprecipitation with mAb TS151R, and the presence of
7 was tested
by immunoblotting. Immunoprecipitations prepared from lysates of K562 cells
that were transfected with cDNAs encoding the
3- or
V-subunits
served as positive and negative controls, respectively. As shown in
Fig. 7, the
3 and
7 integrins were only precipitated by P48 and not by TS151R. The
Vß1 integrin was not precipitated by either of the antibodies.
Thus, the TS151R epitope is not only masked when CD151 is associated with
3ß1 but also when it is bound to
7ß1.
|
Next we compared the distribution of the CD151 epitope recognized by TS151R
with that by P48 (Table 1).
Kidney sections incubated with TS151R (Fig.
8A,C) show hardly any fluorescent staining in the glomerular cells
that express 3ß1 (Fig.
8B,C), whereas in the tubuli expressing
6ß1, staining
is comparable to that of mAb P48. In the skin, where CD151 is not only
associated with
3ß1 but also with
6ß4
(Fig. 8E,F,H,I), the staining
by TS151R (Fig. 8G,I) is weak
compared with that by mAb P48 (Fig.
8D,F). Staining of the different muscle tissues expressing
7ß1 varied: all three muscle tissues were strongly stained by P48
(Fig. 1C,
Fig. 9A,B), smooth muscle cells
were also strongly stained by TS151R (Fig.
9C), cardiomyocytes only weakly at the intercalated discs
(Fig. 8J,L), and striated
muscle cells were not stained at all by this antibody
(Fig. 9D). Staining of
cardiomyocytes for
7 expression is shown in
Fig. 8K,L. For further
comparison and the possible recognition of CD151 epitopes involved in the
binding of integrins, all the above tissues were incubated with a panel of
other antibodies against CD151. This resulted in staining patterns comparable
to those produced by P48 (not shown). Thus, binding of CD151 to
laminin-binding integrins leads to masking of the TS151R epitope in many, but
not all, types of cells in vivo.
|
|
Level of CD151 cell surface expression is integrin dependent
Previously it has been shown that expression of 3ß1 in K562
cells results in an increase in the surface expression of CD151
(Yauch et al., 1998
). To
determine whether this ability of
3ß1 to induce CD151 expression
is a general property of laminin-binding integrins, the surface expression of
CD151 was assessed by flow cytometry (FACScan) of transfected K562 cells
stained with mAb P48. As shown in Fig.
10, the expression of transfected
V had little or no effect
on the expression of CD151. However, expression of
3ß1,
6ß1,
6ß4,
7X1ß1 or
7X2ß1 in
K562 cells increased the expression of CD151 three- to sixfold. Staining of
the cells with TS151R revealed that the CD151 epitope, which is recognized by
this antibody, is masked, and it is not dependent on the type of
laminin-binding integrin that the K562 cells were transfected with. These
results are consistent with the results of the above biochemical study showing
that the TS151R epitope is not expressed on K562 cells expressing different
laminin-binding integrins. However, also no CD151 was detected with TS151R on
untransfected K562 cells. We therefore cannot exclude non-reactivity of this
antibody in fluorescence studies, because of low affinity when the number of
available epitopes is restricted. Furthermore, the results indicate that the
surface levels of CD151 on cells are strongly influenced by the expression of
the laminin-binding integrins.
|
Lack of complex formation between CD151 and the Lutheran molecule or
dystroglycan
The colocalization of CD151 with the different kinds of laminin receptors,
DG, LU and integrins, may suggest a role for CD151 in linking these three
kinds of laminin receptors to each other. To investigate whether CD151, DG and
LU are not only colocalized but also complexed, cultured human podocytes and
proximal tubular epithelial cells (PTEC) were used for immunoprecipitation
studies (Fig. 11). FACS
analysis showed that both cell lines express 3ß1, CD151, DG and
LU, although LU is only weakly expressed by podocytes. These expression
profiles correspond with those of podocytes and PTECs in vivo. Lysates from
cells prepared with 1% CHAPS were incubated with antibodies against
3,
CD151, LU or ß-DG. Subsequently the presence of
3 (light chain, 30
kDa) or ß-DG (43 kDa) in the precipitates was assessed by immunoblotting
using specific antibodies. Protein bands corresponding to
3 could be
detected on the immunoblots of the immunoprecipitates containing
3ß1 or CD151 from both cell lysates (left panels). LU or DG could
not be coprecipitated with
3ß1 with any of the cell lysates. P48
against CD151 and antibodies against
3 or LU did not coprecipitate DG
(right panels) either. This demonstrates that in the tested cell lines, CD151
bound to the laminin-binding integrins does not associate with the other
laminin receptors LU or DG and therefore cannot serve as a link between
them.
|
![]() |
Discussion |
---|
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---|
As indicated by this study, there are different distribution patterns of
CD151. In the first pattern most of the CD151 is colocalized with the
laminin-binding integrins present and thus is mainly expressed at the site of
cell-matrix interactions where there is a basement membrane containing at
least one laminin isoform (Miner et al.,
1997; Aumailley and Rouselle,
1999
). Thus, in glomerular cells, CD151 and
3ß1 are
colocalized at the GBM side of cells. In renal tubular epithelial cells, CD151
and
6ß1 are concentrated at the tubular basement membrane, and in
skin CD151 and
3ß1 and
6ß4 are present at the
dermo-epidermal junction. Finally in myocytes, CD151 together with
7ß1 is expressed at the sarcolemma. The second pattern is seen in
cells such as endothelial and most hematopoietic cells in which the amount of
laminin-binding integrins is limited. As shown by others, these cells contain,
in addition to a considerable pool of unbound CD151, CD151 that is weakly
associated with
5ß1 and other surface molecules
(Hasegawa et al., 1998
;
Fitter et al., 1999
;
Sincock et al., 1999
). The
third distribution pattern is seen in fibroblasts (stromal cells) and K562
cells, which contain CD151 but no laminin-binding integrins.
In addition to the previously reported association between CD151 and the
laminin-binding integrins 3ß1,
6ß1 and
6ß4, we demonstrate here that CD151 also interacts with
7ß1. Like CD151-
3ß1 complexes, CD151-
7ß1
complexes remained intact after Nonidet P-40 lysis of transfected K562 cells,
which classifies their interaction as level 1. Also using transfected K562
cells, it was shown that CD151-
6ß1 and CD151-
6ß4
(Sterk et al., 2000
) interact
at level 2, the interaction not being resistant to Nonidet P-40 treatment. A
correlation between the presence of the laminin-binding integrins and CD151
expression was established by FACS analysis, which showed an increase of CD151
expressed at the cell surface of K562 cells after transfection of either of
the laminin-binding integrins.
CD 151 does not link the different laminin receptors with each
other
Two non-integrin receptors for laminin are DG and LU. DG was originally
isolated from skeletal muscle as a component of the large oligomeric DGC,
which amongst other proteins contains sarcospan and dystrophin
(Durbeej and Campbell, 1999).
The third type of laminin receptor is the molecule expressing the LU antigens,
which is able to bind to the laminin
5 chain present in the
subendothelial matrix (Parsons et al.,
2001
).
Our in vivo results show a considerable overlap in the distribution of
these different types of laminin receptors. Hence, a tight regulation and
precise coordination of the signaling mechanisms by the different receptors
seems essential. An example of an interaction between the different laminin
receptors is shown in glomerulogenesis, during which a switch occurs in
expression of the laminin-binding integrins from 6ß1 to
3ß1 simultaneously with a switch in the deposition of laminin
isoforms, that is, from laminin-1 to laminin-11
(Sterk et al., 1998
). Others
have shown that the symptoms induced by the absence of dystrophin, which
disturb the function of the laminin receptor DG, can be partly compensated by
increasing the expression of
7ß1
(Burkin et al., 2001
).
Moreover, myocytes of patients with Duchenne's disease lacking dystrophin and
of dystrophin-deficient mice express an increased amount of
7ß1
(Hodges et al., 1997
). This
indicates that when one laminin receptor is lacking, another laminin receptor
can (partially) compensate for it. A protein present in the DGC that is
homologous to tetraspanins is sarcospan
(Crosbie et al., 1999
).
Although sarcospan is a component of the DGC and has a function in maintaining
the integrity of muscle tissue, sarcospan-deficient mice have only mild
symptoms of muscular atrophy (Lebakken et
al., 2000
). Moreover, epithelial cells expressing DG do not
contain sarcospan (Durbeej and Campbell,
1999
). These data suggest that in sarcospan-deficient mice and
epithelia expressing DG, other tetraspanins may compensate for the absence of
sarcospan. We demonstrate that, except in the skin, in all tissues tested, DG,
the laminin-binding integrins and CD151 are co-distributed. Furthermore, this
study shows that CD151 is the only known tetraspanin in the glomerulus that is
present at the side of the basement membrane, together with DG. Therefore and
analogous to the sarcolemma, the sarcospan function in the glomerulus could be
taken over by CD151, thereby connecting the integrin family with the DGC.
However, despite their co-distribution, coimmunoprecipitation studies could
not demonstrate a physical association between
3ß1-CD151 complexes
and DG or LU in two different cell lines obtained from the kidney, that is,
human podocytes and proximal tubular epithelial cells.
Neither the variants of the extracellular or cytoplasmic domains of
integrin subunits nor the cleavage of
6 affect binding of CD151
to integrins
The binding between tetraspanins and integrins is established via the
extracellular domains of the tetraspanin and the integrin subunit
(Yauch et al., 2000
).
Theoretically, splicing of the mRNA for the extracellular or cytoplasmic
domains of integrin subunits or maturation-induced cleavage of the
chain could influence the association of tetraspanins with integrins and
thereby alter integrin signaling. However, biochemically no such differences
could be demonstrated for the cytoplasmic A and B variants variants of the
integrin
3 and
6 subunits or for the extracellular variants X1
and X2 of
7. Furthermore, mutational changes in the extracellular
domain of
6 preventing cleavage seemed to have no influence on the
formation of a complex with CD151. This implies that switches of splice
variants as described during development
(Collo et al., 1993
;
Thorsteindóttir et al.,
1995
; Sterk et al.,
1998
) or cleavage of the
subunit during maturation have no
effect on CD151-integrin binding.
All laminin-binding integrins may bind to a single site on CD151
The reactivity of CD151 with TS151R and P48 in immunohistochemistry
indicates that masking of the TS151R epitope detected in the biochemical
experiments and FACS analysis also occurs in vivo. Glomerular cells expressing
3ß1, basal keratinocytes expressing
3ß1 and
6ß4 and cardiac and skeletal muscle cells expressing
7ß1 either did not or only weakly react with TS151R, although
these cell types were strongly stained by mAb P48. A likely interpretation of
this finding is that in these tissues most if not all of the CD151 is bound to
a laminin-binding integrin, leading to masking of the TS151R epitope. However,
renal tubular and smooth muscle cells, which express
6ß1 and
7ß1, respectively, were stained equally well by the two
antibodies. The reason for this observation is not clear, but there are
several explanations. First, the strength of the interaction between CD151 and
integrins might be regulated from within the cell and/or by the
microenvironment, by which the tightness of the binding may be influenced and
thus the exposure of the TS151R epitope. Second, CD151 may not directly
interact with one of the laminin-binding integrins but indirectly by forming a
tetraspanin web with other molecules. In fact, different binding sites have
recently been described on CD151 for tetraspanins and integrins
(Berditchevski et al., 2001
).
Third, CD151, in addition to being complexed with a laminin-binding integrin,
could also be bound to other cell surface molecules, as has been described for
different tetraspanins and MHCII, DRAP27 and HB-EGF
(Angelisova et al., 1994
;
Nakamura et al., 1995
). This
binding should, in that case, involve another part of the binding site than
that which interacts with integrins. Fourth, in kidney sections from HIV
patients, we found that the amount of CD151 reactive with TS151R is greatly
increased in the glomeruli and comparable with that reactive with P48
(L.M.T.S., unpublished). This indicates that under certain pathophysiological
conditions the TS151R epitope on CD151, which is normally complexed with
3ß1, becomes exposed. Finally, a difference in affinities of the
two antibodies for CD151, as suggested by our FACS data, may have influenced
their reactivity.
In summary, we demonstrate extensive co-distribution of various kinds of
laminin receptors in different tissues. We confirm previous findings that
CD151 binds to the laminin-binding integrins 3ß1 and
6ß1 and show for the first time that it also interacts with
7ß1. CD151 does not bind to the laminin receptors DG or LU.
Moreover, we show that variants of the cytoplasmic or extracellular domains
bind equally well to CD151 and that cleavage of the integrin
subunits
does not alter their binding. Studies with the mAb TS151R, which has
previously been shown to recognize an epitope at the binding site of CD151
with
3ß1, indicate that the same site is involved in the binding
to
7ß1 and possibly also to
6ß1 and
6ß4.
The finding that in immunohistochemical studies few cells and tissues,
irrespective of the laminin-binding integrins they express, reacted with
TS151R, suggests that the strength of interaction of CD151 is subject to
regulation either from within the cell and/or by the cell's environment.
![]() |
Acknowledgments |
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