From the Tumor Immunology Programme, 0710, German Cancer Research
Center, D-69120 Heidelberg, Federal Republic of Germany, the
Laboratory of Immunology and Vascular Biology, Department
of Pathology and Digestive Disease Center, Stanford University,
Stanford, California 94305, and the Center of Molecular Biology in
Medicine, Veterans Administration Medical Center,
Palo Alto, California 94304
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ABSTRACT |
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The amino acid motif LDV is the principal binding
site for 4 integrins in fibronectin, and
homologous motifs are recognized in vascular cell adhesion molecule-1
and MAdCAM-1. Three conserved LDV motifs (LDV-1 to 3) occur in the
ectodomain of the human and mouse
4-subunit, the
functions of which are unknown. We demonstrate here that
4-transfected fibroblasts with mutation in LDV-1 (D489N) behaved like
4-wild type but that LDV-2 (D698N) and
LDV-3 (D811N) mutants were impaired in binding and spreading on
4-specific substrates. On the RGD-containing fibronectin
fragment FN-120 there was an inverse behavior; now the
4-wild type and the LDV-1 mutant could not adhere
whereas the two other mutants could. The
1 chain was
critical for the differential integrin response. Biochemical analysis
demonstrated that the LDV-2 and -3 mutations reduced the strength of
the
4
1 association, favored the formation of
5
1, and prevented the expression of
4
7 on the cell surface. Our results
indicate that LDV-2 and LDV-3 are critical for the formation of a
functional heterodimer. The presence of similar amino acid motifs in
ligands and the
4-subunit suggest that metal coordination plays an important role in integrin-ligand binding as well
as for heterodimer formation.
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INTRODUCTION |
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Integrins are a class of heterodimeric cell-surface molecules that are important mediators of cell function. In addition to providing the necessary mechanical stability for cell-cell or cell-matrix contact, integrins can transduce signals out of cells and into the cell from the microenvironment. The cytoplasmic portion of integrin chains can interact with signal- and cytoskeleton-associated proteins and thereby influence cellular properties like shape, adhesion, motility, growth, and differentiation (for review see Refs. 1 and 2).
An essential feature of integrins is their complex regulation of
function which despite their biological importance as adhesive structures is not fully understood. Both subunits form a non-covalently linked heterodimer that appears to be important for transport to the
cell surface as well as for ligand binding. It is believed that
integrins contain multiple ligand contact sites, and several regions
and residues have been identified in both and
chains. All of
the identified contact sites carry amino acid sequences with oxygenated
side chains that can potentially bind divalent cations. It has been
estimated that integrin heterodimers can bind up to five divalent
cations. The observation that all integrin ligands possess a critical
aspartate or glutamate residue in their contact site to the integrin
has led to the proposal that a metal ion provides a bridge between
ligand and receptor. In addition to ligand binding, divalent cations
are believed to contribute to the regulation of receptor conformation
(for review see Refs. 3-5).
The 4 integrins,
4
1 and
4
7, are adhesion molecules that play an
important role in hematopoiesis (6, 7), lymphocyte migration (8, 9),
mouse skeletal muscle formation (10), placental or cardiac development
(11), and possibly tumor metastasis (12, 13). VLA-4
(
4
1) mediates the adhesion of cells to
fibronectin (14-16) or the cytokine inducible endothelial cell ligand
VCAM-11 (17, 18). The
heterodimer
4
7 is a homing receptor that mediates the lymphocyte entry into the gut-associated lymphoid tissues
(8, 19). The ligand for
4
7 is the
addressin MAdCAM-1 (20). Only
4
7-positive
cells can bind to MAdCAM-1, whereas both
4
1- and
4
7-positive cells can bind to fibronectin
and VCAM-1 (21, 22). Molecular studies have identified amino acid sequences in each ligand that are recognized by
4
integrins. A dominant binding site in fibronectin involves the LDV
motif in the HepII/IIICS region, and the peptide surrounding and
encompassing these residues has been termed CS-1 peptide (23, 24). A
homologous sequence IDS, present in domains 1 and 4 of VCAM-1, is
essential for the binding to
4 integrins under static
conditions (25-27) or flow (28), and an LDT sequence in the first
domain of MAdCAM-1 is important for
4
7
binding (29). Three conserved LDV motifs occur in the extracellular
sequence of the
4-subunit in mouse and man which have
been termed LDV-1-3 (30).
We previously reported that the purified 4-subunit as
well as an LDV-containing peptide derived from the 80-kDa N-terminal portion could support the binding of lymphocytes via
4
1 or
4
7 (30) which could be important for
4-mediated homotypic
cell aggregation (22, 31, 32). In another study Ma et al.
(33) have suggested that the LDV sites, in particular LDV sites 1 and 2, may serve as additional cation binding motifs that are required for
cell adhesion. To understand further the functional role of the LDV
motifs in the
4-subunit, we have mutated the central aspartic acid to asparagine in each site in a consecutive fashion and
have examined the effect on cell adhesion and spreading. Our results
suggest a role for LDV-2 and -3 but not LDV-1 in the formation of a
functional
heterodimer. It is therefore possible that both
ligand binding and the dimerization of
- and
-subunits follow the
same principal rule of metal ion coordination.
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EXPERIMENTAL PROCEDURES |
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Cell Culture--
Balb/3T3 fibroblasts were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, 2 mM L-glutamine, and 10 mM HEPES. CHO cells transfected with human full-length
VCAM-1 or a 1-3 domain deletion mutant of VCAM-1 (1-3) were
obtained from Dr. J. Clements (British Biotech, Oxford, UK) and
maintained as described (34). All cells were kept at 37 °C, 5%
CO2, and 100% humidity.
Antibodies--
mAb 5/3 against 4 integrins has
been described before (30). mAbs HM
5-1 and HM
-1 are blocking
hamster mAbs against mouse
5 integrin and the
1-subunit, respectively (35). These mAbs were obtained
from Dr. H. Yagita, Tokyo, Japan. mAbs Fib 30 and Fib 504 are blocking
antibodies against the
7-subunit (36). mAb RMV-7 is a
blocking antibody to mouse
v (37). mAb 9EG7 (38) was kindly provided
by Dr. D. Vestweber (ZMBE Münster, Germany). mAbs were used in a
purified form or as hybridoma supernatants.
Peptides-- The RGDS peptide was purchased from Sigma (Taufkirchen, Germany). The CS-1 peptide CEILDVPST was synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry and purified by preparative high pressure liquid chromatography. It was characterized further by analytical high pressure liquid chromatography and mass spectroscopy.
Cytofluorography-- The cell-surface staining of cells with saturating amounts of mAbs, either hybridoma supernatants or purified antibodies, and phycoerythrin-conjugated goat antibodies to rat Ig (SERVA, Heidelberg, FRG) or hamster Ig (Dianova, Hamburg, FRG) has been described elsewhere (39). For the detection of cytoplasmic staining cells were first incubated on ice with 0.5% formaldehyde in PBS. After washing in PBS cells were permeabilized with 0.05% saponin (Sigma) for 10 min at room temperature. The cells were washed again and stained as above.
Stained cells were analyzed with a FACScan fluorescence-activated cell analyzer (Becton Dickinson, Heidelberg, FRG). For enrichment ofSite-directed Mutagenesis and DNA Transfection--
The mouse
4-cDNA clone in Bluescript SK+ was obtained from Dr.
B. Holzmann (University of Munich, Germany) and was used as template
for site-directed mutagenesis with the Stratagene Chameleon kit
(Stratagene, Heidelberg, Germany). In successive rounds of mutagenesis
the Asp in positions Asp-489, Asp-698, and Asp-811 were changed to Asn
as indicated in Fig. 1. Mutations were confirmed by DNA sequence
analysis. Wild-type and mutated
4-cDNAs were subsequently cloned in the pcDNA3 expression vector (Invitrogen) and transfected into Balb/3T3 fibroblasts using LipofectAMINE (Life
Technologies, Inc., Eggenstein, Germany). Transfectants were selected
for similar expression levels using FACS sorting (see above). The
sorting of transfectants was repeated when the expression level of
4 integrin dropped by more than 20 mean fluorescent units.
Cell Adhesion and Spreading-- Fibronectin or its fragments FN-120 or FN-40 (Life Technologies, Inc.) were coated to LABTEK glass chamber slides (Nunc, Wiesbaden, Germany) at a concentration of 10 µg/ml or as otherwise indicated for 16 h at 4 °C. Wells were blocked with 1% bovine serum albumin in PBS for 1 h at room temperature, washed with HBSS containing 10 mM HEPES, 2 mM Ca2+, and 2 mM Mg2+ (binding buffer) and used for the assay. For adhesion, cells (5-10 × 106/ml) were suspended in the same buffer, and 0.2-ml aliquots were added to the coated slides. The binding assay was performed for 10 min at 37 °C without shaking, and the slides were fixed in 2% glutaraldehyde/PBS after briefly dipping into PBS. For antibody or peptide blocking studies, cells were preincubated with purified antibody (10 µg/ml final concentration, or as otherwise indicated) or peptides (500 µg/ml or otherwise indicated) for 10 min at room temperature and then transferred to the chamber slides. For Mn2+ activation, the Ca2+ and Mg2+ ions in the buffer were substituted with the indicated concentrations of Mn2+. Cell binding was measured by counting six independent 10 × fields by video microscopy using IMAGE 1.55 software. To analyze cell spreading, the plated cells were incubated at 37 °C at a microscope stage, and pictures were taken for 2 h every 10 min and stored on an optical disc. Pictures were analyzed, and the percentage of spread versus non-spread cells in each frame was determined. Clearly visible pseudopodia formation served as criterium for spread cells.
Binding of cells to human VCAM-1-transfected CHO cell monolayers was done using Eu3+-labeled cells (40). Briefly, cells were loaded with EuCl3 for 1 h at 4 °C, washed with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, and resuspended in Hanks' balanced salt solution plus 2 mM Ca2+ and Mg2+. 100 µl of the cell suspension (5 × 105 cells/ml) were allowed to bind to the monolayer of VCAM-1-CHO cells in a 96-well plate for 30 min at 37 °C. Following the binding assay bound and non-bound cells were separated on the basis of buoyant density using Percoll as described (44). For detection of the bound Eu3+-labeled cells, the plates were inverted to remove the Percoll and fixative, then washed once in PBS, and refilled with 50 µl of PBS, and 50 µl of Europium enhancement solution (LKB Wallac, Turku, Finland) was added. The fluorescence was measured in a time-resolved fluorometer (Arcus 1230, LKB-Wallac, Turku, Finland) after 30 min.Isolation of RNA and Reverse Transcriptase-PCR Analysis--
The
isolation of total RNA from cells has been described in detail
elsewhere (41). Total RNA (6 µg) was transcribed into cDNA using
Moloney murine leukemia virus reverse transcriptase (Promega,
Heidelberg, Germany) and oligo(dT)20 for priming. The RNA/DNA hybrid was treated with RNase H and used as template for PCR
using an annealing temperature of 60 °C and 35 cycles of 80 s.
The following primers for the 7 integrin subunit were
used: forward, ATAGGTTTTGGCTCCTTCGTG; reverse,
AGTGGAGAGTGCTCAAGAGTCACAGT. PCR products were separated on a 1%
agarose gel containing 0.5 µg/ml ethidium bromide. The mouse
7 cDNA clone was obtained from G. Krissansen,
University of Auckland, New Zealand.
Biochemical Analysis--
Lactoperoxidase-catalyzed iodination
of intact cells was carried out as described (39). Following the
labeling reaction, the cells were washed three times in PBS and lysed
at 4 °C for 15 min in Tris-buffered saline containing either 0.3%
CHAPS (Sigma), 1% IGEPAL CA-630 (Sigma) or 1% IGEPAL CA-630 in the
presence of 2 mM Ca2+ and Mg2+
ions. Lysates were prepared by centrifugation in an Eppendorf centrifuge at 4 °C for 15 min and precleared with 30 µl of packed rat Ig coupled to Sepharose. Immunoprecipitations were carried out
using mAb 5/3 coupled to Sepharose or mAbs HM-1 preadsorbed to
Protein G-Sepharose for 1 h at 4 °C. The precipitates were washed 4 times in the respective lysis buffers and eluted from the
Sepharose by boiling for 2 min in non-reducing SDS sample buffer.
SDS-polyacrylamide gel electrophoresis was performed on 7,5% slab
gels. Gels were dried and exposed to x-ray-sensitive films (Kodak
Biomax-MS) using the Biomax MS intensifying screen.
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RESULTS |
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Expression of Wild-type and Mutant
4-Subunits--
The LDV motifs in the mouse
4-subunit were mutated in positions Asp-489, Asp-698,
and Asp-811 by changing Asp to Asn as outlined in Fig.
1. The wild-type, single, double, and
triple mutated
4-subunits (referred to as mut 1, mut 12, and mut 123) were transfected into
4-negative Balb/3T3
fibroblasts and selected for equal cell-surface expression by FACS
sorting. As shown in Fig. 2 the level of
expression as detected by fluorescent staining with an
4-specific mAb was comparable for all transfected cells.
As revealed by staining with the respective mAbs, the expression levels
of VLA-5,
v-integrins, as well as the
1 integrin
subunit were not changed significantly following transfection. There
was, however, induction of
7 surface expression in
4-wt and mut 1 cells and little or undetectable expression in mut 12 and mut 123 cells, respectively. Induction of new
chains upon transfection was also observed in another study
(42).
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4-Transfectants Are Altered in Adhesion--
We
investigated the adhesion of the different cells to immobilized
fibronectin. As shown in Fig.
3A all cells could readily bind. The binding of non-transfected 3T3 cells is due to the expression of other integrins than
4 that can support adhesion to
fibronectin (
5
1,
v integrins see
below). The binding site for
4 integrins is located on
the FN-40 chymotryptic cleavage fragment of fibronectin. As shown in
Fig. 3B the binding of
4-wt and mut 1 cells
to FN-40 was of similar magnitude and blocked in the presence of the
mAb to the
4-subunit. A partial blocking was obtained
with mAbs against the
1- or the
7-subunit
suggesting that the binding was mediated by
4
1 and
4
7
integrins. The mut 123 cells, and to a lesser extend mut 12 cells,
showed a significant reduction in binding ability. Untransfected 3T3
cells did not bind.
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Altered Cell Spreading on FN-40--
We studied the spreading of
4-transfectants on FN-40, and the results are depicted
in Fig. 6. After 40 min nearly all
4-wt and mut 1 cells had spread on the FN-40 substrate,
and this spreading was prevented in the presence of the
4-specific mAb 5/3. The mut 12 cells, although partially
able to adhere, were unable to spread on FN-40, and the spreading of
mut 123 cells was similarly decreased when compared with
4-wt cells. As expected, 3T3 fibroblasts could not
spread on FN-40.
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Presence of the 4-Subunit Affects Spreading of Cells
on FN-120--
We next analyzed the behavior of the cells on FN-120,
which contains the RGD-binding site in fibronectin. Fig.
8 shows that 3T3 fibroblasts were able to
spread on FN-120. Unexpectedly, the
4-wt cells and mut 1 cells could not or only poorly spread on FN-120. In sharp contrast, in
mut 12 and mut 123 cells spreading was again observed.
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Modulation of the 9EG7 Epitope by Soluble Ligand--
To
demonstrate a differential activity of either the
4
1 or RGD-binding integrins also in
response to soluble ligands, the expression of the 9EG7 epitope, which
is a conformation-dependent epitope of the
1
chain (38), was investigated. The expression of this epitope can be
enhanced by Mn2+ ions or soluble ligand (43). The binding
site for mAb 9EG7 has been mapped to the cysteine-rich membrane
proximal site of the
1 chain (43). As shown in Fig.
10A, the
4-wt
transfected cells and mut 1 cells showed staining with mAb 9EG7 which
was of similar magnitude as 3T3 cells. In contrast, both mut 12 and mut
123 cells showed a consistently higher staining suggesting that the
epitope was more accessible. These differences were not seen with mAb
HM
-1 which binds to a site distinct from the 9EG7 epitope on the
1 chain.
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LDV-2 and -3 Mutations Affect the Stability of the
4
1 Heterodimer--
The functional data
had indicated that mutations in the
4-subunit were able
to regulate the integrin response in the transfected cells and that the
common
1 chain might be of crucial importance. To
address this question further biochemical analyses were carried out.
Immunoprecipitation with the
4-specific mAb showed the
typical pattern of
4 integrins on SDS-polyacrylamide gel
electrophoresis consisting of the 150-kDa band representing the intact
4 chain and the smaller bands of 80 and 70 kDa that are
proteolytic cleavage fragments of the
4 chain (see Fig.
11A). The mutations slightly affected the electrophoretic mobility of these fragments.
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LDV-2 and -3 Mutations Prevent Surface Expression of
4
7--
Finally we reinvestigated the
failure of mut 12 and mut 123 cells to express
7 at the
cell surface following transfection. We used mAb DATK32 that recognizes
a conformational epitope of
4
7 when it is
associated (34). Fig. 12A
shows that
4-wt and mut 1 cells showed cell-surface
staining with this mAb, whereas mut 12 and mut 123 cells were negative.
This was not due to the lack of
7 message that was
detectable by PCR analysis of reverse-transcribed cDNA in all
transfected cell lines as well as in 3T3 cells (Fig. 12B).
Indeed, cytoplasmic staining of permeabilized cells with a
7 mAb revealed the presence of
7 protein
in all transfectants (not shown). We concluded that the mutations in
the
4-subunit most likely affected the formation of the
4
7 heterodimer or its transport to the
cell surface.
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DISCUSSION |
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The goal of the present study was to assess the functional role of
the three LDV sites in the mouse 4-subunit by
site-directed mutagenesis. We find that Asp
Asn substitution in
LDV-2 and -3 but not in LDV-1 affects adhesion and spreading of
transfected fibroblasts on
4-specific substrates. The
4 mutant cells were differentially affected in adhesion
and spreading on FN-40. Whereas the mut 12 cells were mainly
compromised in spreading, the mut 123 cells were affected in both
adhesion and spreading suggesting that the mutation of the LDV-3 site
was giving an additional effect. In the presence of Mn2+
ions the defects of mut 12 and mut 123 cells could be largely restored,
and the lack of spreading on FN-40 was overcome by prolonged assay
times. This suggested that the receptor was not completely non-functional and that LDV-2 and -3 were probably the most important in events early after
4
1 receptor
engagement. Ma et al. (33) have performed similar
mutagenesis studies using K562 cells transfected with human
4-subunits in which the LDV sites were mutated to LEV.
These authors demonstrated impaired adhesion of D489E and D698E mutants
(equivalent to LDV-1 and -2) on FN-40 and a 1-3 domain form of VCAM-1.
There was, however, no effect in D811E mutants (33). The reasons for
the discrepancies between the previous study and our data are not
entirely clear but could be due, at least in part, to the choice of
amino acid exchanges. Whereas Asp
Glu mutations retain the negative
charge in the amino acid side chain, our Asp
Asn mutations
abolished it. Also the type of mutagenesis (single versus
accumulating mutations) is different, and cell spreading was not
analyzed by Ma et al. (33). Based on sequence comparison it
was proposed that Asp-698 and possibly -489 (identical with LDV-2 and
-1) may be putative metal-binding sites that might be required to
stabilize a protein-protein interaction. The reason why these sites
might be important for
4 integrin function remained
unclear.
Our results provide suggestive evidence that the LDV-2 and -3 sites are
important for a functional interaction with the chain. We postulate
that in the presence of a transfected
4-subunit the
formation of a functional
4
1 heterodimer
is dominant. The formation of other heterodimers, i.e.
5
1, is suppressed presumably due to the
lack of available
1 chains, and these
-subunits reach the cell surface by other means. The mutations of LDV-2 and -3 impair
the ability of
4 to interact with
1 and
favor again the formation of
5
1 at the
cell surface.
Evidence for this hypothesis came from several independent approaches.
First we studied the binding of 4-transfectants to FN-120. We found that in the presence of a transfected
4-wt or mut 1 subunit the fibroblasts were unable to
adhere and spread via
5
1 on FN-120. In
contrast, mut 12 and mut 123 cells could again spread and adhere and
behaved similar to the parental 3T3 cells. We could exclude the
possibility that the observed differences in the binding were the
results of phenotypic changes in the transfectants since the expression
levels of
5,
v,
4, and
1 were comparable. Furthermore, back selection of
transfectants for
4 loss or low expressing variants
fully restored the ability to spread on FN-120, supporting the view
that the suppression was due to the presence of the
4-subunit and not an intrinsic failure of the cells.
Importantly, the dependence of the phenomenon on amino acid
substitutions in the extracellular part of the molecule argued against
an involvement of cytoplasmic proteins that may be required for
integrin function. It rather appeared that cis-type of interactions
with other proteins at the membrane surface was the reason.
An important observation was that in the binding of cells both to FN-40
and to FN-120 the 1 chain was involved. In particular, the inverse behavior of the transfectants on both types of substrates was striking. This led us to consider that the
1 chain
might be a decisive factor in regulating the integrin response in the mutants. That indeed the
1 chain was crucial was
supported by studies on the 9EG7 epitope that has been characterized as
a ligand-induced binding site of the
1 chain (43).
Interestingly, the epitope for this mAb was located in the
cysteine-rich site of the
1-subunit (43) which is
juxtaposed to the site on the
4-subunit where Asp-698
and Asp-811 are located. The level of staining for this epitope in the
absence of any ligand was consistently higher on mut 12 and mut 123 cells than on
4-wt or mut 1 cells. Thus, the LDV-2 and
-3 mutations in the
4 chain induced a change in the accessibility of this particular
1 chain epitope that is
usually up-regulated in the presence of bound ligand or activating
divalent cations but also by the addition of EDTA (41). To study the effect of ligand binding on the 9EG7 epitope, we exposed the
4
1 heterodimer to CS-1 peptide in
solution. A clear-cut up-regulation was seen in
4-wt
cells. In contrast, there was only little change in mut 123 cells
indicating that the epitope was already fully exposed in the first
place and could not be up-regulated much further. These data
demonstrated that the phenotype of the
4-subunit, either
mutated or not, was mirrored in the 9EG7 epitope presumably indicating
a conformational change of the
1-subunits imposed by the
respective
4 chain. Thus, the 9EG7 epitope acted as an indicator of a change in the
conformation.
More direct proof that the LDV-2 and -3 mutation affected the
conformation of the heterodimer came from biochemical studies. By using different detergents for the solubilization of the cells, we
noticed a much decreased stability of the
4
1 heterodimer in mut 12 and mut 123 cells. In the absence of divalent cations the heterodimer could not
resist the detergent milieu as evidenced by the reduced presence of
1 chains in the
4-specific precipitates and the failure to detect the
4 80 and
4
70-kDa fragments in anti-
1-specific precipitates of mut
12 and mut 123 cells. This could mean that the complex was only very
poorly associated or even free
4 chain was present at
the surface of these cells. Due to the lack of heterodimer-specific
mAbs in the mouse, at the present we cannot distinguish between these
two possibilities. Precipitation analysis of
1-associated integrin subunits also revealed that the
5 chain was detectable only in mut 12 and mut 123 cells
and not present or only weakly present in
4-wt and mut 1 cells. Thus, the precipitation analysis reflected the results from the
functional analysis of mutant cells.
Further evidence for a role of LDV-2 and -3 in heterodimer formation
came from studies on the expression of 4
7
in mutant cells. At the cell surface a
4
7
heterodimer was only seen in
4-wt and mut 1 cells,
whereas in mut 12 and mut 123 cells it was not detectable. Despite this
the
7-subunit was available in all mutant cells as
detected by reverse transcriptase-PCR and cytoplasmic staining. It is
likely that the mutant
4-subunits had a decreased
ability to interact with the
7 chain thus preventing the
assembly of the heterodimer or its transport to the cell surface.
The 4 and other integrins can physically interact with
transmembrane-4 superfamily proteins like CD81 (TAPA-1) and others in
the cell membrane of different cell lines (44). The binding site for
TAPA-1 in the
4 chain is not entirely clear but has been
mapped outside the
4 cytoplasmic tail (44).
Transmembrane-4 superfamily proteins can associate with several
integrins, but a direct role in the regulation of
4
integrin function has so far not convincingly been demonstrated.
Although a possible interaction of these molecules with
4 in our transfected fibroblasts has to be considered,
we do not regard this as a reasonable mechanism to explain our
results.
Sanchez-Aparicio et al. (45) have reported that stimulation
of human cells with the 1-specific mAb TS2/16 could not
only activate
4
1 but also led to
recognition of the RGDS sequence in fibronectin. Thus, the
conformational change induced in
4
1 by
the mAb resulted in the ability to recognize the RGD sequence. Could
the mutated
4-subunit in our transfectants induce a
similar change? The data presented in Fig. 9 argue against this
possibility. It is evident that the adhesion of mut 12 and mut 123 cells to FN-120 was dependent on
5
1 since
it was blocked by the respective mAb but not by the mAb against the
4 integrin.
Divalent cations regulate integrins in a complex way. All
integrin-ligand interactions are divalent cation-dependent,
and putative metal ion-binding sites have been identified in - and
-subunits (see Refs. 3-5). The
-subunit of all integrins contain 3-4 divalent cation binding modules with homology to the EF-hand Ca2+-binding motifs (46). In the I domain which is present
in many but not all
-subunits a metal ion-dependent
adhesion site is present that coordinates Mg2+ (47), and a
metal ion-dependent adhesion site-like motif is also
present in the most conserved region of
integrin subunits (47). All
these regions are involved in ligand binding implying that the
ligand-binding pocket is complex and involves both subunits. Since the
ligand epitopes recognized by integrins are often short acidic peptide
motifs with central oxygenated amino acids, it has been suggested that
receptor-bound cation might act as an integrin-ligand bridge. Recently
a model was proposed for the
chain 7-fold repeats that represent
about 40% of the extracellular portion of the
chains (48). This
model predicts a
-propeller domain composed of seven blades made out
of
-sheets around a central axis (48). The I domain is inserted into
the
-propeller between the second and the third blade and is
predicted to sit on the upper rim ot the
-propeller domain (49). The
-propeller model and the crystal structure of the
-subunit I
domain from CD11a and CD11b (47, 49) have allowed for the first time to propose a dynamic quaternary structure model of integrin-ligand interaction sites (5).
Outside the region of ligand contact the structure and conformation of
integrin subunits is less well defined. It is known that the - and
-subunits form a heterodimer also in the absence of ligand and that
withdrawal of divalent cations by chelating agents destabilizes the
heterodimer (50-52). It is quite possible that the conformation
of the
heterodimer also depends on multiple interactions that,
at least to some part, uses metal ion bridging for stabilization. The
metal ion-dependent adhesion site-like domain of several
integrin
-subunits was suggested to be important for the association
with the
-subunit (53-55). Interestingly, the
-propeller model
predicts that the EF-hand Ca2+-binding motifs are located
near one another on the lower surface of the
-propeller domain and
might be involved in interactions with the
-subunit rather than in
ligand binding (49). The LDV-2 and -3 sites are located on the stalk
region of the
4-subunit. Due to this localization they
are probably not directly involved in ligand binding but may be
necessary to establish a proper association with the
chain. The
lack of this putative association affects the affinity of the receptor
and leads to defects that seem to be important in the early phase of
ligand binding. These defects in receptor function of mutant cells were
restored, although not completely, in the presence of Mn2+
ions and by prolonged assay times. It is possible that the presence of
Mn2+ enforced additional binding sites. Collectively, our
data suggest that LDV-2 and -3 sites represent important contact sites
between
4 and its
-subunits. The presence of similar
amino acid motifs in ligands and the
4-subunit suggests
that metal coordination plays an important role in integrin-ligand
binding as well as for heterodimer formation.
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ACKNOWLEDGEMENTS |
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We thank Volker Schirrmacher for support and stimulating discussions. We are grateful to Dr. Rüdiger Pipkorn for peptide synthesis and Drs. Dietmar Vestweber and Hideo Yagita for generous gifts of antibody.
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FOOTNOTES |
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* This work was supported by a grant from the Deutsche Forschungsgemeinschaft (to P. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Tumor Immunology Programme, 0710, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Federal Republic of Germany. Tel.:06221-423713; Fax: 06221-423702; E-mail: p.altevogt{at}dkfzheidelberg.de.
1 The abbreviations used are: VCAM-1, vascular cell adhesion molecule-1; mAb, monoclonal antibody; PBS, phosphate-buffered saline (lacking Ca2+ and Mg2+); FACS, fluorescence-activated cell sorter; wt, wild type; mut, mutant; CHO, Chinese hamster ovary; PCR, polymerase chain reaction; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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