(Received for publication, October 2, 1996, and in revised form, December 23, 1996)
From the Ludwig Institute for Cancer Research, R. Prof. Antonio Prudente, 109, 4 A, 01509-010, São Paulo, SP,
Brazil, the § Department of Cell Biology, Federal University
of Parana, Curitiba, Centro Politécnico, Seter de
Ciências Biológicas, Jardim des Americas, 81531-990, Curitiba, PR, and the
Department of Biochemistry,
Federal University of São Paulo, Universidade Federal de
São Paulo, Escola Paulista de Medicina, rea 3 de maio, 100 4 andar, 04044-020, São Paulo, SP, Brazil
Cell-fibronectin interactions, mediated through
several different receptors, have been implicated in a wide variety of
cellular properties. Among the cell surface receptors for fibronectin, integrins are the best characterized, particularly the prototype 5
1 integrin. Using
[125I]iodine cell surface labeling or metabolic
radiolabeling with sodium [35S]sulfate, we identified
5
1 integrin as the only sulfated integrin among
1 integrin heterodimers expressed by the human
melanoma cell line Mel-85. This facultative sulfation was confirmed not only by immunoprecipitation reactions using specific monoclonal antibodies but also by fibronectin affinity chromatography,
two-dimensional electrophoresis, and chemical reduction. The covalent
nature of
5
1 integrin sulfation was
evidenced by its resistance to treatments with high ionic, chaotrophic,
and denaturing agents such as 4 M NaCl, 4 M
MgCl2, 8 M urea, and 6 M guanidine
HCl. Based on deglycosylation procedures as chemical
-elimination,
proteinase K digestion, and susceptibility to glycosaminoglycan lyases
(chondroitinase ABC and heparitinases I and II), it was demonstrated
that the
5
1 heterodimer and
5 and
1 integrin subunits were
proteoglycans. The importance of
5
1
sulfation was strengthened by the finding that this molecule is also
sulfated in MG-63 (human osteosarcoma) and HCT-8 (human colon
adenocarcinoma) cells.
Proteoglycans are complex molecules formed by a core protein to which one or more glycosaminoglycan (GAG)1 chains are linked. This basic definition, although true, hides the molecular complexity shown by these molecules. They encompass an exceptionally large range of structures involving different core proteins, different classes of GAGs, and different numbers and lengths of individual GAG chains. Other post-translation modifications such as N- and O-glycosylation increase the complexity of these molecules (for review see Refs. 1 and 2).
The biological functions of proteoglycans are numerous. They have been
involved in several biological effects (1, 3-5), such as extracellular
matrix (ECM) assembly (6) and cell surface-ECM receptors for growth
factors and hormones (2, 5, 7) or have had a role in biological
processes such as cell-cell recognition (8) and control of cell growth
(9). The fact that several ECM proteins, such as fibronectin (10),
laminin (11), thrombospondin (12), vitronectin (13), type IV collagen
(14), and tenascin (15), have GAG binding sites adds credence to the
postulated multiple roles of proteoglycans. Supporting the idea of
proteoglycans as ECM receptors, syndecan type I binds fibronectin,
thrombospondin, collagens (5), and tenascin (16); the heparan sulfate
proteoglycan of Schwann cells binds laminin (17); a cell surface
chondroitin sulfate proteoglycan is apparently involved in cell
adhesion to laminin (18); and a cell surface phosphatidyl
inositol-anchored heparan sulfate proteoglycan mediates melanoma cell
adhesion to fibronectin (19). Strong corroboration for these
proteoglycan-ECM interactions comes from the presence of a heparan
sulfate proteoglycan that co-localizes with 1 integrins
as a widespread component of focal adhesion (20).
Among the several ECM molecules that bind proteoglycans, the role of fibronectin should be emphasized not only because of its GAG binding domains but also because of the adhesive properties conferred to this molecule by these domains together with the RGD cell-binding fragment (21-23). Cells devoid of proteoglycans or bearing proteoglycans with altered GAG chains have a reduced capacity of adhesion to fibronectin and have a defective focal adhesion plaque formation in response to this molecule (24, 25).
The best studied receptors for fibronectin that bear adhesiveness and
focal adhesion plaque formation are integrins that are /
heterodimers widely expressed by almost all animal cells (26, 27).
Integrins represent good examples of how post-translational modifications can alter the structure of a molecule, thus modulating its biological activity. Integrin glycosylations represent a kind of
regulation by which a wide variety of these receptors have their
specificity and affinity modulated in several cell lines (28-30).
However, the versatility of cells to modulate the binding properties of
integrins is not restricted to glycosylation. Integrin functions can be
modulated by acylation of membrane lipid (31), by divalent metal
binding (32), and, for the cytoplasmic domain, by tyrosine
phosphorylation, which is the best understood example of this type of
biological modification, especially in leukocytes and platelets (27,
33).
In the present study, we characterize 5
1
integrin as a part-time proteoglycan containing both heparan and
chondroitin sulfate, which per se could affect cell adhesion
to both fibronectin RGD and GAG binding domains.
Human fibronectin was purified from
fresh plasma (obtained from Hospital A. C. Camargo, São Paulo,
Brazil) by gelatin affinity chromatography as described (34).
Monoclonal antibody A-1A5 that recognizes the 1 integrin
subunit (35) and B-5G10 that reacts with the
4 integrin
subunit (36) were provided by Dr. Martin E. Hemler (Dana-Farber Cancer
Institute, Harvard Medical School, Boston, MA). Monoclonal antibody
II-F5, which specifically recognizes the
3 integrin
subunit (37), was a gift from Dr. Renata Pasqualini (The Burnham
Institute, San Diego, CA). Monoclonal antibodies against
2 integrin subunit CLB-thromb/4 and
5
integrin chain SAM-1 were purchased from the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (Amsterdam, the Netherlands). Rabbit polyclonal antibody against the cytoplasmic domain
of the
7 integrin subunit (38) was a gift from Dr.
Stephen J. Kaufman (Department of Cell and Structural Biology,
University of Illinois, Urbana, IL), and rabbit polyclonal antiserum
against the
5
1 integrin molecule (RB3847)
that in immunoblotting reacts only with the
1 integrin
subunit2 was provided by Dr. Kenneth M. Yamada (National Institute for Dental Research, Bethesda, MD).
Monoclonal antibody against chondroitin sulfate chains (CS-56) was
purchased from Sigma.
A human melanoma cell line (Mel-85) was provided by Dr. Stephan Carrel (Ludwig Institute for Cancer Research, Lausanne, Switzerland). A human osteosarcoma cell line (MG-63) was given by Dr. Eva Engvall (Burnham Institute, San Diego, CA), and a human colon adenocarcinoma cell line (HCT-8) was given by Dr. M. M. Brentani (Department of Oncology, School of Medicine, São Paulo University, Brazil). All cells were grown in RPMI 1640 medium (Sigma) supplemented with 10% fetal calf serum (Cultilab, Campinas, Brazil) and gentamicin (50 µg/ml) at 37 °C, 5% CO2 in humidified conditions. Cells were harvested using divalent cation-free phosphate-buffered saline containing 2 mM EDTA. For [35S]sulfate incorporation, cells were labeled in the presence of sodium [35S]sulfate (240 µCi/ml of medium) for 24 h.
Immunoprecipitation ReactionsCell surface expression of
1 integrin heterodimers in Mel-85 cells was probed
through immunoprecipitation reactions of cells that were surface
labeled (using [125I]iodine) by the
lactoperoxidase-H2O2 method as described
previously (39). After washing, cells were solubilized by lysis buffer (50 mM Tris-HCl, pH 7.3, 1% Triton X-100, 50 mM NaCl, 5 mM CaCl2, 5 mM MgCl2, 1 mM
phenylmethanesulfonyl fluoride, and 2 µg/ml of aprotinin) for 15 min
at 4 °C. The extract was clarified by centrifugation for 10 min at
13,000 - g, and the supernatant was preincubated with either
normal mouse or rabbit serum followed by precipitation with protein
A-Sepharose (Sigma). Mel-85 extract (at the same mass
of protein, 1 mg) was incubated respectively with antibodies against
different integrin subunits (as shown above), and for B-5G10 (an
IgG1 molecule), rabbit IgG was preincubated against mouse
IgG followed by protein A-Sepharose. Affinity beads were washed with
lysis buffer, and the immunoprecipitates were eluted by boiling for 5 min with Laemmli buffer.
[35S]Sulfate-labeled Mel-85 cell extracts were
immunoprecipitated using the same mono- or polyclonal antibodies as
above. Immunoprecipitates were analyzed by 7.5% SDS-PAGE (40) followed
by electrotransference onto nitrocellulose membranes (41) and exposure
to x-ray films (Kodak, Rochester, NY). The same procedure was used to
study HCT-8 and MG-63 cell extracts with an anti-5
integrin antibody.
The [35S]sulfate-labeled
immunoprecipitates obtained with anti-5 integrin
monoclonal antibody were incubated with 4 M NaCl, 4 M MgCl2, 6 M guanidine HCl, and 8 M urea for 2 h at 37 °C. Mixtures were then boiled
for 5 min, and
5
1 integrin was separated
from Sepharose beads by centrifugation for 1 min at 13,000 × g. Supernatants were dialyzed against water, concentrated in
a speed vaccum concentrator, subjected to 7.5% SDS-PAGE under
nonreducing conditions, and electrotransferred onto nitrocellulose
membranes that were then exposed to x-ray films at room temperature for
10 days.
Fibronectin-affinity chromatography of [35S]sulfate-labeled Mel-85 cell extract was performed using purified human plasma fibronectin coupled to CNBr-activated Sepharose (Pharmacia Biotech Inc.) as detailed elsewhere (30).
Gel Electrophoresis, Purification ofSDS gel
electrophoresis was performed as described (40). Samples under reducing
or nonreducing conditions were analyzed on 5 or 7.5% polyacrylamide
gels, and proteins were transferred overnight to nitrocellulose filters
as described (41). Molecular mass markers (myosin, 205 kDa;
-galactosidase, 116 kDa; and phosphorylase b, 98 kDa;
albumin, 67 kDa) were purchased from Sigma. For
two-dimensional electrophoresis, samples were separated in the first
dimension by isoelectric focusing (42) using an ampholyte gradient (pH 4.0-6.5, six parts and pH 3.0-10.0, four parts, Pharmacia) followed by 7.5% SDS-PAGE in nonreducing conditions.
Glycosaminoglycan analysis was performed using agarose gel electrophoresis in 0.05 M 1,3-diaminopropane acetate buffer pH 9.0 (Aldrich). After the electrophoretic run, compounds were precipitated in the gel using 0.1% Cetavlon for 2 h at room temperature (43). After drying, the gel was stained with toluidine blue and exposed to x-ray films (X-Omat, Kodak) for 10 days at room temperature. GAG standards used were heparan sulfate from bovine pancreas (44), dermatan sulfate from pig skin, and chondroitin sulfate from shark cartilage (Seikagaku, Kogyo Co., Tokyo, Japan).
To study the specific pattern of glycosylation of 5 and
1 integrin subunits, [35S]sulfate-labeled
Mel-85 cell lysate was immunoprecipitated using a monoclonal antibody
against the
5 integrin subunit as already described, and
the precipitate was submitted to a preparative 7.5% SDS-PAGE under
nonreducing conditions using prestained
-galactosidase (116 kDa)
that comigrates with the
1 integrin subunit as a
standard. Autoradiography of separated
5 and
1 subunits was done in identical conditions as above and
used as a guide. Gel pieces were then cut off in the positions of
separated
5 and
1 subunits, and proteins
were excised and eluted from the gel by incubation in 50 mM
Tris-HCl, pH 7.3, containing 0.1% Triton X-100 overnight at 4 °C.
The mixtures were then filtered through 0.45-µm filters (Nalgene,
Rochester, NY) to remove polyacrylamide, and the solutions containing
extracted proteins were dialyzed against water and concentrated
20-fold. Purified
5 and
1 integrin
subunits were then submitted to
-elimination reaction to release
free GAG chains, which were incubated with chondroitinase ABC,
heparitinases I and II, or a mixture of these enzymes (see below), and
the digests were analyzed by agarose gels.
Immunoblotting reactions using Rb3847 (a rabbit polyclonal antibody
that only reacts with the 1 integrin subunit but not with the denatured
5 integrin subunit) and a monoclonal
antibody against chondroitin sulfate chains (CS-56) were performed as
described previously (30).
The GAG
chains from [35S]sulfate-labeled
5
1 integrin, purified by
immunoprecipitation using a monoclonal antibody against the
5 integrin subunit, were liberated by digestion of the
protein core using excess proteinase-K (50 µg;
Sigma) at 58 °C overnight or by a
-elimination
reaction (treatment overnight at 37 °C with 0.1 M NaOH
in the presence of 2 M NaBH4;
Sigma). The products obtained were analyzed by agarose
gel electrophoresis.
-Eliminated materials were submitted to
digestion with chondroitinase ABC from Proteous vulgaris
(Seikagaku, Kogyo Co, Tokyo, Japan), heparitinases from
Flavobacterium heparinum (45), or all enzymes and analyzed by agarose gel electrophoresis.
Because integrins are substrates for several
different post-translational modifications, we decided to determine
whether they could function as substrates for sulfation. We decided to
address this question using [35S]sulfate labeling of the
cells, immunoprecipitation, and blotting experiments. As shown
in Fig. 1, Mel-85 cells in culture
efficiently incorporate [35S]sulfate. The cell lysate was
submitted to immunoprecipitation with a monoclonal antibody against the
1 integrin subunit, and a [35S]sulfated
1 integrin molecule dimer was detected. This suggests that
1 integrin is a substrate for post-translational
sulfation.
After the demonstration that 1
dimer integrin is a sulfated molecule, our next experimental procedure
was to identify the
subunit complementing the
1
subunit in this particular integrin heterodimer. To investigate this,
Mel-85 cells were surface radiolabeled with [125I]iodine
by the lactoperoxidase method or metabolically labeled with
[35S]sulfate. Both cell lysates were immunoprecipitated
with antibodies against different integrin subunits. We can see in the
[125I]iodine-labeled immunoprecipitates (Fig.
2A) the presence of
1,
2,
3,
4,
5,
7, and probably
1 subunit, a 200-kDa
signal (Fig. 2A, lane 1) that could be
precipitated with the
1 subunit. Neither cell flow
cytometry nor immunoprecipitation showed detectable levels of the
6 integrin subunit in Mel-85 cells (data not shown). Interestingly, Fig. 2B shows that only
5 and
the corresponding
1 subunit are sulfated. These findings
suggested that
5
1 integrin is a
facultative sulfated
1 integrin molecule because none of the other
1 integrin molecules incorporated
[35S]sulfate.
It is known that after reduction of the disulfide bonds by
-mercaptoethanol (chemical reduction), the
5 integrin
subunit comigrates with the
1 subunit (36). The
immunoprecipitates obtained using monoclonal antibodies to
1 and
5 integrin subunits or a polyclonal
antibody against the
5
1 integrin dimer
from a [35S]sulfate-labeled Mel-85 cell lysate were then
subject to chemical reduction. As shown in Fig. 2C, after
chemical reduction the immunoprecipitates reveal just one band in the
gel, confirming that
5
1 integrin is a
sulfated molecule. It is also known that the
5
1 integrin has fibronectin as the only
ECM ligand (27, 46). Fig. 2D shows that after elution from a
fibronectin affinity chromatography
5
1
integrin is detected as a sulfated molecule.
Data in the literature describe proteoglycans as
5
1 integrin-associated molecules that
complement the requirements involved in cell adhesion to fibronectin.
In addition, in melanoma cells a heparan sulfate proteoglycan of
150/175 kDa has been described to bind fibronectin (19, 22, 47, 48). We
cannot therefore discard the possibility of a physical association
between a third molecule that comigrates with
integrin subunits,
masking the sulfated signals in the autoradiograms. To rule out this
possibility the same [35S]sulfate-labeled Mel-85 cell
extract was again immunoprecipitated by specific monoclonal antibodies
to
5 and
1 integrin subunits and now
submitted to a two-dimensional electrophoresis (Fig. 3, A and B). We can observe that the
immunoprecipitation reactions using either anti-
1
antibody or anti-
5 antibody show only a sulfated signal
of
5
1 dimer.
To corroborate the findings described above and demonstrate that the
sulfate groups in 5
1 integrin are
covalently linked and not adsorbed to this molecule or to the beads
during immunoprecipitation, this integrin was immunoprecipitated from a
[35S]sulfate-labeled Mel-85 cell. After washing with
phosphate-buffered saline, the beads were submitted to different
conditions of elution such as high ionic strength (4 M
NaCl) and chaotrophic agents (6 M guanidine HCl, 4 M MgCl2, 8 M urea). After boiling,
the precipitates were dialyzed against water, submitted to 7.5%
SDS-PAGE, and transferred onto nitrocellulose filters, which were then
exposed to an x-ray film (Fig. 3C). These results show that
the
5
1 integrin bears covalently linked
sulfate groups.
Proteoglycans represent the best characterized
sulfated molecules containing serine-linked sulfated GAG chains as a
result of post-translational modifications of the protein core (2, 7,
10). To determine the site of sulfate substitution in the
5
1 integrin dimer,
[35S]sulfate-labeled Mel-85 cell lysate was
immunoprecipitated using antibody against the
5 subunit
and subjected to
-elimination. This procedure cleaves GAG chains
from the protein core. As shown in Fig. 4A
(lane 2),
5
1 integrin after
-elimination showed two sulfated bands that comigrate
electrophoretically with chondroitin and heparan sulfate standards. An
identical result was obtained when [35S]sulfated
5
1 integrin was submitted to proteinase-K
digestion (a serine protease of broad specificity) (Fig. 4A,
lane 3). These experiments suggest that
5
1 integrin is a hybrid
chondroitin/heparan sulfate proteoglycan. This was further investigated
by degradation of the
-eliminated material from
5
1 integrin with specific enzymes that
degrade chondroitin sulfate (chondroitinase ABC) and heparan sulfate
(heparitinases type I and II). We can see that under these conditions,
both sulfated bands were completely degraded by the specific enzymes
(Fig. 4B), demonstrating that
5
1 integrin is in fact a hybrid
chondroitin/heparan sulfate proteoglycan.
Both
Because in Mel-85 cells
5 and
1 subunits of integrin contain
sulfate, our next goal was to determine the specific pattern of
glycosylation, that is, to which subunit chondroitin and heparan sulfates are linked. Two complementary approaches based on immunologic specificities were used. Fist,
5
1
integrin was immunoprecipitated from a 35S-labeled Mel-85
cell extract as described above. After separation by polyacrylamide gel
electrophoresis, the immunoprecipitate was exposed to an x-ray film,
blotted, and reacted with a monoclonal antibody specific for
chondroitin sulfate chains (Fig. 5A). We can
see that both integrin subunits bear chondroitin sulfate chains. Also,
Mel-85 cell lysate was immunoprecipitated with a anti-chondroitin sulfate monoclonal antibody and blotted with a polyclonal antiserum against the
1 integrin subunit (Fig. 5B).
Interestingly, we can see that only the 116-kDa
1
integrin subunit, which corresponds to the completely glycosylated
form, has chondroitin sulfate chains. In contrast, the
pre-
1 integrin chain (100 kDa), which corresponds to a
high mannose structure, has no chondroitin sulfate chains. Pre-
1 integrin shows the glycosylation profile of a
protein that has not crossed the Golgi. Because synthesis of GAG occurs
in the Golgi, the absence of chondroitin sulfate in the
pre-
1 integrin should be expected (49). This finding was
also substantiated by results shown in Fig. 1 in which the
pre-
1 integrin subunit, although coprecipitated, is not
sulfated. These results demonstrate that the integrin dimer
5
1 is a proteoglycan and that in Mel-85 cells both integrin subunits have covalently linked chondrotin sulfate
chains.
As a second approach we have used successive immunoprecipitation
reactions to isolate 5
1 integrin from a
35S-labeled Mel-85 cell lysate. The purified
5
1 integrin was submitted to preparative
polyacrylamide gel electrophoresis. Using an autoradiogram of the gel
and prestained molecular mass standards as guides, separated
5 and
1 subunits were removed from the
gel and subjected to
-elimination to obtain GAG free chains. These
chains were analyzed by agarose gel electrophoresis before and after
treatment with chondroitinase ABC, heparitinases, and a mixture of
these enzymes (Fig. 5C). The result obtained from this last
set of experiments support the concept that
5
1 integrin is a proteoglycan. It shows that both subunits contain chondroitin sulfate and heparan sulfate, thereby demonstrating that
5
1 integrin is
a hybrid chondroitin/heparan sulfate proteoglycan.
Because all experiments described so far were
performed using the human melanoma cell line Mel-85, which has a
neuro-ectodermic origin, we decided to investigate whether this
5
1 integrin post-translational modification was also present in cell lines of endodermic (HCT-8 cells)
and mesodermic (MG-63 cells) origin. Cells were labeled with
[35S]sulfate, and lysates were immunoprecipitated with
monoclonal antibodies against the
5 integrin subunit and
analyzed by SDS-PAGE followed by electroblotting onto nitrocellulose. A
polyclonal antibody against the
1 integrin subunit (Fig.
6A) was used to detect the integrin. We can
see that both cells display the [35S]sulfate
incorporation into the
5 integrin subunit. The results indicate that GAG substitution of
5
1
integrin has been conserved, suggesting its biological significance.
However, in the case of the MG-63 cells, only the
5
subunit was labeled; the
1 subunit found in MG-63 cells
did not contain sulfate. To corroborate the results described in Fig.
6A and provide more evidence that the glycosylation of
integrin
5
1 integrin as a proteoglycan is
maintained in different tissues, we submitted
[35S]sulfate-labeled
5
1
integrin obtained from HCT-8 and MG-63 cell extracts to a
-elimination reaction. Products were analyzed by agarose gel
electrophoresis. As shown in Fig. 6B, we can see that
heparan and chondroitin sulfates are present in
5
1 integrins of endodermic and mesodermic
origins, as observed for neuro-ectodermic cells. The results not only
confirm the conservative proteoglycan nature of
5
1 integrin from different origins but
also indicate a similar glycosylation pattern.
Working with the human melanoma cell line Mel-85, we have
described 5
1 integrin as a hybrid
chondroitin/heparan sulfate proteoglycan. Based on immunoprecipitation
reactions from cell lysates that were cell surface labeled with
[125I]iodine or metabolically labeled with
[35S]sulfate, we were able to detect
5
1 integrin as the only sulfated integrin
compared with other
(s)
1 heterodimers
present in Mel-85 cells. Sulfation of
5
1
integrin was confirmed not only by immunological methods but also by
fibronectin affinity chromatography, two-dimensional electrophoresis,
and reduction of disulfide bonds of the
5
1 heterodimer leading to comigration of
both
5 and
1 integrin subunits,
characteristic of this integrin as described (36). Based on different
procedures such as chemical deglycosylation by
-elimination,
proteinase-K digestion, immunological methods, and susceptibility to
chondroitinase ABC and heparitinases, we were able to confirm this
integrin as a proteoglycan. These results raise the important question
of which mechanisms determine
5
1 as the
only sulfated integrin. Why are
1 subunits not sulfated in other
1 heterodimers? The existence of alternative
splicing for the
1 integrin subunit as described (50)
(reviewed in Ref. 27) could explain in part such differences. However,
because glycosylation of cell surface proteoglycans is restricted to
extracellular domains (2) and the
1 integrin subunit has
only alternatively spliced cytoplasmic domains (27) this mechanism does
not explain our findings. Oligomerization of
integrin
heterodimers is an event that occurs during transit through the
endoplasmic reticulum (28) and precedes glycosaminoglycan biosynthesis,
which occurs during transit through the Golgi (2). Perhaps the best
explanation for the part-time proteoglycan nature of
5
1 integrin is that the conformation of
the heterodimer exposes the serine residues that are acceptors for the
GAG chains, which does not happen with other
1
heterodimers. This conformational hypothesis is consistent with the
lack of a consensus sequence for proteoglycan biosynthesis initiation
(1, 2) and by the experiments performed with decorin, a proteoglycan in
which the primary structure of the protein core surrounding the sugar
acceptor serine residue can be changed without appreciable modification
in the glycosaminoglycan (51). Considering the findings described
above, it is possible to assume that the same conformational folding of
5
1 that makes this integrin capable of
recognizing the RGD peptide only in fibronectin among several other ECM
molecules (52) is also responsible for a specific GAG synthesis that
complements the molecular requirements involved in the interaction of
this integrin with fibronectin.
The possibility that other integrins can also be sulfated is not ruled
out by the present study because we could not detect 6
1 integrin in Mel-85 cells. This is an
integrin that binds laminin, a molecule with a GAG binding domain
spatially close to the E8 domain corresponding to the
6
1 integrin binding site (53).
Furthermore, the structural relationship of the
5 chain with
IIb and
v (reviewed in Ref. 27)
could suggest other
integrin heterodimers as putative acceptors
for GAG addition. Interestingly, Hayashi, Madri, and Yurchenco (54)
have shown that endothelial cell interaction with the basement membrane
proteoglycan (perlecan) occurs between the core protein of perlecan and
1 and
3 integrins, an interaction
partially RGD-independent and modulated by GAGs. The
1
integrin heterodimer involved in this adhesion resembles the
5
1 molecule and the
3
integrin, the
v
3 vitronectin receptor
(54).
The present study suggests for the first time that integrins such as
5
1 may have two extracellular binding
sites that play a role in fibronectin binding. Previous studies have
implicated a specific involvement of the heparin binding site of
fibronectin with cell adhesion. These data were based on the fact that
the purified fibronectin fragment containing only the heparin binding domain without the RGD peptide can promote adhesion in several different cell models (21, 23). Because our work describes
5
1 integrin as a part-time proteoglycan
compared with other
1 dimers, we can postulate that
the fibronectin-
5
1 integrin interaction,
which occurs primarily through the RGD peptide in fibronectin, is
complemented and stabilized by the secondary interactions of
5
1 chondroitin or heparan sulfate chains
with the fibronectin heparin binding domains. The possibility that
5
1 integrin, an integrin that binds only
fibronectin, has chondroitin and heparan sulfate chains interacting
with the fibronectin heparin binding domains is suggested by the facts
that during ECM assembly the fibronectin heparin binding domain can
also bind chondroitin sulfate or dermatan sulfate proteoglycans (10)
and that soluble proteoglycans can inhibit cell adhesion to fibronectin
(reviewed in Ref. 22) and by the existence of nonintegrin fibronectin
receptors like CD44 (a chondroitin sulfate proteoglycan) and a heparan
sulfate proteoglycan (19, 48) as well as by the recent finding that monoclonal antibodies raised against the fibronectin heparin binding domain (Hep II/IIICS) inhibit cell adhesion and also partially inhibit
integrin binding to fibronectin (55). A model is postulated in which
the RGD and heparin binding sites in fibronectin, although linearly
separated, are spatially close due to fibronectin folding. It is thus
possible to assume that cell surface proteoglycans and integrin
cooperativity during cell adhesion can really be achieved in the case
of
5
1 integrin by two binding sites in the integrin molecule that bind RGD peptide and GAG binding domains in
fibronectin.
We thank Drs. E. Engvall, K. M. Yamada, M. E. Hemler, M. M. Brentani, R. R. Pasqualini, S. Carrel, and S. J. Kaufman for the gifts of reagents described under "Experimental Procedures." C. P. Dietrich, V. Buonassisi, and P. Colburn are gratefully acknowledged for revision of the manuscript.