(Received for publication, February 27, 1995; and in revised form, July 18, 1995)
From the
The integrin family of adhesion receptors consists of at least
21 heterodimeric transmembrane proteins that differ in their tissue
distribution and ligand specificity. The recently identified 8
integrin subunit associates with
1 and is predominantly expressed
in smooth muscle and other contractile cells in adult tissues, and in
mesenchymal and neural cells during development. We now show that
8
1 specifically localizes to focal contacts in cells plated
on the extracellular matrix proteins fibronectin or vitronectin. In
addition we show that human embryonic kidney cells (293), transfected
with
8 cDNA, express
8
1 on their surface and use this
receptor for adhesion to fibronectin and vitronectin. Furthermore,
8
1 binds to both fibronectin- and vitronectin-Sepharose and
can be specifically eluted from either matrix protein by the
arginine-glycine-aspartic acid (RGD)-containing peptide, GRGDSP.
Because fibronectin and vitronectin adhesion appeared to be mediated by
RGD, we examined additional RGD-containing proteins, including
tenascin, fibrinogen, thrombospondin, osteopontin, and denatured
collagen type I. We found that only tenascin was able to mediate
adhesion of
8-transfected 293 cells. By using recombinant
fragments of tenascin in adhesion assays, we were able to localize the
8
1 binding domain of tenascin to the RGD-containing, third
fibronectin type III repeat. These data strongly suggest that tenascin,
fibronectin, and vitronectin are ligands for
8
1 and that this
integrin binds to the RGD site in each of these ligands through
mechanisms that are distinct and separate from
5-and
v-containing integrins.
Integrins are a class of cell adhesion glycoproteins composed of
two noncovalently associated subunits, and
. Each subunit
contains a large extracellular domain, a transmembrane domain, and a
short cytoplasmic domain. Integrins are known to bind to a wide variety
of extracellular matrix proteins, including fibronectin, vitronectin,
collagens, and laminins. The specificity of protein binding is
determined by particular combinations of
and
subunit
pairing. The ligand binding site is formed by the extracellular domain
of both subunits and requires the presence of divalent cations. Many
integrins interact with ligands through the tripeptide
arginine-glycine-aspartic acid (RGD).
The 8 integrin subunit
was originally identified by Bossy et al.(1) in the
chick embryo nervous system and was shown to be a partner for
1.
We have identified human
8, cloned and sequenced the cDNA, raised
antibodies to the predicted cytoplasmic domain sequence, and determined
its distribution in adult mammalian tissues (2) . We found that
8 is predominantly expressed in a variety of visceral and vascular
smooth muscle cells, kidney mesangial cells, and lung
myofibroblasts(2) .
To gain insight into potential functions
of 8
1 in vivo, we sought to determine potential
ligands. We tested various ligands for their ability to direct
8
1 to focal contacts, to bind to
8
1 by affinity
chromatography, and to support adhesion of
8-transfected cells.
All of the results provide strong evidence that
8
1 can
function as an RGD-dependent receptor for tenascin, fibronectin, and
vitronectin.
To block endogenous production of extracellular matrix proteins, cells were incubated with the protein synthesis inhibitor cycloheximide (30 µg/ml) for 3 h in serum-free medium. Cells were then detached with 2 mM EDTA and seeded onto fibronectin- and vitronectin-coated coverslips in the presence of cycloheximide.
For antibody-blocking experiments, cells were
incubated with no antibodies, with P5D2 (anti-1) (1:10), with
P3D10 (anti-
5) (1:5), with L230 (anti-
v) (1:10), with P5D2
and P3D10 or with P5D2 and L230, in a plate precoated with 1% BSA.
After 15 min of incubation at 4 °C, cells were then transferred to
protein coated wells (3 µg/ml of fibronectin, vitronectin, or
TNfn3) and processed as above. All antibodies were used at saturating
concentrations. The data were expressed as the mean absorbance of
triplicate wells ± S.E., minus the mean absorbance of BSA-coated
wells.
Peptide blocking experiments were carried out in an analogous manner. Peptides were used at the following final concentrations: 125 µM RGDGW, 125 µM CRRETAWAC, 1 µM 4C, and 100 µg/ml GRGDSP.
The data reported represent the
results from one clone of mock-transfected and 8-transfected 293
cells. The adhesion assay results were confirmed with four independent
clones of
8-transfected 293 cells and with wild type
(untransfected) 293 cells (data not shown).
Cell lysates were prepared by incubation with
200 mM octylglucoside, 100 mM Tris-HCl, 1 mM divalent cation (MnCl, CaCl
or
MgCl
) for 1 h at 4 °C, followed by centrifugation at
14,000 rpm for 20 min.
Affinity columns were prepared by coupling
the ligand (fibronectin or vitronectin) to cyanogen-bromide activated
Sepharose as described previously(17) . I-Labeled
cell lysates were applied to a 1-ml affinity column. The column was
washed with 10 volumes column buffer (50 mM octylglucoside, 50
mM Tris-HCl, 1 mM divalent cation (MnCl
,
CaCl
, or MgCl
). Elutions were carried out using
4 volumes of GRGDSP peptide (1 mg/ml), followed by 2 volumes of column
buffer, and 4 volumes of 10 mM EDTA in column buffer without
divalent cations. Finally the columns were washed with 4 volumes of 1 M NaCl in column buffer. Fractions (1 ml) were collected and
either analyzed directly by SDS-PAGE or subjected to
immunoprecipitation and then analyzed by SDS-PAGE as described
previously(17) .
Figure 1:
Immunoprecipitation of
8-containing integrins from HISM cells (lane 1), REF
cells (lane 2), 293 cells (lane 3), and
8-transfected 293 cells (lane 4). Aliquots of
I-surface-labeled lysates were immunoprecipitated with
anti-
8 antibody. Proteins were analyzed by SDS-PAGE under
nonreducing conditions. Positions of molecular size markers in
kilodaltons are shown to the right.
HISM cells plated
on extracellular matrix proteins were analyzed by double-labeling
immunofluorescence microscopy using antibodies to vinculin (to detect
focal contacts) and 8 (Fig. 2). Three hours after plating
on fibronectin, vitronectin, or collagen, HISM cells were well spread
and formed vinculin-containing focal contacts. In cells plated on
fibronectin or vitronectin,
8 co-localized to vinculin-containing
focal contacts (Fig. 2, A-D). In contrast, in
cells plated on collagen type I or denatured collagen type I,
8
did not localize to focal contacts, despite the abundance of focal
contacts identified by vinculin staining (Fig. 2, E and F). Cells plated on tenascin adhered, but did not form
vinculin-containing focal contacts (data not shown). Cells plated on
fibrinogen or laminin attached poorly and did not spread or form focal
contacts. No localized
8 staining was detected in these cells
(data not shown). Identical results were obtained using rat embryo
fibroblast cells (data not shown). It is unlikely that
8
localization was due to matrix protein secretion by the cells, because
control experiments using cells pretreated with the protein synthesis
inhibitor, cycloheximide, yielded similar results (Fig. 3). We
were not able to demonstrate focal contact formation by
8-transfected or untransfected 293 cells. However,
8-transfected SW 480 colon carcinoma cells showed new localization
of
8 to focal contacts when plated on fibronectin and vitronectin
(data not shown). Thus, vitronectin and fibronectin, but not collagen,
specifically promote localization of
8 to focal contacts. These
data suggest vitronectin and fibronectin are ligands for
8
1.
Figure 2:
Focal contact formation by HISM cells on
various extracellular matrix proteins. HISM cells were allowed to
spread on 10 µg/ml fibronectin (A and B),
vitronectin (C and D), or collagen type I (E and F) for 3 h then fixed, permeabilized, and
double-labeled with anti-8 antibody (A, C, and E) and anti-vinculin antibody (B, D, and F). Sample focal contacts are identified by arrows.
Figure 3:
Focal contact formation by HISM cells in
the presence of cycloheximide. Cells were preincubated with
cycloheximide (30 µg/ml) for 3 h and then allowed to spread on 10
µg/ml fibronectin (A and B) and vitronectin (C and D) for 3 h in the presence of cycloheximide.
Cells were fixed, permeabilized, and doublelabeled with anti-8
antibody (A and C) and anti-vinculin antibody (B and D).
To determine whether the 8 transfectants have
altered levels of other
1-associated
subunits, we performed
fluorescein-activated cell sorting analysis using a panel of
anti-integrin antibodies (Fig. 4). Mock-transfected and
8-transfected 293 cells contained similar amounts of cell surface
2-,
3-,
5-, and
v-containing integrins. Thus,
changes in adhesive properties of
8-transfected cells are not due
to changes in the surface expression of other integrins.
Figure 4:
Flow cytometry of 8-transfected 293
cells (black bars) and mock-transfected 293 cells (white
bars). The results represent an average of four different clones
of
8-transfected 293 cells and mock-transfected 293 cells. Cells
were incubated with the following antibodies: P1H5 (anti-
2), P1B5
(anti-
3), P3D10 (anti-
5), L230 (anti-
v), and P5D2
(anti-
1) and were analyzed using a FACScan. The y axis
represents fluorescence intensity (in arbitrary
units).
Figure 5:
Adhesion of mock-transfected (black
squares) and 8-transfected (open squares) 293 cells
to increasing concentrations (0.3, 1, 3, 10, and 20 µg/ml) of (A) fibronectin and (B) vitronectin. The y axis represents the absorbance at 595 nm after staining attached
cells with crystal violet.
Figure 6:
Adhesion of 8-transfected (black
squares) and mock-transfected (open squares) 293 cells to
3 µg/ml fibronectin. A, cells plated on fibronectin were
incubated with no antibody, anti-
1 antibody (P5D2), anti-
5
antibody (P3D10), or anti-
1 and anti-
5 antibody in
combination. B, cells plated on fibronectin were incubated
alone (control), or with the peptides CRRETAWAC and
RGDGW.
Figure 7:
Adhesion of 8-transfected (black
squares) and mock-transfected (open squares) 293 cells to
3 µg/ml vitronectin. A, cells plated on vitronectin were
incubated with no antibody, anti-
1 antibody (P5D2), anti-
v
antibody (L230), or anti-
1 and anti-
v antibody in
combination. B, cells plated on vitronectin were incubated
alone (control), or with the peptides 4C and
RGDGW.
Vitronectin adhesion of
mock-transfected and wild type 293 cells was almost completely
inhibited by the blocking anti-v antibody, L230 (Fig. 7A), consistent with previous
reports(15, 20) . In contrast, vitronectin adhesion in
8-transfected cells was only partially inhibited (19%) by the
anti-
v antibody, L230, and inhibited by 41% using the anti-
1
antibody, P5D2. Vitronectin adhesion was completely abolished using
both antibodies in combination. These data suggest that
8-transfected cells are using
8
1 in addition to
v-containing integrins, to adhere to vitronectin.
To further
elucidate the binding characteristics of 8
1 to fibronectin
and vitronectin, adhesion assays were performed in the presence of
three different synthetic peptides (Fig. 6B and
7B). Although integrins can interact through a common RGD site
in the ligand, conformationally constrained peptides can discriminate
between various RGD binding integrins. The cyclic peptide, CRRETAWAC,
has recently shown to be highly selective for
5
1(13) . At concentrations sufficient to block
adhesion of
5
1 to fibronectin, CRRETAWAC does not block
v
1 fibronectin adhesion or
v-mediated vitronectin
adhesion(13) . In an analogous fashion, the cyclic peptide 4C
selectively inhibits
v-mediated adhesion(21) . In
contrast, the peptides GRGDSP and RGDGW are able to block both
5-
and
v-mediated adhesion(12) . We took advantage of these
selective peptides to further define the binding characteristics of
8
1. We found that adhesion of
8-transfected cells to
fibronectin was inhibited by the peptide RGDGW, but not by the
CRRETAWAC peptide (Fig. 6B). The addition of the
5
blocking antibody, P3D10, to CRRETAWAC, did not significantly decrease
fibronectin adhesion (data not shown). In contrast, adhesion of
mock-transfected 293 cells to fibronectin was inhibited by either RGDGW
or CRRETAWAC (Fig. 6B). Adhesion of mock-transfected
and
8-transfected 293 cells to fibronectin was not affected by the
v-selective 4C peptide (data not shown). These results suggest
that
8
1 interacts with fibronectin by mechanisms that are
similar to, but distinguishable from, those used by
5
1.
Adhesion of 8-transfected cells to vitronectin was inhibited by
the peptide RGDGW, but not by the 4C peptide (Fig. 7B).
The addition of the
v blocking antibody, L230, to 4C did not
further inhibit vitronectin adhesion (data not shown). In contrast,
adhesion of mock-transfected 293 cells to vitronectin was inhibited by
either RGDGW or 4C (Fig. 7B). Adhesion of
mock-transfected and
8-transfected 293 cells to vitronectin were
not affected by the
5-selective peptide, CRRETAWAC (data not
shown). Thus, the 4C peptide, at the concentration used, blocks
v-
but not
8
1-mediated adhesion to vitronectin, suggesting that
8
1 interacts with vitronectin through mechanisms distinct
from those used by
v integrins.
Figure 8:
Affinity chromatography of I-surface-labeled lysates from
8-transfected 293
cells on (A) fibronectin-Sepharose column in the presence of 1
mM MnCl
and (B) vitronectin-Sepharose
column in the presence of 1 mM MgCl
. Lane
1, whole cell lysate; lanes 2-8, column buffer
fractions; lanes 9-12, GRGDSP-eluted (1 mg/ml)
fractions; lanes 13-16, 10 mM EDTA-eluted
fractions; lane 17, 1 M NaCl wash. Arrows indicate the positions of putative receptor subunits. Positions of
molecular size markers in kilodaltons are shown to the left.
Figure 9:
Immunoprecipitation of 8- and
1-
containing integrins from (A) fibronectin- and (B)
vitronectin-Sepharose column fractions. Lanes 1-3,
immunoprecipitations with anti-
8 antibody; lanes 1, whole
cell lysates; lanes 2, column buffer fractions; lanes
3, GRGDSP-eluted fractions; lane 4A, GRGDSP-eluted
fractions immunoprecipitated with anti-
5 antibody (P3D10); lane 4B, GRGDSP-eluted fractions immunoprecipitated with
anti-
v
5 antibody (P1F6). Proteins were analyzed by SDS-PAGE
under nonreducing conditions. Positions of molecular size markers in
kilodaltons are shown to the left.
The GRGDSP
eluate of the vitronectin-Sepharose column contained major bands of 150
kDa and 95 kDa (Fig. 8B, arrows). However,
immunoprecipitation of the eluted fractions with anti-8 antibody
demonstrates the 170-kDa/120-kDa complex corresponding to
8
1
was specifically eluted from the vitronectin-Sepharose column by GRGDSP (Fig. 9B, lanes 1-3). As we have shown
previously(15) , the 150-kDa/95-kDa-bands correspond to the
v
5 heterodimer as confirmed by immunoprecipitation of the
GRGDSP eluate with an anti-
v
5 antibody, P1F6 (Fig. 9B, lane 4).
Figure 10:
Adhesion
of 8-transfected (black bars), mock-transfected (white bars) and
3-transfected 293 cells (gray
bars) to 3 µg/ml fibrinogen, denatured collagen type I,
tenascin, thrombospondin, and osteopontin. The y axis
represents the absorbance at 595 nm after staining attached cells with
crystal violet.
Tenascin is a modular protein that contains
several fibronectin type III repeats. An RGD site located in the third
fibronectin type III repeat of tenascin (TNfn3) has been shown to
mediate adhesion of several integrins, including v
3 and
probably
v
6(22) . To determine whether
8
1
was mediating adhesion through this RGD site, we tested the adhesion of
8- and mock-transfected 293 cells to various recombinant fragments
of tenascin (Fig. 11). The
8-transfected cells adhered to
the RGD-containing fragment, TNfn3, in a concentration-dependent
manner. As expected, mock-transfected 293 cells did not adhere to
TNfn3. In contrast,
8-transfected 293 cells did not adhere to a
fragment containing the fourth through sixth fibronectin type III
repeats, TNfn4-6 (Fig. 11). To confirm that
8-transfected cells were binding through the RGD site of TNfn3, we
tested adhesion of
8-transfected cells to a mutant TNfn3 in which
the RGD site had been altered to RAA (TNfn3RAA). We found that this
abolished adhesion of
8-transfected 293 cells (Fig. 11). In
a control experiment,
9-transfected 293 cells, which have been
shown to adhere to TNfn3 at a non-RGD site(14) , adhered well
to TNfn3RAA (data not shown).
Figure 11:
Adhesion of 8-transfected cells to
increasing concentrations of TNfn3 (
), TNfn3-RAA (
),
TNfn4-6 (
), and adhesion of mock-transfected cells to
increasing concentrations of TNfn3 (
). The y axis
represents the absorbance at 595 nm after staining attached cells with
crystal violet.
Adhesion to TNfn3 by
8-transfected cells was abolished by the anti-
1 antibody,
P5D2 (Fig. 12). We also tested the ability of the selective,
synthetic peptides to block adhesion to TNfn3. The peptides, GRGDSP and
RGDGW blocked adhesion of
8-transfected cells to tenascin (Fig. 12). In contrast, the cyclic CRRETAWAC peptide did not
inhibit adhesion and the cyclic 4C peptide only partially inhibited
adhesion to TNfn3 (Fig. 12). Adhesion of
3-transfected
cells to tenascin was abolished by the 4C peptide (data not shown).
Taken in concert, these results suggest that
8
1 is binding to
the RGD site in tenascin. However, this interaction must be somewhat
distinct from the
v-tenascin interactions because it is not
completely inhibited by the peptide 4C.
Figure 12:
Adhesion of 8-transfected cells to 3
µg/ml TNfn3 alone (control) or in the presence of an
anti-
1 antibody, P5D2, the peptides GRGDSP, RGDGW, or the cyclic
peptides CRRETAWAC and 4C. The y axis represents the
absorbance at 595 nm after staining attached cells with crystal
violet.
In summary, we have
demonstrated that 8
1 can bind to tenascin, fibronectin, and
vitronectin by interacting with the RGD sites on these ligands. We also
show that
8
1 is capable of localizing to focal contacts on
fibronectin and vitronectin on fibroblasts and smooth muscle cells.
8
1 is eluted from both fibronectin and vitronectin affinity
columns by an RGD-containing peptide. Our fibronectin adhesion data are
in agreement with the recently published observations that chicken
8
1 is able to support attachment, spreading, and neurite
outgrowth by transfected cells(23) . Two other
1-containing integrins are known to interact with RGD sites:
5
1 and
v
1. These
subunits, along with
IIb, are the most closely related to
8 (42-43% amino
acid identity). In addition, these subunits share several other
structural features with each other including the presence of
post-translational cleavage and the absence of I domains. Thus,
8,
5,
v, and
IIb define a subfamily of
subunits that
have close sequence homology, bind to RGD-containing peptides, are
post-translationally cleaved, and do not contain I domains. Despite the
similarities to
v and
5, the binding specificity of
8
1 is unique. In contrast to
5
1, whose only known
ligand is fibronectin,
8
1 is more promiscuous and can bind to
vitronectin and tenascin as well as fibronectin. In addition, binding
by
8
1 is not affected by the peptide CRRETAWAC, which
efficiently blocks
5
1. When compared to
v
3, the
binding repertoire of
8
1 is more limited. Although both
8
1 and
v
3 bind to tenascin, fibronectin, and
vitronectin,
8
1 does not bind to several other
v
3
ligands, including fibrinogen, thrombospondin and denatured collagen.
Additionally,
8-mediated adhesion is not affected by the peptide
4C. Thus, the binding characteristics of
8
1 are unique and
distinguishable from both
5
1 and
v
3.
In adult
mammalian tissues, 8
1 is prominently expressed in vascular
and visceral smooth muscle cells, kidney mesangial cells, and lung
myofibroblasts(2) . Tenascin, fibronectin, and vitronectin are
thought to play a role in the response to injury and
inflammation(24) . Thus,
8
1 may contribute to the
functional changes that occur in smooth muscle cells during tissue
repair. Since smooth muscle cells also express other fibronectin,
vitronectin, and tenascin receptors, such as
5
1,
v
5, and
v
3(25) , it will be important to
determine the specific functional contribution of
8
1 to
smooth muscle cell behavior.