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175
Integrin Receptors on Aortic Smooth Muscle
Cells Mediate Adhesion to Fibronectin,
Laminin, and Collagen
Ronald
I.
Clyman, Kevin A. McDonald, and Randall H. Kramer
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Extracellular matrix receptors on vascular smooth muscle cells help in anchoring the cells
during contraction and in promoting cellular migration after vessel injury. We found that rat
aortic smooth muscle cells attach to surfaces coated with fibronectin, laminin, and collagen
types I and IV. Cell attachment to these substrates appears to be mediated by members of the
f1 integrin family of extracellular matrix receptors. Antibodies to the PI subunit not only
demonstrated the presence of integrin complexes in focal adhesion plaques but also blocked cell
adhesion to the different substrates. Ligand-affinity chromatography and sodium dodecyl
sulfate-polyacrylamide gel electrophoresis isolated a series of receptor complexes that were
recognized by antisera to Pf integrin receptors. Each of the receptors appeared to be a
heterodimer in which one of several subunits shared a common 120-kDa (nonreduced) P,3
subunit protein. The rat aortic smooth muscle cells had one ca subunit (150 kDa nonreduced,
140 kDa reduced) that bound exclusively to fibronectin. There was a second at subunit (150 kDa
nonreduced, 160 kIDa reduced) that bound exclusively to collagen type I. In addition, there was
a third ca subunit (185 kDa nonreduced, 200 kDa reduced) that was promiscuous and bound to
collagen types I and IV as well as to laminin; the 185-kDa a subunit appeared to bind to
collagen more effliciently than it did to laminin. Thus, smooth muscle cells express multiple
integrin receptors with different ligand specificities that appear to mediate cell interactions
with the extracellular matrix. (Circulation Research 1990;67:175-186)
a
T he mechanisms that regulate smooth muscle
cell migration into the arterial intima play an
important role in arterial wound repair and
atherosclerosis. During arterial remodeling, both
nondividing and proliferating smooth muscle cells
migrate out of the muscle media and through the
internal elastic lamina to increase the intimal smooth
muscle cell number.' Factors that affect the cell's
ability to adhere to the extracellular matrix (ECM)
alter the balance between cell stability and cell
mobility. Components of the ECM that surround
medial smooth muscle cells include fibronectin, laminin, and collagen types I and IV.2-4 These ECM
proteins not only promote cell adhesion and
From the Cardiovascular Research Institute (R.I.C.) and
Departments of Pediatrics (R.I.C.), Anatomy (K.A.M., R.H.K),
and Stomatology (K.A.M., R.H.K.), University of California, San
Francisco, Calif.
Supported in part by US Public Health Service Pulmonary
Special Center of Research grant HL-27356 and American Heart
Association grant 880763.
Address for reprints: Ronald I. Clyman, MD, Box 0544, 1403
HSE, University of California, San Francisco, San Francisco, CA
94143.
Received October 2, 1989; accepted February 16, 1990.
migration5,6 but also influence the phenotypic
appearance of the smooth muscle cell.7
The response of the smooth muscle cell to the ECM
probably depends on specific cell surface receptors. In
vivo, aortic smooth muscle cells form dense adhesion
plaques at the junction between the ECM and the
intracellular contractile microfilament system. These
dense plaques include proteins such as vinculin, talin,
and a-actinin.8 In other cell types, a group of related
cell surface glycoproteins, called integrins, have been
implicated as surface receptors for mediating cell
adhesion to the ECM and to other cells. Each receptor
is a heterodimer in which one of several homologous a
subunits associates noncovalently with a ,B subunit.9"10
The integrins require the presence of divalent cations
to function"-15; the a subunits of the receptors contain several repetitive amino acid sequences that are
similar to the Ca'+ binding sites of other divalent
cation binding proteins.13-'5 Several of the integrins
can interact with a specific amino acid sequence,
Arg-Gly-Asp (RGD), which is located in the cell
binding region of fibronectin and vitronectin and is
also present in laminin, collagen types I and IV, and
other adhesive proteins.10 On the other hand, some
integrin receptors appear not to use the RGD
176
Circulation Research Vol 67, No 1, July 1990
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sequence, although sequences related to RGD may be
important in these interactions.16
In human cells there are at least three major
subfamilies of integrin receptors, which are defined by
their component 3 chains. (There is recent evidence
for a fourth17 and a fifth'8 class of /3 subunits.) The first
subfamily comprises at least six related complexes,
each consisting of a P,I chain with a distinct companion
a chain. Members of the /, subfamily include receptors for fibronectin (a3/3, and a53,1), laminin (al/3,,
a3PI, and a6,1), and collagen types I and IV (a1/l,
a2,13, and a,3p8).19-24 On human cells, these receptors
correspond to the very late activation (VLA) heterodimers first described on T-cells.25,26
The purpose of the present study was to identify rat
aortic smooth muscle (RASM) cell proteins that might
act as cell surface receptors for ECM molecules. We
used affinity chromatography with immobilized ECM
ligands to purify surface receptors followed by immunochemistry to determine their relatedness to the /,1
integrins. We compared the structural and functional
characteristics of the RASM cell surface glycoproteins
with other putative ECM receptors described for cells
of different tissues and species.
Materials and Methods
Cell Culture
Smooth muscle cells were isolated as previously
described27 from rat aortas and characterized by
their morphology and ability to be recognized by
monoclonal antibody against a smooth muscle actin
(Sigma Chemical Co., St. Louis). The cells were
grown in monolayer culture in Dulbecco's modified
Eagle's medium (DME) H-16 (Gibco Laboratories,
Grand Island, N.Y.) supplemented with 1 g/l glucose, 5% fetal calf serum, 100 units/ml penicillin, and
100 ,ug/ml streptomycin. Cells were passaged at
preconfluence after a 2-minute treatment with
trypsin-EDTA (0.25 g/100 ml trypsin and 2 mM
EDTA in Ca2'/Mg2+-free phosphate buffered saline
[PBS] [mM: Na2PO4 15, KH2P04 1.5, NaCl 140, KCl
2]) at room temperature. Cells were used between
passages six and 12.
Antibodies
Anti-hamster /3, (variously called anti-GP140 and
anti-ECMR), a polyclonal antiserum raised in goats
to the 120-160-kDa integrin glycoprotein complexes
purified from BHK cells, was the generous gift of
Drs. K. Knudsen and C. Damsky.28,29 This antiserum
inhibits adhesion of several mouse and rat cell lines
to laminin, fibronectin, and type IV collagen,30,31 but
not to thrombospondin.32 It recognizes the 120-kDa
/, integrin heterodimers.30,31,33 Anti-hamster /,3
serum also has been shown to recognize the /3
subunit of the vitronectin receptor34 in some cell
lines; however, the RASM cells used in our experiments do not express /3 integrins (R. I. Clyman,
unpublished observations, 1989).
A rabbit antiserum to a peptide derived from the
cytoplasmic domain of the /31 integrin subunit, anti-/,
cyt,33 and a rabbit antiserum to a peptide derived
from the cytoplasmic domain of the integrin a5
subunit, anti-a5,33 were the generous gift of Dr. K.
Tomaselli and Dr. L. Reichardt (University of California, San Francisco). Anti-human /,3, a rabbit antiserum that recognizes the /,3 and the a5 subunits of
the fibronectin receptor,35 was the generous gift of
Dr. E. Ruoslahti (La Jolla Cancer Foundation, La
Jolla, Calif.). Heat-inactivated normal rabbit and
normal goat sera were used as controls.
Cell Adhesion Assay
Cell adhesion was measured using a previously
described assay.30 Cells were labeled for 18-24 hours
with 2 ,Ci/ml 5 -[1251]iodo-2'-deoxyuridine ([1251]
IUdR, ICN Biomedicals, Irvine, Calif.). In some
experiments, protein synthesis was inhibited by preincubating the cultures with cycloheximide (25 ,g/
ml) for 3 hours before cell harvest. Cycloheximide
was included in the culture media used in the subsequent adhesion assay. The cycloheximide experiments were performed to show that cell adhesion was
not due to newly synthesized ECM proteins. The
cells were removed from the culture plates by brief
incubation (2 minutes) at room temperature with
trypsin-EDTA; this method has been used previously
in studying integrins.36,37 The trypsin was inactivated
by washing the cells with DME plus 5% fetal bovine
serum (FBS) at 40 C. The cell pellet was resuspended
in DME plus 1 mg/ml bovine serum albumin (BSA)
for the assay. In some experiments, the cells were
suspended in a divalent cation-free medium (mM:
NaCl 140, KCl 5.4, D-glucose 5.56, HEPES 10; pH
7.4; 1 mg/ml BSA) to study the effects of divalent
cations on cell adhesion.
The wells of uncharged polystyrene 96 well microtiter plates (Serocluster, Costar Corp., Cambridge,
Mass.) were precoated with poly-L-lysine (M,
500,000, Sigma), wheat germ agglutinin (Calbiochem
Corp., La Jolla, Calif.), fibronectin,38 laminin,39 type
IV collagen,39 type I collagen (Collagen Corp., Palo
Alto, Calif.), gelatin (Sigma), or with BSA in sterile
Ca'+/Mg2e-free PBS for 1 hour at 370 C. The wells
were then washed with PBS, and nonspecific adherence to the coated wells was blocked with 1 mg/ml
BSA in DME (or divalent cation-free medium) for
1 hour at 370 C. Appropriate dilutions of antibodies
and peptides (Gly-Arg-Gly-Asp-Ser-Pro [GRGDSP]
and Gly-Arg-Gly-Glu-Ser-Pro [GRGESP], Peninsula
Laboratories, Belmont, Calif.) were added to the
wells. The 'MI-labeled cells (2 x 10 cells per well) were
allowed to attach to the wells for 15 minutes at 370 C.
Cell Surface Radioiodination and Metabolic Labeling
With [3H]Glucosamine
To metabolically label glycoproteins, cells were
incubated for 48 hours in DME plus 5% FBS and 50
liCi/ml [3H]glucosamine (New England Nuclear,
Boston). After the culture plates were washed four
Clyman et al Integrin Receptors on Smooth Muscle Cells
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times with PBS, the cells were solubilized in cold lysis
buffer (0.5% NP40 [Calbiochem], 25 mM Tris-HCI,
pH 7.4, 1 mM EDTA, 150 mM NaCI, 1 mM phenylmethylsulfonyl fluoride, 2 ug/ml aprotinin) at 40 C
for 1 hour. The lysates were then centrifuged at 700g
for 10 minutes, transferred to new tubes, and centrifuged at 10,OOOg for 10 minutes.
For surface labeling, the cells were removed from
the tissue culture plates with trypsin-EDTA solution
and inoculated into 10-cm bacteriologic culture
dishes. Collagen-coated microcarrier beads (Cytodex-3, Pharmacia LKB Biotechnology, Piscataway,
N.J.) that had previously been swollen and washed in
culture medium were added to the plates so that
approximately four to eight cells bound to each
microcarrier bead. We used 3 ml of swollen beads
(approximately 106 carriers) in 30 ml of culture
medium. The cells attached to the beads rapidly, and
the plates were gently rocked 15 times per minute for
24 hours at 370 C. The microcarriers with cells
attached were then washed three times with cold PBS
and resuspended in an equal volume of iodination
buffer (mM: Tris-HCI 50, pH 7.4, NaCl 150, MnSO4
0.1, glucose 20). lodination was initiated by adding
glucose oxidase (Sigma), lactoperoxidase (Calbiochem), and carrier-free Na 125I (Amersham Corp.,
Arlington Heights, Ill.) at final concentrations of
0.1 unit/ml, 100 ,ug/ml, and 500 ,uCi/ml, respectively.
The suspension was mixed by rotation on ice for 25
minutes; the reaction was terminated with excess
glucose-free iodination buffer. The microcarriers
were recovered by centrifugation and extracted with
cold solubilization buffer for 1 hour: (mM) octyl
,B-D-glucopyranoside 200 (Boehringer Mannheim,
Indianapolis), Tris-HCl 50, pH 7.4, MnSO4 1. Protease inhibitors (1 mM phenylmethylsulfonyl fluoride
and 2 ,ug/ml aprotinin [Sigma]) were added during
solubilization and throughout the subsequent procedures. The suspension was centrifuged for 10 minutes
at 2,000g, and the pellet was extracted a second time.
The supernatants of the solubilized whole-cell
extracts were combined and used for immunoprecipitation or affinity chromatography. In some experiments, the collagen-coated microcarrier beads, without any attached cells, were iodinated and extracted
with solubilization buffer; the solubilized extract of
the microcarrier beads was used as a control for the
whole-cell extracts during the affinity chromatography experiments.
Affinity Chromatography
Affinity columns were prepared by coupling the
ligand (BSA, type I collagen, type IV collagen, laminin,
or gelatin) to CNBr-activated Sepharose (Pharmacia
LKB). The fibronectin affinity column was made from
the 120-kDa fragment of fibronectin.40 The solubilized
whole-cell extract from (7x 106)+(4x 106) cells (mean±
SD) was applied to a BSA-Sepharose precolumn (1 x 1
cm) previously equilibrated with 50 mM octylglucoside,
50 mM Tris-HCl, pH 7.4, 1 mM MnSO4, 1 ,g/ml BSA,
and 0.005% NaN3 (wash buffer A). The BSA-
177
Sepharose precolumn was eluted with four column
volumes of wash buffer A, and the eluate was passed
repeatedly through a substrate-Sepharose column (1 x5
cm) over 1.5 hours. The substrate-Sepharose column
was then washed with five column volumes of wash
buffer A (25 ml). Bound receptors were eluted first with
one column volume of GRGDSP (1 mg/ml) followed
by three column volumes of wash buffer A; second, with
four column volumes of 0.2 M NaCI in wash buffer A;
third, with four column volumes of 10 mM EDTA in
wash buffer A without divalent cations; and finally, with
three column volumes of 1 M NaCl in wash buffer A.
The substrate-Sepharose columns (fibronectin, laminin, and type I and type IV collagen) were reused up to
five times. Twenty-two microliters of 1 M MgCl2 was
added immediately to each 1-ml fraction of the EDTA
eluate. In some of the affinity studies, 1 mM MnSO4
was replaced with 1 mM CaCl2 in both the solubilization solution and the wash buffer A.
Immunoprecipitation
Whole-cell extracts in either lysis buffer or solubilization buffer and column eluates were first precleared by incubating 1 ml of the whole-cell extract or
column eluate with 50 jul of packed protein ASepharose beads (Sigma) for 30 minutes at 40 C, then
centrifuging the mixture. Next, 10 gl. of antiserum
was added to the 1-ml supernatants and incubated
for 2 hours at 4° C. Immune complexes were recovered by incubating the supernatants for 2 hours with
50 ,ul of packed protein A-Sepharose. The protein A
beads with bound immune complexes were washed as
described.30 Control immunoprecipitation performed
with preimmune normal goat or normal rabbit serum
produced negligible radioactivity.
Electrophoresis
Eluates from the affinity columns and samples
from the immunoprecipitation experiments were
analyzed by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) according to
Laemmli.Y Samples were solubilized in sample
buffer, reduced with 5% 2-mercaptoethanol, and
heated at 100° C for 4 minutes. Nonreduced samples
were treated as above except that 10 mM
N-ethylmaleimide was substituted for 2-mercaptoethanol. The samples were separated on polyacrylamide gels and detected by autoradiography. 3Hlabeled proteins were visualized by equilibrating the
gel with 2,5-diphenyloxazole, scintillation grade
(National Diagnostics, Manville, N.J.). Prestained
protein standards (Sigma) were used to calculate the
apparent molecular mass and consisted of a2macroglobulin (180 kDa), /3-galactosidase (116 kDa),
fructose- 6-phosphate kinase (84 kDa), pyruvate
kinase (58 kDa), fumarase (48.5 kDa), lactate dehydrogenase (36.5 kDa), and triosephosphate
isomerase (26.6 kDa).
178
Circulation Research Vol 67, No 1, July 1990
Immunofluorescent Staining
100
Glass coverslips were coated with 100 ,g/ml of
fibronectin, laminin, and collagen types I and IV in
PBS for 1 hour at 370 C. Cells were suspended in
serum-free culture medium containing 0.1% BSA
and overlaid (2 x 104 cells) on coverslips. After
3 hours incubation at 37° C, the cells were fixed and
permeabilized as previously described.30 After incubation with 1% normal goat serum, the samples were
incubated for 1 hour with anti-human f1 (1: 200) and
mouse antivinculin (1:100) (ICN). The samples were
then incubated for 1 hour with secondary antisera
coupled to fluorescein isothiocyanate or rhodamine.
The coverslips were mounted in Fluoromount (Fisher Scientific Co., Pittsburgh) and viewed on a Nikon
microscope equipped with epiluminescence optics.
Adhesion
50
0
.1
1
10
100
1000
Concentration, g / ml
10 r
fN
-
aFN
LN
Adhesion
50h
Results
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Cell Adhesion Studies
RASM cells adhered to wells coated with laminin,
fibronectin, and types I and IV collagen in both a
dose-dependent and a time-dependent manner
(Figure 1). RASM cells also readily attached to
poly-L-lysine and wheat germ agglutinin (Figure 1).
In contrast, the cells attached poorly to wells coated
with gelatin and failed to attach appreciably to wells
coated with BSA.
Figure 2 examines the requirements for divalent
cations in the adhesion of RASM cells to different
substrates. Without any divalent cations in the incubation medium, there was negligible cell binding to
laminin or collagen type I or IV; adhesion to fibronectin was also markedly reduced. The requirement
for divalent cations in the medium was specific for
ECM-derived substrates; adhesion of RASM cells to
poly-L-lysine or to wheat germ agglutinin was not
affected by their absence. Both Mn2' and Mg2' were
equally effective in promoting adhesion to fibronectin,
laminin, and collagen types I and IV. The presence of
Ca2' alone was 70-90% as effective as Mn21 in
supporting adhesion to fibronectin and laminin; however, Ca2+ alone could not meet the divalent cation
needs of RASM cells adhering to collagen I and IV.
RASM cell attachment to laminin, fibronectin, and
collagen I and IV was studied in the presence of a
hexapeptide, GRGDSP, derived from the cell binding domain of fibronectin, and a control hexapeptide,
GRGESP, with glutamic acid substituted for
arginine.42 When RASM cells were exposed to 1 mg/
ml soluble GRGDSP peptide, cell adhesion to
fibronectin-coated wells was decreased by 27+±7%
(mean±SD; p<0.05, paired t test, n=6). The
GRGESP peptide had no detectable effect on cell
binding to fibronectin. Neither peptide had a noticeable effect on RASM cell attachment to laminin or
collagen type I or IV.
A 1% concentration of the anti-hamster I,3 serum
inhibited cell adhesion to fibronectin and to collagen
I and IV by greater than 90% and inhibited adhesion
to laminin by 71% (Figure 3). The same concentra-
-
1
0
Gel
a
_
_
.
30
r
=
O
w~~~~~~
-
60
BSA
Minutes
rat aortic smooth muscle cell
FIGURE 1. Dependency of
adhesion on different coating concentrations of extracellular
matrix proteins and time course of cell adhesion. Cells were
labeled with 5-[l25I]iodo-2'-deoxyuridine and exposed to 25
/gIml cyclohexamide for 3 hours before the experiment.
Upper panel: Wells were precoated with different concentrations of fibronectin (FN), type I collagen (I), type IV collagen
(V, gelatin (Gel), or laminin (LN). Cells were allowed to
attach to the wells for 15 minutes at 370 C. In this experiment,
49±8% of the radiolabeled cells adhered to poly-L-lysine (10
pg/ml), 57±8% adhered to wheatgerm agglutinin (10 pglml),
and 0±0% adhered to bovine serum albumin (BSA) (1
mglml). Values represent the mean percentage (±SD) of the
radioactivity that adhered to the well in triplicate wells. This
experiment was repeated four times and produced the same
findings. Lower panel: Wells were precoated with fibronectin
(8.8 pg/lml), type I collagen (5 ugIml), type IV collagen (0.5
pg/ml), gelatin (5 pg/ml), laminin (20 pglml), or BSA (1
mg/ml). Cells were allowed to attach to the wells for 5, 15, 30,
and 60 minutes at 370 C. Values represent the mean percentage (±SD) of the radioactivity that adhered to the well in
triplicate wells. This experiment was repeated twice and produced the same findings.
tion of normal goat serum did not inhibit adhesion.
The effects of anti-hamster 81 were concentration
dependent. The inhibition produced by anti-hamster
,81 was specific for ECM-derived substrates because
attachment to poly-L-lysine was not affected by similar concentrations of the antiserum.
Immunofluorescent Staining with Anti-Human
f31
Serum
Using anti-human 131 antibody, we performed
immunofluorescence staining of cells adhering to
fibronectin, laminin, and collagens I and IV (Figure
4). Intense staining was localized in linear deposits at
the marginal edge of the cells on all four protein
Clyman et al Integrin Receptors on Smooth Muscle Cells
LN
T
FN
loo 50s
0
l
4 1I
Adhesion
L
lv
PLL
WGA
100
so _
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OL
*Z& _Z
Caimug-o
ECCa=2mM
Elfg=2mM
MCa-0.1 mM
@MW-0.1 mM
substrates. This pattern of staining was most distinct
on the cells adhering to fibronectin. These condensations of 131 receptor complexes colocalized with
sites of vinculin positive adhesion plaques.
Immunoprecipitation of Proteins Recognized by
Anti-Hamster f3 Serum
Analysis of the whole-cell detergent extracts of
RASM cells by SDS-PAGE under nonreducing
conditions identified numerous radioiodinated membrane proteins (Figure 5, lane 1). When the wholecell extracts were immunoprecipitated with antihamster f31, we found a limited series of major,
100 .
o
50-
FFN
FIGURE 3. Effect of anti-hamster PI, serum
on rat aortic smooth muscle cell attachment
to extracellular matrix proteins. Cells were
LN
PLL
lv
labeled with 5-[125I]iodo-2'-deoxyuridine and
* al Goat sw
Ant PI Serum
I : 100
* 1:200
EN 1:400
50-
a t:800
0-
L
FIGURE 2. Rat aortic smooth muscle
cell adhesion requirements for divalent
cations. Cells labeled with 5-f'25Iliodo2'-deoxyuridine were suspended in
divalent cation-free medium and
plated into wells precoated with bovine
serum albumin (BSA) (1 mg/ml),
fibronectin (FN) (8.8 pg/ml), laminin
(LN) (20 pg/ml), type I collagen (I) (5
pg/ml), type IV collagen (IV) (0.5 pg/
ml), poly-L-lysine (PLL) (10 pg/ml),
or wheat germ agglutinin (WGA) (10
pg/ml). CaCl2, MgCI2, and MnSO4 or
MnCI2 were added to the wells to make
the final concentrations shown in the
figure. Cells were allowed to attach to
the wells for 15 minutes at 37 C.
Values represent the mean percentage
(+SD) of the radioactivity that
adhered to the well in triplicate wells.
The percentage of cells adhering to
BSA -coated wells (in 1 mM CaCI2 and
1 mM MgCl2 solutions) was 0±0%.
This experiment was repeated twice and
produced the same findings.
closely migrating proteins with apparent molecular
masses centered at 120, 140, 165, and 185 kDa
(nonreduced) (Figure 5, lane 2). Two minor bands
were seen at 83 and 97 kDa. When the immunoprecipitated whole-cell extracts were examined under
reducing conditions (Figure 5, lane 4), the major
120-kDa band exhibited decreased mobility, and the
120- and 140-kDa bands appeared to merge into a
diffuse band centered at 140 kDa. There was also
decreased mobility under reducing condition of the
major bands at 165 kDa (to 175 kDa) and 185 kDa
(to 200 kDa). In immunodepletion experiments, antiserum to the cytoplasmic domain of the integrin (,
L
I
Adhesion
100-
fjMnw2mM
X P* w-0.1 mM
179
plated into wells precoated with bovine serum
albumin (BSA) (1 mglml), fibronectin (FN)
(8.8 p4g/ml), laminin (LN) (20 pgIml), type I
collagen (I) (0.8 pg/ml), type IVcollagen (IV)
(0.5 pglml), or poly-L-lysine (PLL) (10 pg/
ml). The assay was performed in thepresence
of control medium, goat anti-hamster f3j
serum (diluted 1:100, 1 :200, 1:400, 1:800),
and normal goat serum (diluted 1 :100). Cells
were allowed to attach to the wells for 15
minutes at 370 C. The percentage of cells
adhering to BSA-coated wells was 1±0%.
Values represent the mean percentage (+SD)
of the radioactivity that adhered to the well in
triplicate wells. This experiment was repeated
three times and produced the same findings.
180
Circulation Research Vol 67, No 1, July 1990
1
2
116
4
5 6
7
8 9 10 11 12 13
kD
kD
180-
3
e
180-
116-
1
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FIGURE 5. Immunoprecipitation of solubilized whole-cell
extracts with anti-hamster ,81 serum and affinity chromatography on fibronectin-Sepharose columns. Cells were surface
labeled, solubilized in octyl f3-D-glucopyranoside, and immunoprecipitated with anti-hamster 61 serum. Samples were
analyzed under nonreducing (lanes 1 and 2) or reducing
conditions (lanes 3 and 4) by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (7%). Immunoprecipitates
from the whole-cell extracts are shown in lanes 2 and 4. The
cell extract also was applied to the fibronectin-Sepharose
column and successively eluted with wash buffer A (see
'Materials and Methods'), 0.2 M NaC1 buffer, GRGDSP
(1 mglml), and 10 mM EDTA buffer. Column fractions
(2 ml) were examined under nonreducing (lanes 5 and 6)
conditions. Material eluted with GRGDSP: lane 5. Material
eluted with EDTA (lane 6) was subjected to immunoprecipitation with antiserum to the a5 subunit (lanes 7 and 8) and
with anti-hamster 1% serum (lanes 9 and 10). The immune
complexes were examined under nonreducing (lanes 7 and 9)
and reducing conditions (lanes 8 and 10). After treating a
sample of the EDTA eluate at 1000 C in the presence of
1 g!100 ml SDS, the sample was subjected to immunoprecipitation with anti-hamster f3, serum (lane 11), antiserum to the
/3i cytoplasmic subunit (lane 12), and antiserum to the a5
cytoplasmic subunit (lane 13) and examined under nonreducing conditions (lanes 11-13). Molecular mass markers in
kilodaltons are shown on the left of each gel
FIGURE 4. Localization of /3, integrin receptors in focal adhesion plaques. Rat aortic smooth muscle cells were permitted to
adhere to coverslips coated with fibronectin (panels A and B),
laminin (panels C and D)% collagen IV (panels E and F), or
collagen I (panels G and H) as described in "Materials and
Methods. "At the time offixation, the cells were in various stages
of spreading and migration. The cell shapes were not unique to
any of the coating substrates. After the samples were fixed and
permeabilized, they were stained with antibodies to vinculin
(panels A, C, E, and G) and human P,I (panels B, D, F and H).
Arrows point to representative adhesion plaques identified by
antibodies to human
/3.
subunit recognized the same protein bands as the
anti-hamster /,3 serum (data not shown).
When RASM cells were labeled with [3H]glucosamine, the anti-hamster P, serum specifically recognized a major complex of polypeptides with molecu-
lar masses of 120, 140-160, and 185 kDa under
nonreducing conditions (data not shown). In addition, there were minor bands at 97, 200, and 220 kDa.
The 165 -kDa band observed on the surface-iodinated
cells was not detected in the metabolically labeled
cells. Under reducing conditions, the 120- and 140160-kDa complex appeared to migrate as a single
diffuse band of 140 kDa; the 185-kDa band migrated
at 195 kDa, and the 200-220-kDa band migrated at
240 kDa. The [3H]glucosamine-labeled cell extracts
were also subjected to immunoprecipitation with
antibodies to the human fibronectin receptor, antihuman I31 antiserum. The recovered radiolabeled
proteins comigrated with those immunoprecipitated
with the anti-hamster ,% antiserum under both
nonreducing and reducing conditions; however, the
minor bands at 200 and 220 kDa (nonreduced conditions) were not observed with the anti-human ,3
antiserum (not shown).
Clyman et al Integrin Receptors on Smooth Muscle Cells
1
2
3
kD
180
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kD
180_
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7 8 9 10 11121314
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i
181
FIGURE 6. Affinity chromatography of detergentsolubilized rat aortic smooth muscle cells on type WV (lanes
1-3) and type I (lanes 4-6) collagen-Sepharose columns
and laminin-Sepharose columns (lanes 7-16). Proteins
eluted from the type IVcollagen-Sepharose column with 10
mM EDTA buffer were immunoprecipitated with antihamster f31 serum (lane 2, nonreduced). The original
sample was examined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (7%) under nonreducing (lane 1) and reducing (lane 3) conditions. Proteins
eluted from the type I collagen-Sepharose column with 10
mM EDTA buffer were immunoprecipitated with antihamster 13, serum. The original sample was examined
under nonreducing conditions (lane 4), and the immune
complex (lanes 5 and 6) was examined under nonreducing
(lane 5) and reducing (lane 6) conditions. Lanes 7-16:
Proteins eluted from the laminin-Sepharose columns with
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10 mM EDTA buffer. Aliquots from the first four 2-ml
fractions from the EDTA buffer elution were analyzed
under nonreducing (lanes 7-10) and reducing (lanes 1114) conditions. EDTA eluates in lanes 11 and 12 were
pooled (lane 15, reduced) and immunoprecipitated with
anti-hamster 13 serum (lane 16, reduced). Molecular mass
markers (in kilodaltons) are shown on the left of each geL
Identification of Fibronectin-Binding Proteins
'251-labeled proteins eluted from a fibronectin
affinity column were identified by SDS-PAGE and
autoradiography (Figure 5). No labeled proteins
were eluted from the fibronectin column by the 0.2 M
NaCI buffer wash (data not shown). Bound material
was eluted with the RGD-containing hexapeptide
(Figure 5, lane 5). After separation under nonreducing conditions, two broad polypeptide bands of 120
and 150 kDa were detected. These bands were
immunoprecipitated by the anti-hamster /31 serum
(data not shown) and corresponded to the 120- and
140-160-kDa bands in the anti-hamster /,3 and antihuman
immunoprecipitates from the whole-cell
extracts (compare with Figure 5, lane 2). Subsequent
washing with EDTA buffer eluted a similar dimeric
protein complex (Figure 5, lane 6) that had the same
apparent molecular mass as the complex eluted with
GRGDSP and was immunoprecipitated with antisesubunit
rum to the cytoplasmic domain of the
(Figure 5, lanes 7 and 8) and with anti-hamster 13l
serum (Figure 5, lanes 9 and 10). After reduction
with 2-mercaptoethanol, the apparent molecular
mass of the two subunits shifted so that they comigrated at 140 kDa (Figure 5, lanes 8 and 10), as did
the corresponding bands on immunoprecipitation of
the whole-cell extract (see Figure 5, lane 4). If the
dimeric protein complex, eluted with EDTA, was
treated at 1000 C in the presence of 1 g/100 ml SDS
to disrupt noncovalent associations, then antihamster ,B3 serum (Figure 5, lane 11) and antiserum
to the cytoplasmic domain of the P, subunit (Figure
5, lane 12) precipitated only the 120-kDa band
(nonreduced); antiserum to the cytoplasmic domain
a5
of a5 precipitated only the 150-kDa band (nonreduced) (Figure 5, lane 13).
No additional labeled proteins could be eluted
with 1 M NaCI buffer.
Identification of Type WV Collagen-Binding Proteins
GRGDSP eluted a polypeptide from the type IV
collagen-Sepharose column that had an apparent
molecular mass of 58 kDa (nonreduced) and 84 kDa
(reduced). Similarly, 0.2 M NaCI buffer eluted a
broad band with apparent molecular mass of 58 kDa
(nonreduced). Neither the 58-kDa polypeptide
released by GRGDSP nor the material eluted with
0.2 M NaCI buffer was immunoprecipitated by antihamster /% serum. In addition, when 1251-labeled
proteins from microcarrier beads alone (without any
cells) were loaded onto type IV collagen-Sepharose
columns and cluted with 0.2 M NaCl buffer, the same
bands were observed (58 kDa, nonreduced, and 84
kDa, reduced; data not shown).
When the type IV collagen-Sepharose column was
eluted with EDTA, two polypeptides were recovered
with apparent molecular mass (nonreduced) of 120
and 185 kDa (Figure 6, lane 1). Upon reduction,
these peptides migrated at 135 and 200 kDa (Figure
6, lane 3). The set of proteins eluted with EDTA was
recognized by the anti-hamster IA serum (Figure 6,
lane 2) and appeared to correspond to the 120- and
185-kDa bands seen on the immunoprecipitation of
the whole-cell extract (Figure 5, lane 2). The high
affinity binding of the 120/185-kDa proteins to the
type IV collagen-Sepharose column required the
presence of Mn2+ and was not detected in buffers
182
Circulation Research Vol 67, No 1, July 1990
containing Ca2+ without the presence of
Mn2' (data not shown).
Mg2e
or
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Identification of Type I Collagen-Binding Proteins
When cell extracts were loaded onto a type I
collagen-Sepharose affinity column, a different elution pattern was obtained. Both GRGDSP and the
0.2 M NaCI buffer failed to elute any radiolabeled
material (data not shown). Three bands were eluted
from the type I collagen-Sepharose column with
EDTA (Figure 6, lanes 4 and 5): two major bands at
185 and 120 kDa and a minor band at 150 kDa. On
reduction, the 185- and 120 -kDa bands shifted to 200
and 135 kDa, respectively. The band at 150 kDa
shifted to 160 kDa on reduction (Figure 6, lane 6).
All three bands eluted with EDTA were recognized
by the anti-hamster ,X1 serum (Figure 6, lane 5).
EDTA effectively removed all of the polypeptides
that specifically bound to the collagen type ISepharose column because no further material in this
region could be eluted with buffer containing 1 M
NaCl (not shown). The polypeptides that bound to
collagen type I-Sepharose were specific for native
collagen because they bound only minimally (if at all)
to gelatin-Sepharose columns.
To demonstrate that the 185/120-kDa material in
the EDTA eluate from the collagen type I-Sepharose
was the same complex as found in the EDTA eluate
from the type IV collagen-Sepharose column, we
first passed the whole-cell extract over a type I
collagen-Sepharose column and then passed the void
and wash volume from the type I-Sepharose column
over a type IV collagen-Sepharose column. After the
whole-cell extract was passed over the collagen type I
column, there was no 185/120-kDa (EDTA-eluted)
set of proteins binding to the type IV collagenSepharose column; this was consistent with the
removal of the 185/120-kDa material by the type I
collagen column (not shown).
Identification of Laminin-Binding Proteins
Application of 0.2 M NaCI buffer to a lamininSepharose affinity column released a diverse group of
polypeptides with apparent molecular masses less
than 90 kDa. The major band observed had a nonreduced mobility of 58 kDa, which shifted to 84 kDa on
reduction (data not shown). These proteins were also
observed when the extracts of microcarrier beads,
without cells, were passed over laminin-Sepharose
columns and eluted with 0.2 M NaCl buffer.
GRGDSP released a negligible amount of labeled
polypeptides (data not shown), which had the same
electrophoretic mobility as the material released by
0.2 M NaCl buffer. None of the low molecular mass
peptides released by GRGDSP or 0.2 M NaCl buffer
was immunoprecipitated by anti-hamster ,B1 serum.
When the column was eluted with EDTA, at least
five diffuse polypeptide bands were eluted: (nonreduced conditions) 49, 56, a broad band of 120, 185,
and 210 kDa (Figure 6, lane 7). On reduction, the
electrophoretic mobilities shifted to 57, 60, 100, 110,
a major band at 135, 200, and 240 kDa (Figure 6, lane
11). When the EDTA eluate from the laminin column was immunoprecipitated with either antihamster f,, anti-human 3,, or anti-fl, cyt sera and run
on SDS-PAGE under reducing conditions, the sharp
band at 240 kDa and the minor bands at 57 and 100
kDa (Figure 6, lane 16) were not precipitated. The
major band at 135 kDa (reduced) as well as the bands
at 60, 110, and 200 kDa (reduced) were precipitated
by the antisera (Figure 6, lane 16, data not shown for
anti-human 81 or anti-X31 cyt sera). When the EDTA
eluate from the laminin column was run on a twodimensional PAGE gel (first dimension nonreduced;
second dimension reduced), we found that a sharp
125-kDa band (nonreduced), which comigrated with
the broad 120-kDa band, corresponded to the 110kDa band reduced (not shown); similarly, the 5060-kDa bands (nonreduced) corresponded to the
bands around 60 kDa (reduced). The bands at 120
and 185 kDa (nonreduced) migrated at 135 and 200
kDa (reduced), respectively.
EDTA removed the polypeptides from the
laminin-Sepharose column with different efficiencies.
The 185-kDa band (nonreduced) was eluted in the
earlier EDTA fractions (Figure 6, lanes 7 and 11),
whereas the minor 125/110-kDa (nonreduced/
reduced) band was eluted in subsequent EDTA
fractions (Figure 6, lanes 12-14).
Discussion
The RASM cells attached avidly to the ECM proteins laminin, fibronectin, and collagen types I and IV
(but not denatured collagen), as well as to non-ECM
substrates (poly-L-lysine and wheat germ agglutinin)
(Figure 1). Differences in adherence between the
substrates may represent differences in the affinities of
the cells for the substrates or differences in the
binding of the substrates to the assay wells.43
Several features of RASM cell attachment to ECM
proteins were compatible with its mediation by integrinlike receptors of the P1, family: their requirement
for divalent cations and their inhibition by the antihamster (31 serum. The presence of divalent cations was
necessary for RASM cell attachment to the ECM
proteins, but not to the non-ECM substrates (Figure 2).
Although Ca'4 alone would permit cell attachment to
laminin and fibronectin, it would not support attachment to collagen types I and IV. As was observed in
other cell lines,1"4445 there was greater cell attachment in the presence of MI12 and Mg`+ than Ca'+
(Figure 3). These results are compatible with the
observation that the known integrin receptors require
divalent cations for binding to their ligands."""246
The specific peptide sequence RGD found in many
ECM proteins has been implicated as a recognition
sequence for fibronectin,16 laminin,47 48 and
collagen.49 Although mammalian-RGD-directed
receptors are typically among the integrin superfamily,10 non-integrinlike binding proteins that appear
to bind to the RGD sequence also have been found.49
The soluble RGD hexapeptide GRGDSP did inhibit
Clyman et al Integrin Receptors on Smooth Muscle Cells
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RASM cell attachment to fibronectin; however,
RASM cell adhesion to fibronectin was inhibited by
only 27%. In addition, high concentrations of the
hexapeptide GRGDSP were required to inhibit cell
attachment. This has been observed by others. In
general, cell surface receptors have only a moderate
affinity for fibronectin and other adhesive proteins
(approximately 10-6 M).50 When the size of the cellbinding fragment has been reduced to the size of the
GRGDSP region, its affinity for the cell surface is
decreased significantly.50 The synthetic RGD peptide
did not have a noticeable effect on adhesion to laminin
or collagen types I and IV. Our data are consistent
with the hypothesis that other receptors, not dependent on RGD-like sequences, may play a role in cell
anchorage12'51'52 or that sequences outside of the
minimum RGD recognition site may play important
roles in the efficient binding of RASM cells to ECM
proteins.50,53 Before any conclusion is drawn about the
lack of specificity of the RGD sequence in mediating
cell attachment, however, a more extensive panel of
peptides should be tried.16,49
Anti-hamster PI serum interfered with RASM cell
adhesion to fibronectin, laminin, and collagen types I
and IV. The anti-hamster /,1 serum is a polyclonal
antibody that probably binds to several sites on the /,
subunit. Although the exact antigenic determinants
are not known, presumably the receptor site (or sites)
is included in the antigenic site(s), or its conformation is changed when the antibody binds to the /,
subunit. It is unlikely that antibody-induced cytotoxicity was involved because prior studies with several
different rodent cell lines have shown that the inhibitory effects of anti-hamster /1 serum are completely
reversible.28'29'30'33 In addition, in the presence of
antiserum, the RASM cells were able to attach to the
non-ECM substrate poly-L-lysine (Figure 3).
To identify adhesion receptors for fibronectin,
collagen types I and IV, and laminin, we looked at
the `PI-abeled cell surface proteins that both bound
to the respective affinity columns (with dependence
on divalent cations) and were recognized by the
anti-hamster P,B serum. Several groups have examined the effectiveness of moderate salt concentrations in eluting the low-affinity receptors50 that bind
to the ECM proteins.20'24'49'54-56 We found that when
integrinlike cell surface proteins were bound to substrate affinity columns in the presence of Mn'+, they
could not be eluted with 0.2 M NaCl alone. Other,
low molecular weight proteins that were not recognized by anti-hamster /3, serum were frequently
eluted from the columns with 0.2 M NaCl. However,
these low molecular weight proteins were probably
derived from proteins on the microcarrier beads or
serum proteins trapped within the beads during the
iodination. To test whether additional surface proteins on RASM cells might bind to the specific
ECM-affinity column in a divalent cation-independent manner, we washed the affinity columns with
EDTA buffer and followed this with 1 M NaCl. No
new proteins were detected in the 1 M NaCI eluates.
183
Specificity of binding to the ECM ligand affinity
columns was tested using resin coupled with BSA.
None of the polypeptides recognized by the antihamster /, serum bound to the BSA column; in
contrast, they could be selectively and completely
removed from the solubilized cell extracts by passing
them over a series of laminin, fibronectin, and collagen types I and IV affinity columns.
Both the GRGDSP peptide and EDTA eluted a
broad protein dimer complex from the fibronectin
affinity column that was recognized by anti-hamster
/31 serum and had a molecular mass of 120 and 150
kDa (nonreducing conditions). In our hands, EDTA
was much more effective than GRGDSP (1 mg/ml) in
eluting the protein from the column (not shown).
This may have been due to our use of Mn2+, rather
than Ca2+ or Mg2`, as the divalent cation in our
solutions." This protein complex had strong similarities to the classical fibronectin receptor (a5/38).19'35'57
When the complex was dissociated by SDS, antisera
to the cytoplasmic domains of the /81 and as subunits
precipitated the 120- and 150-kDa bands, respectively. On reduction, the 120-kDa band of the fibronectin binding complex demonstrated a disulfidedependent upward shift in molecular mass; this is
typical of the / subunit of the integrin class of cell
surface receptors28 and suggests the presence of
multiple intramolecular disulfide-rich domains in this
protein band. The fibronectin receptor complex isolated from RASM cells bound only to fibronectin.
This behavior is similar to that of the as3, receptor
and differentiates the RASM cell fibronectin receptor from promiscuous receptors like the aC,31 receptor, which also binds to fibronectin but binds to
laminin and collagen types I and IV as well.20'2'
A heterodimer complex (185/120 kDa), recognized
by anti-hamster /3, serum, was eluted from the type
IV collagen affinity column with EDTA. This integrinlike receptor bound to collagen in the presence of
Mn2+ but not in the presence of Ca2+. The effect of
Ca2' and Mn2+ on the protein binding to the affinity
column parallels the ability of these divalent cations
to alter RASM cell adhesion to immobilized collagen
type IV (Figure 2). This 185/120-kDa complex
appears to be a promiscuous receptor because it
could also bind to collagen type I as well as laminin
(Figure 6). On reduction, the 120-kDa /,1 chain
increased its apparent molecular mass to 135 kDa (as
it did in the fibronectin receptor); the 185-kDa a
band increased to 200 kDa. By analogy, this 185 -kDa
protein has approximately the same SDS-PAGE
mobilities in both reducing and nonreducing conditions as the a subunit of the VLA-1 (a1131) heterodimer that has been characterized in human cell
lines.25 The VLA-1 integrin dimer recently has been
found to bind to both type IV and type I collagen in
human melanoma cells.23
Neither the VLA-1 collagen receptor in melanoma
cells nor the 185/120-kDa collagen/laminin receptor
complex from rat cells could be eluted from the
collagen/laminin affinity columns with the amino acid
184
Circulation Research Vol 67, No 1, July 1990
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sequence RGD (see "Results" and Figure 6).23,24
This indicates that the 185/120-kDa VLA-1-like integrin interacts specifically with collagen and laminin
and that a unique recognition signal other than RGD
may be involved.
A unique set of radiolabeled proteins was observed
to bind to collagen type I affinity columns. In addition
to the 185/120-kDa integrin complex that bound to
collagen IV and to laminin, there was also a second
integrin heterodimer complex (150/120 kDa) that
only bound to type I collagen and could be eluted
with EDTA. This 185/150/120 -kDa complex of receptors that bound to collagen type I was observed to
bind efficiently to native collagen but poorly to
gelatin. This correlates with the poor adhesion of the
cells to gelatin substrates. The RASM cell 150-kDa
protein has approximately the same SDS-PAGE
mobility pattern under both reducing and nonreducing conditions as the a subunit of the a,2P, heterodimer that has been characterized in human cell
lines.25 In the past, the a23, receptor has been found
to bind to both collagen type I and type IV.20,21,23,58,59
We found that this a2-like subunit preferentially
bound to collagen type I.
The 185/120- and 150/120-kDa integrin aIf31 heterodimers described in the present work differ from
other collagen binding proteins: Wayner and Carter20
and, more recently, Gehlsen et al60,61 have shown that
another /1 integrin, a,31, can mediate human cell
adhesion to collagen and laminin as well as to
fibronectin. Although the RASM cell 185/120-kDa
complex may mimic the a,3,1 receptor in its ability to
bind to collagen and laminin, neither its pattern of
electrophoretic migration on reduction nor its inability to bind to fibronectin are consistent with the af3,1
receptor. Dedhar et a149 identified three surface
proteins (Mr 250,70, and 30 kDa) on human osteosarcoma cells that might mediate cell adhesion to type I
collagen; these collagen-binding proteins could be
inhibited by a specific Gly-Arg-Gly-Asp-Thr-Pro
(GRGDTP) peptide, but were not related to the
integrins. Gullberg et a162 found a glycoprotein in rat
hepatocytes that mediated adhesion to collagen type
I but not to type IV; however, the protein complex
they identified had an M, of 115/105 kDa under
nonreducing conditions and 130/115 kDa under
reducing conditions, which differed from those
reported here.
RASM cells appear to have several proteins that
bind to laminin. The anti-hamster /1 serum inhibited
most of the cell binding to laminin, which suggests that
the major cell receptors for laminin are integrinlike
proteins or that 3,1 integrins are essential for mediating
cell adhesion. Several integrinlike proteins of the 13,
subfamily have been shown to act as potential laminin
receptors in other cell lines. Cell substrate attachment
antigen, located on avian cells, is reported to bind to
laminin as well as to fibronectin and collagen.63 In
mammalian cells, the a,3,1 integrin binds to laminin as
well as to fibronectin and collagen types I and
IV.20,21,25,60,61 Sonnenberg et a122 have found that ca6,81,
which is present in a variety of human and rodent cell
types, appears to mediate adhesion to laminin. Rat
B50 cells use an al,81-like receptor to bind to
laminin.24 Recently, Kramer and Marks64 observed in
human melanoma cells a new member of the Plintegrin family that binds to laminin.
The major protein band that elutes with EDTA
from the laminin-Sepharose column is the 120-kDa
/3, subunit. The 185/120-kDa al,81-like complex that
binds to collagen I and IV also binds to laminin.
Recently, Ignatius and Reichardt24 have identified a
200/120-kDa laminin receptor in rat B50 cells that
also appears to behave like an al,/1 dimer. This
"laminin receptor" from rat B50 cells also binds to
collagen type I and therefore may be the same
receptor as the 185/120-kDa complex reported here
(M.J. Ignatius, L.M. Goetzl, L.F. Reichardt, personal
communication, 1988). We have found that in the
RASM cells, the al,31-like receptor binds much more
efficiently to collagen than it does to laminin (unpublished observations). In human cells, al,/1 also preferentially binds to collagens23 but does exhibit some
affinity for laminin (R.I. Clyman, unpublished
results, 1989). A sharp protein band of 210 kDa
(nonreduced) was also consistently eluted from the
laminin column. This band did not immunoprecipitate with either the anti-hamster /31, the anti-human
I31, or the anti-fl1 cyt sera. The failure to precipitate
this band may mean that this putative a subunit is
only loosely associated with the /31 subunit so that the
complex dissociated during immunoprecipitation24,65,66or that it is another, non-f, integrin protein similar to those described by Kleinman et al55 or
Smalheiser and Schwartz.56
Two other bands were weakly recognized by the
anti-hamster /31 and anti-human /1 sera (56 and 125
kDa, nonreduced conditions, or 60 and 110 kDa,
reduced conditions). These proteins appear to be
similar to those seen on human melanoma cells,64 but
additional studies will be needed to characterize
these bands. Although the 60-kDa (reduced) protein
has a similar electrophoretic pattern when run under
nonreducing conditions as the nonintegrin, 68-kDa
monomer,54,67,68 its ability to be immunoprecipitated
by the anti-hamster /31 serum makes it unlikely that
they are the same protein.30 Although we do not
believe that there is a 68-kDa laminin binding protein on RASM cells, we have not tried to elute the
columns or block cell adhesion on laminin with the
pentapeptide Tyr-Ile-Gly-Ser-Arg.67
ECM receptors on smooth muscle cells play an
important role in anchoring the cells during vasoconstriction. The expression of several integrin-related
receptor complexes specific for different ECM components would also provide a means for regulating
the smooth muscle cell migration that occurs after
injury to the arterial wall. We have identified a series
of /,1-integrinlike complexes on RASM cells that may
be responsible, in part, for the cellular interactions
with fibronectin, laminin, and collagen types I and
IV. These receptors appear to be similar to members
Clyman et al Integrin Receptors on Smooth Muscle Cells
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of the 813-integrin family of ECM receptors. Our
conclusions are based on the recognition of these
glycoproteins by several antibodies to the 81 subunit,
by their substrate affinities, and by their electrophoretic mobilities on SDS-PAGE. This series of receptor complexes shares a common 120-kDa (nonreduced) fl1-subunit protein. There is a 185-kDa
(nonreduced) a subunit that is promiscuous and
involved in binding to collagen types I and IV as well
as to laminin. Interestingly, this has strong parallels
to the a1 subunit in its mobility and relative affinity
for laminin and collagen types I and IV. In these
smooth muscle cells, the 185-kDa (nonreduced) a
subunit appears to bind to collagen much more
efficiently than it does to laminin. In addition, there
is a 150-kDa (nonreduced) a subunit that binds
exclusively to collagen type I and a 150-kDa (nonreduced) a5 subunit that binds exclusively to fibronectin. We have also identified two proteins that are
associated with the /31 subunit and bind only to
laminin. By immunofluorescent staining, the /31 integrins were localized in linear deposits at the marginal
edge of the cells on all four of the substrates tested.
The clustering of these receptors in focal adhesion
plaques (demonstrated by the costaining of these
sites with antibodies to vinculin) suggests that these
receptors are important in cell adhesion to the different substrates. Further studies will reveal what
role these different glycoprotein receptor complexes
have in the adhesion process.
Acknowledgments
We thank Ms. Mai Vu and Esther Cheng for their
help in developing the methods used in these studies
and Mr. Paul Sagan and Ms. Evangeline Leach for
their expert editorial assistance.
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KEY WORDS * adhesion proteins * smooth muscle cells
integrins * arteriosclerosis * vascular smooth muscle
-
Integrin receptors on aortic smooth muscle cells mediate adhesion to fibronectin, laminin,
and collagen.
R I Clyman, K A McDonald and R H Kramer
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Circ Res. 1990;67:175-186
doi: 10.1161/01.RES.67.1.175
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