 |
INTRODUCTION |
Integrins are heterodimeric cell surface receptors that are
present in multicellular animals and serve as a major mechanical link
to hold cells and tissues together (1). In most biological contexts,
integrin receptors exist in an environment of ligand excess, and hence,
binding between receptor and ligand is usually controlled by
intracellular signals rather than by receptor or ligand availability.
During normal development, 
5
1 integrin becomes dispensable for basal epithelial cells undergoing
differentiation to keratinocytes and for myoblasts differentiating into
myotubes. In both cases there is a down-modulation of integrin function that precedes the down modulation of its synthesis (2, 3). Modulation
of integrin function is important for cell migration to produce
differences in functional integrin states at the leading and trailing
edges (4). A highly specialized control of integrin function has
evolved in platelets and lymphocytes to mediate the rapid response to
injury or parasitic invasion (1, 5). The ability of integrin-mediated
adhesion to be regulated by intracellular cues is critical to many
facets of biology.
The activation of integrin-ligand binding requires metabolic energy and
the actin cytoskeleton, but the exact mechanisms are poorly understood.
In platelets and lymphocytes, G-protein-coupled receptors can serve as
co-stimulatory receptors to activate integrin binding through
intracellular signaling pathways (5). Outside the hemopoietic system,
Ha-Ras and R-Raf have been identified in transfection assays as
suppressors of integrin activation as measured by the binding of the
PAC-1 monoclonal antibody to integrin chimeras containing the
IIB and 3 extracellular domains (6). R-Ras was identified as an activator of integrin function (7). The
tetraspan protein CD98 has been implicated in the activation of
31 integrin (8). Despite the
identification of intracellular signals, which can affect the
activation state of integrins, the link between these pathways and the
integrin molecules remains obscure.
Active integrins concentrate in focal adhesions that also contain both
protein kinases and high concentrations of phosphorylated proteins (9).
The cytoplasmic domains of both 1 and 3
integrin show a high degree of homology and contain two tyrosines
located in domains that are important for the binding of focal adhesion proteins and the regulation of integrin function (10, 11). Substitutions of alanine for tyrosine at these sites or introduction of
structure-perturbing mutations at adjacent sites results in loss of
integrin function (12-15). Nevertheless, demonstrating a role for
tyrosine phosphorylation in the regulation of integrin function has
been more problematic. Measurement of integrin phosphorylation has been
technically more difficult than for other focal adhesion-associated proteins. Hence, most reports have relied on tyrosine to phenylalanine mutations to probe this issue. In most integrin function assays, these
mutants were indistinguishable from wild type (12, 13, 15), although
they could be distinguished in clot retraction and cell motility assays
that involve complex cytoskeletal functions in addition to integrin
function (15, 16). To more directly address the role of phosphorylation
of the cytoplasmic domain of integrins in the regulation of ligand
binding function, we developed a system for conditional modulation of
integrin phosphorylation and applied a newly developed method to
measure directly the effect of this phosphorylation in the strength of
the v3-fibronectin bond (17, 18).
| |
MATERIALS AND METHODS |
Cell Lines and Reagents--
Human osteosarcoma cells (HOS
cells)1; ATCC, Manassas,
VA) were cultured in Dulbecco's modified Eagle's medium with
10% fetal calf serum (Mediatech, Herndon, Virginia) and
penicillin-streptomycin. Human plasma fibronectin and cell culture
media were purchased from Invitrogen. Monoclonal antibody,
LIBS-1, was a gift from M. Ginsberg. AIIB2 and BIIG2 hybridomas were
gifts from C. Damsky. Monoclonal antibodies LM609, P1D6, P1B5 and
PM6/13 were purchased from Chemicon (Temecula, California). Fluorescein
isothiocyanate-conjugated goat anti-mouse secondary antibody was
purchased from Jackson ImmunoResearch (West Grove, Pennsylvania), and
phycoerythrin-conjugated goat anti-rat antibody was purchased from
Sigma. To generate HOSnsrc cells, HOS cells were transfected with the
temperature-sensitive UP-1-v-Src mutant using the HIT retroviral
vector system (19, 20). Stable transfectants were selected with
Geneticin. WT 3, 3(Y747F),
3(Y759F), and 3(Y747F,Y759F) DNAs were
cloned into pREP9 (Invitrogen), and v DNA was cloned
into pCDM8 (Invitrogen) (from S. Blystone). The ptreLuc vector
expressing hygromycin resistance was a gift from P. Bates. HOSnsrc
cells were transfected with a mixture of a 3 vector, an
v vector and ptreLuc using LipofectAMINE Plus
(Invitrogen). Stable and transient transfectants were selected by
hygromycin resistance.
Spinning Disc Assay--
The spinning disc assay was performed
essentially as described (17, 21, 22). Briefly, the cells were allowed
to adhere for 7 min to fibronectin on glass coverslips in the chamber
of the spinning disc device, which was partially filled with spinning buffer pre-warmed to 37 °C to keep the cells near this temperature during the adhesion period. Cells were spun for 5 min, fixed with 3.7%
paraformaldehyde, and stained with ethidium homodimer. Cell density at
different radial positions was determined by using a motorized stage
and Phase 3 image analysis software Version 3.0, and the shear stress
corresponding to 50% cell detachment (
50) was
calculated using SigmaPlot software version 5.0 (17).
Wash Adhesion Assay--
Corning tissue culture microtiter
plates were coated with different densities of fibronectin type III
repeats 7-10 (23) and blocked with 1% bovine serum albumin. Cells
were labeled with calcein AM, trypsinized, and plated in
triplicate at 104 cells/well. After 1 h, the cells
were washed 3 times using a microplate washer and shaken on a Vortex
Genie (Fisher) at a setting of 3-5 min of shaking between washes. The
shaking is the most stringent step in this procedure and, hence,
regulates the strength of the washing. This produced plates with a
uniform distribution of cells in the wells rather than a donut-shaped
clearing due to differential shear at different points in the well.
Plates were read using a modified Dynatech MicroFluor plate reader.
Flow Cytometry--
Cells were trypsinized, resuspended in
fluorescence-activated cell sorter buffer (0.1% bovine serum
albumin and 0.01% sodium azide in phosphate-buffered saline), and
incubated on ice for 15 min. Anti-5 and
-1 hybridoma supernatants BIIG2 or AIIB2, respectively,
were added at 1:5 dilutions or P1B5 or LM609 purified monoclonal
antibodies were added at 10 µg/ml. For LIBS-1 binding studies, cells
were treated with 2 mM GRGDSP and stained with the
monoclonal antibody LIBS-1 at a 1:200 dilution in
fluorescence-activated cell sorter buffer. Incubation of the primary
antibody was carried out at 4 °C for 30 min with shaking.
Fluorescein isothiocyanate-conjugated anti-mouse or
phycoerythrin-conjugated anti-rat antibodies (for AIIB2 and BIIG2) were
added at a dilution of 1:100 and incubated for 30 min at 4 °C. Cells
were analyzed by flow cytometry.
Cross-linking of Bound Integrins--
Cells were plated on 2 µg/ml fibronectin-coated dishes for 1 h, cross-linked with 1 mM
Bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (Pierce)
in phosphate-buffered saline for 30 min. The cells were extracted with
0.1% SDS in phosphate-buffered saline containing protease inhibitors.
The extracted protein concentration was determined by a protein assay
reagent (Pierce) to ensure that same total numbers of cells were
attached to the substrate. The cross-linkers were cleaved in carbonate
buffer (50 mM Na2CO3, 0.1% SDS, pH
11.6) for 2 h at 37 °C, and the cross-linked pool of integrins
was analyzed by Western blotting using polyclonal antibodies to the
cytoplasmic domains of integrins 5, v,
1, and 3 (Chemicon). Blots were developed
with ECL (Amersham Biosciences, Inc.) and analyzed using a Fuji
LAS-1000 system and ScienceLab 2.5 software.
Detection of 3 Phosphorylation--
Normal and
transformed HOSnsrc cells were grown at 35 or 39.5 °C for 72 h
before treatment. The plates were washed, fresh Dulbecco's modified
Eagle's medium containing 0.2% fetal calf serum and 75 µM sodium orthovanadate was added, and incubation was
continued at 35 or 39.5 °C for 2 h. Cells were then lysed in
CHAPS buffer (1% CHAPS, 10 mM Tris-HCl, pH 7.6, 2 mM sodium orthovanadate, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2,
0.01% NaN3, 10 µg/ml aprotonin, 350 µg/ml
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 0.5 mg/ml DNase I)
at 20 °C for 10 min. Lysates were spun at 15,000 × g at 4 °C for 10 min and precleared overnight with 50 µl of goat anti-mouse IgG beads (ICN Pharmaceuticals, Costa Mesa,
CA). Lysates were then immunoprecipitated with a mixture of
anti-5 antibody P1D6 (Chemicon), anti-1
antibody TS 2/16, and goat anti-mouse beads. The supernatant fraction
was immunoprecipitated with anti-3 antibody PM6/13
(Chemicon) and goat anti-mouse beads. The beads were washed 3 times
with radioimmune precipitation buffer containing 2 mM
sodium orthovanadate, separated on reducing 8% SDS-PAGE, transferred
to a polyvinylidene difluoride membrane (Millipore), and blotted with
either 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY) for
phosphotyrosine or rabbit polyclonal antibody to either
1 cytoplasmic domain (24) or 3
cytoplasmic domain (AB 1932, Chemicon). Blots were developed with ECL
(Amersham Biosciences, Inc.) and analyzed using a Fuji LAS-1000 system
and ScienceLab 2.5 software.
| |
RESULTS |
v-Src Induces Phosphorylation of 3
Integrin--
HOS cells were transfected with a vector containing the
temperature-sensitive mutant of v-Src (UP1 (19)) and neoR. This mutant
is temperature-sensitive for kinase activity, and the protein remains
stable at the non-permissive temperature (Ref. 19 and data not shown).
Geneticin-resistant colonies were selected and screened for
temperature-sensitive expression of v-Src kinase activity. Two clones
that exhibited minimal phosphorylation of pp60v-Src at the
non-permissive temperature of 39.5 °C and high levels of pp60v-Src
phosphorylation at the permissive temperature of 35 °C were chosen
for further analysis (Fig.
1A). HOS cells growing at
35 °C and expressing active v-Src kinase will hereafter be referred
to as HOSnsrc35, and HOS cells grown at 39.5 °C expressing a
inactive v-Src kinase will be referred to as HOSnsrc39.5.
View larger version (36K):
[in this window]
[in a new window]
|
Fig. 1.
Expression of v-Src kinase in HOS cells
causes an increase in phosphorylation of 3
integrin. A, Two independent clones of HOSnsrc cells
incubated at 39.5 °C (second and fourth lanes)
(non-permissive temperature) or 35 °C (third and fifth
lanes) (permissive temperature) and parental HOS cells
(lane 1) blotted for phosphotyrosine. B,
1 Integrin was immunoprecipitated (IP) from
HOSnsrc35 and HOSnsrc39.5 cells, and 3 integrin was
immunoprecipitated from the supernatant of the 1
immunoprecipitate. The immunoprecipitates were analyzed by Western blot
with anti-phosphotyrosine (P-Tyr) and anti-1
or anti-3 antibodies.
|
|
Src-dependent Phosphorylation of 1 and
3 integrin subunits was analyzed by comparing the levels
of phosphotyrosine in 1 and 3
immunoprecipitates of HOSnsrc35 and HOSnsrc39.5 cells. Fig.
1B shows that activation of v-Src resulted in a >5-fold
increase in the level of 3 phosphorylation. Tyr-747 in
the cytoplasmic domain of 3 integrin is a likely site
for this phosphorylation since it has been shown to be phosphorylated
in vivo and is in a site homologous to Tyr-788 in chicken
1, which can be phosphorylated by v-Src in
vitro (25) (26). In contrast, 1 integrin showed a
moderate level of phosphorylation in HOSnsrc39.5 cells, and this level
did not appear to increase substantially after the activation of v-Src
kinase in HOSnsrc35 cells. The increased background seen in the
HOSnsrc35 1 integrin blot is likely due to the increase in phosphorylation of many proteins after v-Src expression. This differs from previous reports using 32P-labeling that
report increased phosphorylation of 1 in
v-Src-transformed chicken cells (27-29). The HOSnsrc cells express
lower levels of v-Src compared with v-Src-transformed chicken embryo
fibroblasts. Because Src co-localizes selectively with
v3 as opposed to
51 (30), the reduced expression of v-Src
would produce a selective phosphorylation of 3 over
1.
v-Src Modulates v3-Fibronectin Bond
Strength--
A modified wash-type adhesion assay was used to
determine which integrins were responsible for adhesion of HOSnsrc35
and HOSnsrc39.5 cells to fibronectin. In the modified assay, the
stringency is controlled by the mechanical shaking device rather than
by the force of the buffer during fluid changes. This provided a more reproducible assay and reduced the effects of the well geometry on the
assay. The remaining cells following the washing procedure were
uniformly distributed over the well rather than the donut-shaped detachment pattern common to wash assays. Adhesion was determined for a
range of fibronectin densities, and the proportion of cells remaining
was plotted as a function of fibronectin density (as determined
previously (31)). Decreases in binding affinity or adhesion are
reflected in a rightward shift of the sigmoid plot (analogous to the
interpretation of enzyme-linked immunosorbent assays). Fig.
2A shows that treatment with
anti-3 antibody had no effect on adhesion, whereas
anti-1 antibody reduced the adhesion about 60%, and
combined anti-1 and anti-3 reduced
adhesion to background levels with no dependence on fibronectin
density. Fig. 2B shows that the adhesion of HOSnsrc35 cells
was reduced about 60% relative to HOSnsrc39.5, as seen in the
rightward displacement of the curve; again, anti-3 had
no effect but either anti-1 or the combination of
anti-1 and anti-3 reduced adhesion to background levels. Fig. 2C shows the analysis of adhesion in
the presence and absence of blocking antibodies for a single
fibronectin density. Note that this form of the analysis does not
distinguish between HOSnsrc35 and HOSnsrc39.5 cells in the absence of
antibodies. These data suggest that expression of v-Src produces a
reduction in total adhesion to fibronectin possibly through an effect
on v3.
View larger version (19K):
[in this window]
[in a new window]
|
Fig. 2.
Wash-type adhesion assay. Hosnsrc39.5
cells (A) and HOSnsrc35 cells (B) were tested for
adhesion to different densities of purified fibronectin type III
repeats 7-10 in microtiter plates in the absence or presence of
antibodies to 1 integrin (AIIB2), 3
integrin (LM609), or both AIIB2 and LM609 together (Both).
C, HOSnsrc39.5 cells (black bars) and HOSnsrc35
cells (gray bars) were assayed on purified fibronectin type
III repeats 7-10 coated at 320 ng/cm2 in the presence and
absence of antibodies as shown. The error bars are the S.D.,
n = 3.
|
|
To provide a better resolution of the contributions of
v3 and 51
to HOSnsrc35 and HOSnsrc39.5 cell adhesion to fibronectin, we used the
spinning disc device. This device exposes the cells to a linear
hydrodynamic shear gradient and measures the forces required for cell
detachment under different conditions. Previous experiments demonstrate
that the detachment force is directly proportional to both the number
of receptor-ligand bonds and the strength of those bonds (17, 21). The
strength of the individual integrin ligand bonds depends on activation
processes within the cell (18, 32). Thus, this approach provides a
quantitative measure of the relative strength of the integrin-ligand
bonds. Fig. 3A shows a
combined cell detachment profile for HOSnsrc35 and HOSnsrc39.5 cells
from fibronectin as a function of applied shear stress at 7 min after
plating. The data show about a 40% reduction (leftward shift, cells
detach at lower shear stress) in adhesion strength as a result of
temperature-dependent activation of v-Src. Analysis of the
levels of cell surface expression of integrin by flow cytometry showed
no difference in the levels of 3, 5,
v, 1, or 3 between
HOSnsrc35 and HOSnsrc35 cells (data not shown). Thus the reduction of
adhesion after the activation of v-Src cannot be explained by altered
integrin expression levels. To determine whether the difference was due
to differences in incubation temperature, HOSnsrc and parental HOS
cells incubated at 35 and 39.5 °C were assayed using the spinning
disc. Fig. 3B shows a summary of the 50
values (shear stress for 50% adhesion) for several experiments similar
to that shown in Fig. 3A. Incubation temperature had no
effect on the adhesion of parental HOS cells but showed a 40%
reduction for HOSnsrc cells at 35 °C.
View larger version (19K):
[in this window]
[in a new window]
|
Fig. 3.
Modulation of
v3-fibronectin
bond strength by v-Src. A, Cell detachment profile for
HOSnsrc35 (circles) and HOSnsrc39.5 (triangles)
plated on fibronectin (50 ng/cm2) using the spinning disc
device at the permissive and inhibitory temperatures for v-Src. Filled
figures ( and  ) represent normalized cell density data points as
a function of applied shear stress; open figures ( and  )
represent a sigmoid curve fit. The mean (50%) cell detachment force
(50) for cells with active and inactive v-Src kinase
were 90.4 and 176.8 dynes/cm2, respectively. B,
Comparison of 50 for parental HOS cells incubated at
39.5 °C versus 35 °C and HOSnsrc39.5 versus
HOSnsrc35 plated on fibronectin (50 ng/cm2). C,
50 from a series of plots as in A at
different fibronectin densities. D, Comparison of
50 for HOSnsrc35 and HOSnsrc39.5 untreated and treated
with anti-1 (AIIB2), anti-5 (P1D6),
anti-v3 (LM609), or anti
v3 + anti-1 (AIIB2 + LM609) plated on fibronectin (50 ng/cm2). *, experiment not
done. The error bars represent the S.D.
|
|
For cells that express the same number of integrin receptors,
differences in the slope of the mean cell detachment force (as determined form analyses shown in Fig. 3A) as a function of
fibronectin density reflect differences in the strength of the
integrin-ligand interaction. Fig. 3C shows that the
HOSnsrc35 and HOSnsrc39.5 cells had different slopes. For the
HOSnsrc39.5, there is a suggestion of a two-component curve as it
deviates from linearity at higher fibronectin densities, again raising
the possibility that the two receptors with different binding strengths
were involved. To determine which integrin receptors were responsible
for these differences, spinning disc experiments were performed on
HOSnsrc cells treated with antibodies to 5 (BIIG2),
1 (AIIB2), or 3 (LM609) integrin. Fig.
3D shows that the adhesion of HOSnsrc35 cells was reduced to
background levels by either anti-5 or
anti-1 but was unaffected by anti-3. In
contrast, HOSnsrc39.5 cells showed a significant but partial reduction
in the presence of either anti-5 or
anti-3 and required the mixture of anti-1 and anti-3 to reduce adhesion to background levels.
Thus, both 51 and
v3 mediated the adhesion of HOSnsrc39.5
cells. Specific adhesion strengths do not appear to be directly
additive; i.e. the sum of the adhesion strength in the
presence of anti-5 representing v3-mediated adhesion plus the adhesion
strength in the presence of anti-3, representing
51-mediated adhesion, was more than the
adhesion strength in the absence of antibody. This result is not
unexpected since cross-talk between these receptors had been described
(33). Unlike the HOSnsrc39.5 cells, the HOSnsrc35 cells showed no
v3-mediated adhesion to fibronectin.
Attempts to use vitronectin to provide an alternative means of
distinguishing v3- from
51-mediated adhesion were limited by the
expression of vitronectin receptors by the HOSnsrc cells in addition to
v3. Thus, activation of v-Src in the
HOSnsrc cells results in the inactivation of
v3 integrin function as determined by its
ability to support a mechanical connection to fibronectin. This
v3 function could be switched on and off
by switching the incubation temperature of the cells, thereby
activating or inactivating the v-Src kinase enzymatic function.
Ligand-Bound 51 and
v3--
Chemical cross-linking has been
used for the analysis of many receptor-ligand interactions. We have
taken the approach of using cell-impermeant cross-linkers to cross-link
cell surface integrins to substrate-immobilized ligands. After
cross-linking with
Bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone, the cells were
extracted with a strong detergent, leaving the cross-linked integrin
behind. The cross-linker was cleaved at high pH, releasing the
cross-linked integrin for analysis by Western blot. Control experiments
have shown that the recovery of integrin from the cross-linked fraction
requires the proper ligand and that the integrin be activated (17, 24).
In addition, for 51, the amount of
cross-linked integrin recovered was directly proportional to the
strength of the interaction measured by the spinning disc assay and the
number of receptor-ligand bonds formed (31). This cross-linking assay
provides an alternative assay for the presence of specific
integrin-ligand bonds. The data in Fig. 4
show both the supernatant fraction (non-cross-linked) and the
cross-linked fraction for each integrin subunit. Based on quantitation
of three independent experiments, the higher v-Src kinase activity in
the HOSnsrc35 cells had no significant effect on the level of
cross-linked 5 or 1. This is consistent
with the adhesion data, which showed that v-Src activation had minimal
effect on 51-mediated adhesion. In
contrast, the level of v was reduced about 2-fold, and
the level of 3 was reduced about 4-fold in the HOSnsrc35
cells compared with the HOSnsrc39.5 cells. The levels of
v in the cross-linked fraction are low, and the antibody
used does not give as clear a Western blot as the others. Nevertheless,
these results support the conclusion that
v3 function is reduced in the HOSnsrc35 cells.
View larger version (60K):
[in this window]
[in a new window]
|
Fig. 4.
Biochemical analysis of ligand-bound
53
and
v3.
HOSnsrc35 cells and HOSnsrc39.5 cells were plated on fibronectin.
Soluble (S) and cross-linked (X-L) integrins were
analyzed for levels of 1, 3,
5, and v integrin subunits by Western
blot. m is the mature 1; p is the
precursor (only seen in the soluble fraction).
|
|
Restoration of v3 Binding by Tyr 
Phe Mutants of 3--
Previous experiments show that
activation of v-Src kinase in HOS cells increased the level of
3 phosphorylation and blocked the ability of
v3 to mediate adhesion to fibronectin.
Both the use of a temperature-sensitive v-Src kinase activity mutant
and the relatively low level of v-Src expressed in these cells
contribute to the argument that the effect on adhesion is specific to
v-Src kinase function. Nevertheless, the correlation between the
increase in 3 phosphorylation and reduction of
v3 function could be due to the
phosphorylation of 3 integrin-associated proteins rather
than 3 integrin itself by v-Src kinase. To determine
whether phosphorylation of 3 itself was critical for the
reduction of v3-mediated adhesion,
non-phosphorylatable mutants of 3 integrin were
transfected into HOSnsrc35 cells. Both transient transfections and
selection of stable transformants were performed. There are two
tyrosines in the cytoplasmic domain of 3 at positions
747 and 759 (in the human sequence). Both of these tyrosines are
embedded in sequences that are similar to the Tyr-788 region in the
cytoplasmic domain of chicken 1 (Tyr-783 human)
integrin, which can be phosphorylated by v-Src kinase in
vitro (26). It is likely that both Tyr-747 and Tyr-759 in
3 integrin can be phosphorylated in vivo, but definitive data exist only for Tyr-747 (25).
The transient transfection assays gave the best expression levels at
48 h after transfection, but we found that the cells recovering
from various transfection protocols were too fragile to be assayed in
the spinning disc assay at 48 h. As an alternative, the relative
proportion of active-fibronectin-bound 3 integrin was
determined using the chemical cross-linking demonstrated above. HOSnsrc35 cells were transiently transfected with WT 3
or 3(Y747F) and analyzed 48 h later by flow
cytometry for expression of 3 and by chemical
cross-linking to fibronectin for v3
function. Fig. 5 shows that both WT
3 and 3(Y747F) transfectants had
increased 3 expression about 2-fold. Analysis of
cross-linked 3 showed about 4-fold higher levels of
3 in the 3Y(747F) mutant compared with WT
3. By blocking the ability of the cells to phosphorylate Tyr-747 in 3 integrin, more 3 integrin
was incorporated into the ligand-bound fraction. Thus the mutant was
able to complement the defect in 3 integrin function
caused by v-Src kinase activation.
View larger version (38K):
[in this window]
[in a new window]
|
Fig. 5.
Rescue of
3 binding by the
expression of a 3
phosphorylation mutant. WT 3 and
3(Y747F) mutant expression plasmids were transiently
transfected into HOSnsrc35 along with an expression plasmid for
v integrin. Flow cytometry was used to
analyze the level of 3 expression on the surface of WT
3 transformants (A), 3 (Y747F)
transformants (B), and parental HOSnsrc35 cells
(C). MFI is mean fluorescent index or geometric
mean of 3 fluorescence-negative control (secondary
antibody alone)/negative control. D, Western blot showing
the level of cross-linked 3 for WT 3 and
3 (Y747F) transient transfectants of HOSnsrc35
cells.
|
|
In two separate experiments, stable transfectants were isolated for
HOSnsrc35 cells expressing WT 3,
3(Y747F), 3(Y759F), and
3(Y747F,Y759F). Transfected clones were analyzed using
the spinning disc assay in the presence of antibody to block
51 integrin and permit an independent
measurement of v3 function. Table
I shows that WT 3 produced
no significant increase in the strength
v3-mediated adhesion, whereas mutants
containing the Y747F mutation showed a 5-fold increase, and the single
Y759F mutant showed a small increase. Thus, the non-phosphorylatable Y747F mutation was able to restore
v3-mediated adhesion in the presence of
an active v-Src kinase. This complementation was seen for both the
transient and stable transfectants and demonstrates that the
phosphorylation of Tyr-747 in 3 is the dominant
mechanism by which v3-mediated adhesion
to fibronectin is blocked in the v-Src-transformed HOS cells. Because
endogenous v3 is still expressed by these
cells, the Y747F mutation is a dominant suppressor of v-Src-modulated
adhesion to fibronectin in the HOSnsrc cells.
View this table:
[in this window]
[in a new window]
|
Table I
Analysis of adhesion strength for stable transfectants
T50 gives the 50% detachment shear stress in dynes/cm2 showing
mean and S.D. for three independent measurements.
|
|
v-Src Reduces LIBS-1 Recognition of
v3--
The mechanism by which
v-Src-mediated phosphorylation of Tyr-747 on 3 integrin
modulates the function of v3 was examined by looking for changes in the conformation of the extracellular domain
of v3 in response to the activation of
v-Src kinase. Differences in v3
conformation were assayed using flow cytometry to analyze the binding
of LIBS-1 monoclonal antibody, which recognizes the ligand-occupied
state of 3 integrins (34). Table
II shows that inactivation of v-Src by
incubation of the HOSnsrc cells at 39.5 °C increased the level of
LIBS-1 binding about 2-fold, whereas this temperature-shift had no
effect on the binding of LIBS-1 to parental HOS cells lacking the v-Src
gene. In addition, the two v-Src kinase inhibitors herbimycin A and PP2
produced a significant increase in the level of LIBS-1 binding, whereas the inactive PP3 had only a modest effect. The 2-fold difference in
binding is similar to that reported for the effect of Ras on the
binding of the PAC-1 monoclonal antibody to
IIb3 (6). Thus, the phosphorylation of
3 cytoplasmic domain led to a detectable change in the
conformation of the extracellular domain of
v3.
View this table:
[in this window]
[in a new window]
|
Table II
Analysis of v3 conformation by LIBS-1 antibody
binding
MFI is the geometric mean fluorescent intensity. Activation index is
normalized MFI to HOSnsrc35 for four separate experiments done at
different times.
|
|
| |
DISCUSSION |
The ability of integrin-mediated adhesion to be regulated by
intracellular signals is critical to the function of integrins in
processes that include cell migration, cell differentiation, cell
survival, and cell proliferation (1). Direct analysis of the mechanisms
by which the integrin function is controlled have been limited by both
the biological systems that have been studied and by the assays used.
We developed a quantitative assay to measure the relative strength of
the integrin-ligand bonds in intact cells as a means of providing a
more direct measurement of integrin activation than was available using
"activation-specific" monoclonal antibodies (17, 34). Using that
approach we have identified additional states of the integrin-ligand
binding interaction that could not be identified using previous
approaches (17, 18). In this study, we also developed a cell line that
expresses a temperature-sensitive Src kinase and that displays a
temperature-sensitive phosphorylation of 3 integrin.
This provides the first instance in which the phosphorylation of a
specific integrin can be controlled without using specific
phosphorylation-blocking integrin mutants. This is important because
this phosphorylation event is likely to be transient, as suggested by
the difficulty of observing phosphorylation of either 1
or 3 integrin in the absence of vanadate pretreatment. It is possible that the regulation is accomplished through a
phosphorylation-dephosphorylation cycle. The second critical element of
the model system is the focus on 3 rather than
1 integrin (discussed below). This is the first report
of v-Src-induced phosphorylation of 3 integrin. Remarkably, blocking 3 phosphorylation of Tyr-747 by
mutation complemented the adhesion defect showing that failure to
phosphorylate 3 resulted in failure to block
3 function. This is also the first case of dominant
suppression of a v-Src function by a single Tyr Phe mutation in a
candidate v-Src target.
Sequence analysis of 1, 2,
3 integrin revealed a high degree of homology in their
cytoplasmic domains. The presence of tyrosines at 783 and 795 in human
1 and 747 and 759 in human 3 correspond
to phenylalanines in 2 integrin (35). Conformational disruption by the introduction of mutations into the domains containing these tyrosines in 1 and 3 integrin
compromises its function in adhesion and/or spreading assays,
suggesting that these sequences are critical for normal function (36,
37). In most analyses, the Tyr Phe mutations had a weaker phenotype
than Tyr Ala mutants, and mutations near Tyr-747 had a stronger
phenotype than mutations near Tyr-759. (13, 14). The complementation
data presented here also show a stronger phenotype for the Y747F as compared with the Y759F mutant. The absence of tyrosines in
2 integrin cytoplasmic domain suggests that the
mechanisms of regulation of 2 integrin-mediated adhesion
will be different from 1 and 3. Despite
the sequence homology between 3 and 1
integrin, several lines of evidence suggest that they are regulated differently.
First, analysis of the compartmentalization of phosphorylated and
non-phosphorylated 1 integrin in v-Src-transformed
chicken embryo fibroblasts demonstrated that the increase in
1 phosphorylation caused by v-Src expression was mostly
in the soluble and not the adhesion-associated pool of 1
(28). Second, mutants of 1 in which the cytoplasmic
tyrosine corresponding to Tyr-747 in 3 was mutated to
glutamate to simulate the phosphorylated form were distributed away
from focal adhesions, whereas the phenylalanine substitutions tended to
accumulate in focal adhesions (10). Third, analysis of the function of
phenylalanine substitutions in 1 using GD25 cells, which
express no endogenous 1 integrin, led to defects in the
organization of focal adhesions, cytoskeleton, and cell motility (15).
Each of these studies led to a model in which phosphorylation of
1 resulted in the dissociation of connections between
1 cytoplasmic domain and the cytoskeleton and
dissociation between 1 and its ligand. The results are
also consistent with a model in which 1 integrin is
phosphorylated outside the focal adhesion, and the phosphorylated forms
do not cycle into focal adhesions. Hence, phosphorylation would reduce the available 1 integrin pool. This interpretation would
be favored, at least for the case of Src kinase, by the demonstration
that Src colocalizes with 3 but not with
1 in mouse fibroblasts (30). In the present study,
varying the level of v-Src kinase activity had no substantial effect on
the level of 1 phosphorylation but had a large effect on
3 phosphorylation. Does this difference between
1 and 3 extend to cells other than the
HOSnsrc cells? GD25 cells lacking 1 integrin adhere to
fibronectin use v3 (38). Transformation
of the GD25 cells by v-Src resulted in the complete loss of adhesion to
fibronectin (29). Also, chicken embryo fibroblasts plated on
fibronectin cannot be detached by the addition of CSAT monoclonal
antibody, which blocks 1 integrin function;
however, after transformation by v-Src, these cells can be detached by
CSAT (39). Because the chicken embryo fibroblasts also express
v3, inactivation of
v3 by v-Src could lead to detachment by
CSAT. Thus, 3 integrin phosphorylation as a result of
Src kinase appears to have a direct effect on its ability to mediate
adhesion to fibronectin, whereas the effects of Src kinase on
1 integrin appear to be less direct. In chicken embryo
fibroblasts, expression of v-Src had no effect on the strength of the
51-fibronectin bond in the short term (15 min) assays. The longer term reductions in adhesion could be explained
by increases in both protease secretion, resulting in ligand removal,
and hyaluronic acid, which insulates 51
from the surface-bound fibronectin (39). There is no evidence that
either of these effects can be mediated through effects of the
activation levels of 51 by v-Src.
The functional assays used in this report rely on the analysis of the
strength and relative number of integrin-ligand bonds. For cultured
cells, strong adhesion is maintained by a linkage from the inert
plastic or glass substrate to the adsorbed extracellular matrix
proteins, including predominantly fibronectin, to integrins, and
to the actin cytoskeleton. The two regulated links in this chain are
the integrin-ligand and the integrin-cytoskeleton connections. Interestingly, the regulation of these two linkages appears to be
coupled. First, disruption of the actin cytoskeleton with agents such
as cytochalasin D cause a reduction in adhesion strength (17, 40).
Second, changes in the mechanical strength of the integrin-ligand bond
correlate with changes in the mechanical strength of the
integrin-cytoskeleton linkage (18, 41). Thus, changes in the mechanical
strength of the interactions of the integrin cytoplasmic domain with
cytoskeletal-associated proteins could affect the strength of the
integrin-ligand bond. Indeed, such changes are thought to provide the
basis for the regulation of integrin function by intracellular signals
(9, 11).
The cytoskeletal proteins talin and filamin bind to the cytoplasmic
domains of 1, 2, and 3
integrins, and the binding to 1 and 2
integrins can be inhibited by a Tyr Ala mutation in the
membrane-proximal tyrosine (37, 42-44). This suggests that talin and
filamin binding may be regulated by phosphorylation of Tyr-747.
Phosphorylated 1 integrin, produced by overexpression of
v-Src kinase in vivo, showed a reduced binding affinity for talin in a gel filtration assay (26). Myosin was shown to bind to a
phosphorylated 3 peptide, suggesting that the binding of myosin to 3 could also be affected by 3
phosphorylation (45). This list is unlikely to be inclusive, and other
cytoskeletal proteins have been found to bind 3
cytoplasmic domain peptides, but the specific binding sites have not
yet been mapped, e.g. skelemin (46). These proteins can also
serve to link other cytoskeletal and cytoskeletal regulatory proteins
including vinculin, zyxin, Mena/VASP, and FAK (11).
Although phosphorylated Tyr-747 and Tyr-759 in 3
integrin could provide sites for the binding of specific cytoskeletal
proteins, it must be noted that the phosphorylated forms are likely to
be quite transient since they are only easily visualized after
treatment of the cells with phosphatase inhibitors for several hours
before lysis. In contrast, stable integrin-mediated adhesion requires that the cytoskeletal connections be maintained. We propose a model in
which the role of 3 phosphorylation is to alter the conformation of the cytoplasmic domain and provide for the dissociation of one protein complex and the formation of a new protein complex. 3 integrin containing a phosphorylated Tyr-747 would
represent a transition state, and hence, it would not be attached to
the cytoskeleton. The absence of the cytoskeletal connection would limit the strength of the mechanical linkage required for cell adhesion
and could retain v3 in an inactive
conformation. A precedent for this can be found in analyses of
2 integrin (42). In the absence of ligand, talin was
bound to the cytoplasmic domain of 2. After the binding
of 2 to ligand, talin was cleaved, released from
2, and replaced by -actinin.
3 Integrin Controls Ligand
Binding Strength*
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Dept. of
Microbiology, University of Pennsylvania, Philadelphia, PA 19104-6076. Tel.: 215-898-8792; Fax: 215-898-9557. E-mail:
boettige@mail.med.upenn.edu.
