(Received for publication, November 10, 1994; and in revised form, May 24, 1995)
From the
The interactions of
Integrins are structurally related cell surface receptors
involved in cell-cell adhesion and cell attachment to the extracellular
matrix. They are noncovalently associated heterodimers of The Divalent cations
are required for integrins to bind to their ligands (1) .
Figure 1:
Aspartic acid
substitutions in
A novel bivalent cation binding site has been
identified in the integrin In an attempt to identify the
ligand binding site in
Figure 2:
Surface expression of
To examine whether the Asp Surface expression of
Figure 3:
Analysis of
Figure 4:
Adhesion of K-WT and K-LEV to VCAM. A, K-WT (
The
avidity of integrins can be regulated through inside-out cell
signaling. The avidity of VLA-4 increases upon activation with phorbol
esters(26, 27) . Both K-LEV and K-WT could respond to
PMA stimulation (Fig. 5A). However, PMA failed to fully
restore the capability of K-LEV to bind VCAM. In addition, the
Figure 5:
Effect of PMA stimulation on adhesion of
K-WT and K-LEV. A, adhesion of K-WT (WT) and K-LEV (LEV) in the absence and presence of PMA (10 ng/ml) to
recombinant VCAM1-3 at coating concentrations of 2.5 and 5
µg/ml. B, K-WT, K-LEV, and K562 were tested for adhesion
to microtiter plates coated with the indicated amounts of FN-40 in the
presence of PMA.
The ability of K-LEV
to adhere to fibronectin was also tested. FN-40 is a chymotryptic
fragment of human fibronectin(28) . It contains the sequence
essential for
Figure 6:
Analysis of surface expression of
The mutant
Figure 7:
Adhesion of K-WT (
It is believed that the ligand binding
sites on
Figure 8:
Binding
of VCAM to wild-type and mutant
Figure 9:
Comparison of the new putative cation
binding motif in
We have demonstrated that glutamic acid substitutions at
Asp-489 and Asp-698 severely impair the ability of
To investigate the effect of D489E and D698E
substitutions on gross conformation of The mutant The ability of K-LEV, K-D489E, and K-D698E to
adhere to VCAM or fibronectin was severely impaired as demonstrated in Fig. 4, 5, and 7. To determine whether impaired adhesion was due
to the inability of mutant Both Asp-489 and Asp-698 are in an
One mechanism of impairing integrin
function is disruption of divalent cation binding sites. Divalent
cations are required for integrins to bind to their
ligands(1, 35, 36) . Masumoto and Hemler (13) demonstrated the importance of the three putative divalent
cation binding sites for In summary, we have identified two
aspartates, Asp-489 and Asp-698, in
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
integrin
with vascular cell adhesion molecule (VCAM) and fibronectin play
important roles in many physiological and pathological processes. To
understand the mechanism of
integrin-mediated cell adhesion, we made mutant
constructs. Three aspartic acid (Asp) residues in
, Asp-489, Asp-698, and Asp-811, were replaced with
glutamic acids (Glu). The wild-type and mutant
constructs were transfected into K562 cells, and stable
transfectants with similar levels of
surface
expression were established. The Asp
Glu substitutions did not
affect
association or heterodimer
formation as demonstrated by immunoprecipitation analysis. However, the
glutamate substitutions at Asp-489 and Asp-698 severely impaired cell
adhesion to VCAM and fibronectin, whereas the substitution at Asp-811
had no detectable effect on cell adhesion. In contrast to these
results, isolated
, containing the
D489E or D698E substitution, was able to bind to VCAM, suggesting that
these two residues are not critical for ligand recognition. In
searching for a mechanism to explain inhibition of adhesion by Asp-489
and Asp-698 mutations, we found that the sequences flanking Asp-698
resemble the DxxxxxD-S-Sx divalent cation/ligand
binding motif in
integrins and the I-domains of
integrins.
This suggests that Asp-698 in the
integrin, which
does not possess an I-domain, may also be involved in cation binding
and may be part of a sequence functionally similar to that found in the
I-domains of other
integrins.
and
subunits (1) . Many different
subunits are able to
associate with the same
subunit to form
/
dimers with
distinct ligand specificity. The ligand specificity of integrins are,
therefore, believed to be determined largely by the structures of the
subunits.
integrin
(VLA-4, CD49d/CD29) is expressed on lymphocytes, monocytes, bone marrow
progenitors, and on some tumor cell
lines(2, 3, 4, 5) . The ligands for
the
integrin are vascular cell
adhesion molecule (VCAM) (
)and fibronectin. The interaction
of
with its ligands is important in
T cell activation, cell adhesion, inflammation, myogenesis, and tumor
metastasis(5, 6, 7, 8, 9) .
Furthermore, the
integrin is critically involved in
disease pathogenesis and progression in many animal models such as
experimental autoimmune encephalomyelitis, autoimmune diabetes, and
late phase antigen-induced
asthma(10, 11, 12) . Elucidation of the
mechanisms of
-ligand interaction is
important in understanding these physiological or pathological
processes and may have great therapeutic potential.
has a large extracellular domain and short
cytoplasmic and transmembrane domains. The N-terminal half of the
extracellular domain contains 7 homologous segments, which are
conserved among all the integrin
subunits (Fig. 1). The
5th, 6th, and 7th homologous sequence segments from the N terminus have
been identified as putative divalent cation binding
sites(1, 13) . Masumoto and Hemler (13) demonstrated the importance of these divalent cation
binding sites for
function. Mutations at each of the
three divalent cation binding sites (N283E, D346E, and D408E) of
resulted in significant loss in the ability of
to bind VCAM and fibronectin, whereas mutations at
Cys-278, Cys-717, Cys-767, and Cys-828, downstream from the cation
binding sites, did not affect interaction of
with
VCAM (13) .
. Boxes indicate highly
conserved regions in
integrins. M
indicates a putative divalent cation binding
site.
subunits(14, 15) .
The aspartic acid in this cation binding sequence, conserved among
subunits, is required for ligand
binding(15, 16) . A similar cation binding motif is
also found in the I-domain containing
subunits(15, 18) . Mutation at the aspartic acid
residue in this cation binding site abolishes cell-ligand adhesion and
cation binding(15, 17, 19) . Therefore, in
addition to the essential role of the classical integrin cation binding
sites in cell adhesion,
subunits and the I-domains in
subunits also appear to be critically involved in the interaction of
integrins with cations and ligands.
, we made synthetic peptides
derived from
and examined their abilities to inhibit
cell adhesion to VCAM. Our preliminary experiments showed that
peptides, containing residues 482-491,
691-700, and 804-813, specifically inhibited the
interaction of
with recombinant VCAM (20) , and the aspartic acid in these peptides appeared to be
critical for inhibition. (
)In this study, we have replaced
the three aspartic acid residues, Asp-489, Asp-698, and Asp-811, with
glutamic acid and examined the function of the mutated
integrins in cell adhesion.
Materials
Restriction enzymes, T4 DNA ligase,
and Klenow fragments were purchased from New England Biolabs, and the
Sequenase 2.0 Sequencing Kit was obtained from United States
Biochemical Corp. Taq DNA polymerase was obtained from
Perkin-Elmer Cetus. Phorbol 12-myristate 13-acetate (PMA) was purchased
from Sigma, and the 40-kDa fragments of human fibronectin (FN-40) were
from Life Technologies, Inc. PBS (Bio-Whittaker) contains 10 mM phosphate (pH 7.0), 138 mM NaCl, 2.7 mM KCl, 0.5
mM MgCl, and 1 mM CaCl
.
Antibodies
Antibodies used were as follows: mouse
anti-human monoclonal antibodies C212.7(21) ,
HP2/1 (Amac, Inc., (4) ), B-5G10 (Upstate Biotechnology, (3) ), CIII376.2, a mouse monoclonal antibody raised against
peptide LQEENRRDSWSYINSKSNDD in the cytoplasmic portion of
, mouse anti-human
monoclonal
antibody K20 (Amac, Inc., (22) ), and goat anti-mouse
IgG-fluorescein conjugate (Tago).
Mutagenesis
Site-directed mutagenesis was
performed by overlap extension in the polymerase chain reaction using
pBWT, a pBluescript SK plasmid containing human -cDNA
inserted at XhoI/XbaI sites as a template as
described (23) . The third base of the aspartic acid (Asp)
codon for Asp-489, Asp-698, Asp-811, or Asp-698 and Asp-811 in
, was mutated from T to A using mutant oligonucleotide
primers to generate the codon for glutamic acid (Glu). The mutant
cDNA carrying the Asp
Glu substitutions were
cloned into pBluescript and sequenced to confirm the presence of the
intended mutation and absence of any Taq polymerase errors.
The four mutant
-cDNAs were constructed in pBluescript
by exchanging the restriction fragment containing the mutation for the
corresponding wild-type sequence. The wild-type and mutant
cDNAs were subcloned into pcDNA3 (Invitrogen) and designated as
pcWT (wild-type), pcD489E, pcD698E, pcD811E (carrying Asp
Glu
substitution at Asp-489, Asp-698, and Asp-811, respectively), and pcLEV
(carrying Asp
Glu triple substitutions at Asp-489, Asp-698, and
Asp-811).
Transfection
K562 cells were maintained in RPMI
1640 media with 5% fetal calf serum. Cells (5 10
)
were transfected with 20 µg of linearized
pcDNA3
constructs by electroporation at 270 V/0.4 cm and 960 microfarads in
0.4 ml of PBS using a Gene Pulser (Bio-Rad). After 48 h, transfected
cells were placed in medium containing 0.4 mg/ml Geneticin (Life
Technologies, Inc.). Geneticin-resistant cells were subjected to
several rounds of fluorescence-activated cell sorting (FACS) using mAb
C212.7 and a FACStar Plus cytometer (Becton Dickinson) to enrich and to
clone
expressing stable transfectants.
Flow Cytometry
Cells were incubated with primary
mouse mAb in PBS containing 10% human serum (Life Technologies, Inc.)
on ice for 20 min. After washing three times in PBS, the cells were
treated with goat anti-mouse IgG coupled to fluorescein (Tago) in PBS
containing 2% fetal calf serum. The cells were washed twice in PBS,
resuspended in the same buffer, and analyzed using a FACScan cytometer
(Becton Dickinson).Immunoprecipitation and Western Blotting
Cells
were washed with cold PBS, and samples were adjusted to equal cell
numbers and cell densities of 10/ml. Cells were then
incubated with PBS containing 0.2 mg/ml sulfosuccinimidobiotin (Pierce)
and 14 mM glucose at room temperature. After 30 min, glycine
was added to a final concentration of 4 mM, and the cell
suspension was incubated on ice for 15 min. Cells were washed three
times in PBS and then lysed on ice in buffer containing 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM
MgCl
, 1 mM CaCl
, 1% Nonidet P-40. Cell
lysates were precleared by centrifugation followed by incubation with
GammaBind G Sepharose (Pharmacia Biotech) and recentrifugation.
Aliquots of the lysates were incubated with specific integrin
antibodies overnight at 4 °C, subsequently absorbed onto GammaBind
G Sepharose, followed by washing in buffer containing 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM MgCl
, 1 mM CaCl
, 0.1% Nonidet
P-40, and 0.05% Tween 20. Absorbed complexes were removed from the
beads by heating in reduced SDS-PAGE sample buffer at 65 °C for 30
min, and the supernatants were analyzed on 4-12% polyacrylamide
gradient gels (Novex). Proteins resolved on gels were transferred onto
nitrocellulose membranes (Schleicher & Schuell), and the membranes
were first incubated in 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5% nonfat dry milk for 1 h and then incubated with
streptavidin-horseradish peroxidase conjugate (Boehringer Mannheim) in
PBS containing 0.1% Tween 20. Biotinylated proteins were detected using
the ECL kit (Amersham Corp.) according to manufacturer-suggested
protocol.
Adhesion Assays
Assays for cell adherence to
ligand proteins were carried out essentially as described(20) .
Briefly, cells were labeled by incubating with 6-carboxyfluorescein
diacetate and were added, with or without PMA or antibodies, to 96-well
microtiter plates coated with recombinant VCAM, consisting of domains 1
to 3 of VCAM (VCAM1-3)(20), or coated with FN-40. Nonspecific
sites on the plates were blocked with 1% bovine serum albumin. After a
25-min incubation at room temperature, unbound cells were removed by
washing and low speed centrifugation with the plates inverted. Cells
remaining bound to the plates were quantitated using a Cyto Fluor 2300
fluorimeter (Millipore). Nonspecific background cell binding was below
2% under these conditions.Integrin-Ligand Binding Assays
Two approaches were
used to assess the ability of integrin to bind to
VCAM. In assay A, purified recombinant human VCAM1-3 was coupled
to Sepharose 4B. 5
10
cells were surface-labeled
with biotin and lysed in 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM MgCl
, 1 mM MnCl
, and 50 mM
-octyl glucoside (Buffer
A). Insoluble material was removed by centrifugation, and the
supernatants were incubated with Sepharose 4B in Buffer A containing 5
mg/ml myoglobin. After centrifugation, the supernatants were then
incubated with VCAM-Sepharose at 4 °C for 3 h. The VCAM-Sepharose
was washed four times in Buffer A. One-half of the VCAM-Sepharose was
incubated in Buffer A containing 20 mM EDTA overnight, and
eluted
was immunoprecipitated with mAb CIII376.2 and
GammaBind G Sepharose. The biotinylated proteins bound to Sepharose
were analyzed on 4-12% SDS-PAGE gels and detected with
streptavidin peroxidase conjugate, using ECL following Western
blotting. In assay B, CIII376.2, a mAb against the C-terminal peptide
of
, was coupled to Sepharose. 1
10
cells were lysed in Buffer A or 50 mM Tris, pH 7.5, 150
mM NaCl, 0.5 mM MgCl
, 1 mM CaCl
, and 50 mM
-octyl glucoside (Buffer
B). After a brief centrifugation, the supernatants were first
precleared with Sepharose 4B and then were incubated with
CIII376.2-Sepharose or Sepharose (control) overnight at 4 °C. After
washing, aliquots of the Sepharose were incubated with various
concentrations of biotinylated VCAM1-3 in Buffer A or Buffer B
containing 5 mg/ml myoglobin at 4 °C for 2 h. The Sepharose was
recovered by centrifugation and washed in the same buffer. Biotinylated
VCAM bound to the Sepharose was analyzed as described above. An aliquot
of the cells (1
10
) was surface-labeled with
biotin, and the cell lysate was immunoprecipitated with mAb CIII376.2
as a control for
surface expression.
Construction and Expression of Wild-type and Mutant
Asp-489, Asp-698, and Asp-811 of (Fig. 1) were substituted with glutamic acid by
site-directed mutagenesis. Stably transfected cell lines expressing
wild-type
(K-WT) and
containing
glutamic acid substitutions at the three aspartic acid residues
Asp-489, Asp-698, and Asp-811 (K-LEV) were established in K562. Five
clones from each transfectant were selected, and the clones showed
comparable levels of
surface expression by
immunofluorescence staining (data not shown). Clones K-WT.1 and K-LEV.5
were characterized in the present study. Surface expression of
integrin was analyzed by flow
cytometry using mAb C212.7. As shown in Fig. 2A,
surface expression on cell line K562 was not
detectable. Both K-WT and K-LEV showed similar levels of
surface expression (Fig. 2, E and I).
The data suggest that the mutations at the three aspartic acid residues
did not have a significant effect on the biosynthesis and surface
expression of
. As shown in Fig. 2D,
K562 expresses abundant
subunits. When transfected
with pcWT, the surface expression of
increased
significantly (Fig. 2H). The pcLEV transfectant also
exhibited elevated
expression (Fig. 2L).
by K562, K-WT, and K-LEV. Flow
cytometric analysis of cell lines K562 (A-D), K-WT (E-H), and K-LEV (I-L) were carried out
using the mAbs C212.7 (A, E, and I), HP2/1 (B, F, and J), B-5G10 (C, G, and K), and K20 (D, H, and L), followed by incubation with fluorescein-labeled goat
anti-mouse IgG. Samples treated with integrin antibodies are
represented as shaded profiles superimposed on the open
profiles representing control
staining.
Glu
triple substitutions cause significant changes in
secondary structure, the mutant
transfectant
was analyzed using different
mAbs for
immunofluorescent staining (Fig. 2). As shown in Fig. 2I, the epitope for
mAb C212.7
was retained on
LEV, indicating that the mutations do
not disrupt the structure of this epitope. Two other
mAbs recognizing different epitopes on
, HP2/1
and B-5G10, were also tested. Both antibodies recognized
LEV (Fig. 2, J and K). It has
been shown that mAb C212.7 and HP2/1 block
binding to
VCAM and
fibronectin
(17, 24, 25) . The
epitopes that mAb HP2/1 and B-5G10 recognize belong to previously
defined B1 and C epitope groups of
,
respectively(24) . The FACS analysis (Fig. 2) suggests
that the mutations at Asp-489, Asp-698, and Asp-811 do not affect the
structures of these epitopes, and, therefore, the mutations probably do
not cause any significant change in the global secondary structure of
.
on K-WT
or K-LEV transfectants was also analyzed by immunoprecipitation (Fig. 3). Both the
WT and
LEV
proteins show identical patterns: they are present on the cell surface
in a cleaved form (80 kDa and 70 kDa) and are associated with the
endogenous
subunits (120 kDa). The results indicate
that the Asp
Glu substitutions do not affect the in vivo cleavage of
,
heterodimer formation, or the stability of the complex.
surface expression on K-WT and K-LEV by immunoprecipitation.
K562, Jurkat, K-WT, and K-LEV were surface-labeled with biotin. Cell
extracts were incubated without (Control) or with various
antibodies as indicated. The lysates were then precipitated with
protein G-Sepharose. Proteins bound to G-Sepharose were analyzed by
SDS-PAGE and Western blotting.
Impaired Adhesion of K-LEV to VCAM and
Fibronectin
Adhesion of K-WT and K-LEV to immobilized
recombinant human VCAM1-3 was compared. As illustrated in Fig. 4A, K-WT bound to VCAM strongly, whereas K-LEV was
much less adherent to the same ligand. At ligand coating concentrations
of 2.5 to 5 µg/ml, adhesion by K-LEV was approximately
4-6-fold lower than adhesion by K-WT. Similar results were
obtained with adhesion to murine L-cell transfectants expressing the
7-domain form of human VCAM (data not shown)(20) . The control
cell line K562, showed little, if any, binding to recombinant VCAM,
indicating that the adhesion was dependent on transfection of
. As demonstrated in Fig. 4B, the
adhesion of K-WT or K-LEV transfectants to VCAM could be blocked
completely by mAb C212.7, which is directed against the
subunit of VLA-4. The adhesion could also be blocked by an
antibody against human VCAM (data not shown). This indicates that the
adhesion of
transfectants to immobilized VCAM is an
- and VCAM-dependent specific interaction.
) and K-LEV (
) were tested for adhesion to
microtiter plates coated with the indicated amounts of recombinant
VCAM1-3. ♦, K562 cells. B, K-WT and K-LEV were
tested for adhesion to microtiter plates coated with recombinant
VCAM1-3 (2.5 µg/ml) in the presence of PMA (10 ng/ml) and the
presence or absence of C212.7 Ab. Control wells were coated with bovine
serum albumin only.
antibody C212.7 triggered homotypic cell aggregation
of K-LEV (data not shown), suggesting that the mutant VLA-4 integrin is
also functional in outside-in cell signaling.
recognition (29) . Adhesion of K-WT to FN-40 required PMA stimulation.
K-LEV failed to adhere to FN-40 under these conditions (Fig. 5B).
Impaired Adhesion of K-D489E and K-D698E to VCAM and
Fibronectin
Mutations at all three aspartic acid residues of the
subunit resulted in decreased cell adhesion to
fibronectin and VCAM. To determine the contribution of each
substitution, mutant
constructs, pcD489E, pcD698E,
and pcD811E, each of which contains a glutamic acid substitution at
only one of the aspartic acid residues, were transfected into K562. The
pcWT construct was used as a control. Stable clonal transfectants
(K-WT, K-D489E, K-D698E, K-D811E) with similar
surface expression were obtained following repeated rounds of
cell sorting. K562, K-WT, K-D489E, K-D698E, and K-D811E were analyzed
for
surface expression (Fig. 6). As expected,
the mutant and wild-type transfectants expressed
at
comparable levels, and the
antibodies C212.7, HP2/1,
and B-5G10 recognized all three mutant
subunits (Fig. 6).
WT,
D489E,
D698E,
and
D811E. FACS analysis of cell lines K562 (A, F, and K), K-WT (B, G,
and L), K-D489E (C, H, and M), K-D698E (D, I, and N), and K-D811E (E, J, and O) was carried out using the mAb C212.7 (A-E), HP2/1 (F-J), and B-5G10 (K-O), followed by incubation with fluorescein-labeled
goat anti-mouse IgG. Fluorescence histograms of anti-
mAb-treated samples are represented as shaded profiles superimposed on the open profiles of control
samples.
transfectants K-D489E,
K-D698E, and K-D811E were compared with K-WT in adhesion assays for
their abilities to bind VCAM and fibronectin. Transfectant/VCAM
adhesion is shown in Fig. 7A. The percentage of K-D811E
cells adhering to VCAM is similar to that of K-WT. However, the binding
of K-D489E and K-D698E transfectants to VCAM was significantly lower
than that of K-WT. Their adhesion to VCAM (at 3.5 µg/ml) was only
10-20% of that of K-WT. Even at a higher concentration of VCAM (7
µg/ml), K-D489E and K-D698E adhered poorly. These results suggest
that aspartate 811 in
is not essential for VCAM
binding. In contrast, Asp-698 and Asp-489 are required for
VLA-4-mediated cell adhesion.
), K-D489E
(▴), K-D698E (▪), and K-D811E (
). A,
transfectants were tested for adhesion to microtiter plates coated with
the indicated amounts of recombinant VCAM1-3. B,
transfectants were tested for adhesion to microtiter plates coated with
the indicated amounts of FN-40 in the presence of
PMA.
for VCAM and fibronectin are identical or
overlapping(24, 25, 30) . To examine the
effect of the Asp
Glu substitutions on fibronectin binding, the
abilities of K-D489E, K-D698E, and K-D811E to adhere to FN-40 were
tested. As shown in Fig. 7B, K-WT adhered to FN-40 upon
PMA activation. Adhesion of K-D811E was comparable to that of K-WT.
However, K-D489E and K-D698E failed to adhere to FN-40 under the same
conditions.
Binding of Wild-type and Mutant
The direct binding of ligand to wild-type and mutant
to
VCAM
isolated from K-WT, K-LEV, K-D489E, and K-D698E was
assessed using either surface-labeled cell extracts and VCAM-Sepharose
or biotinylated VCAM1-3 and affinity-purified
(Fig. 8). As indicated in Fig. 8A,
complexes
from both K-WT and K-LEV cells bound to VCAM-Sepharose in a
cation-dependent manner. The amount of mutant
bound
to VCAM-Sepharose was comparable to that of wild-type. In other
experiments, wild-type or mutant
was
immobilized on CIII376.2-Sepharose and incubated with various
concentrations of biotinylated VCAM1-3. At a VCAM concentration
of 0.06 µg/ml, VCAM binding to either wild-type or mutant
was not detectable (data not shown).
As shown in Fig. 8B, at VCAM concentrations of 0.3 and
above, the mutant
from K-LEV,
K-D489E, and K-D698E was able to bind VCAM even at low concentrations
of cations, and the amounts of VCAM bound to the mutant forms of
were similar to that bound to the
from K-WT. These results suggest
that substitutions at Asp-489 and Asp-698 are not critical for VCAM
recognition.
. A, extracts
of biotin-surface-labeled K-WT and K-LEV cells were precipitated with
VCAM-Sepharose. Proteins bound to VCAM-Sepharose (P1) were
eluted from VCAM-Sepharose in Buffer A containing 20 mM EDTA
and immunoprecipitated with mAb CIII376.2 (P2). Both P1 and P2
were analyzed on 4-12% SDS-polyacrylamide gels. Arrows indicate the 80-kDa and 70-kDa forms of
. B,
in K-WT, K-LEV, K-D489E, and K-D698E
cells was immunoprecipitated with CIII376.2-Sepharose 4B (VLA-4) or
Sepharose (control). 0.3 µg/ml biotinylated VCAM1-3 was
incubated with VLA-4 immobilized on CIII376.2-Sepharose. Biotinylated
VCAM bound was analyzed on 4-20% Tricine SDS-polyacrylamide
gels.
Asp-489 and Asp-698 Flanking Sequences
To
understand the mechanism of inhibition of cell adhesion mediated by
with D489E and D698E substitutions, we compared the
flanking sequences at these sites with corresponding sequences in other
integrins. Comparison of the Asp-489 flanking sequences with
corresponding sequences in other
chains showed that this aspartic
acid is conserved among most integrin
chains. Therefore, it seems
unlikely that this residue plays an important role in discriminating
between different ligands. It may be involved in a common integrin
structure required for cell adhesion. Analysis of the Asp-698 flanking
sequences showed that the DISFLLD
VSSLS sequence resembles
the DxxxxxD-S-Sx cation binding motif found in
subunits and the I-domain of other
integrins (Fig. 9).
to similar sequences in other human
and
integrins. The amino acid sequences are deduced from
the nucleotide sequence data obtained from the GenBank
data base, with the following accession numbers:
(X68742),
(M28249),
(X16983),
(D25303),
(L25851),
(Y00796),
(M18044),
(Y00093),
(X07979),
(M15395),
(M35999),
(X51841),
(M35011),
(M35198),
(M62880), and
(M73780). Alignment was done using the PILEUP program of the
University of Wisconsin Genetics Computer Group. The conserved amino
acid residues are in shaded boxes. The residues, which have
been shown to be required for integrin function, are shaded in
black.
-transfected cells to adhere to VCAM and fibronectin.
A third mutation at Asp-811 had no detectable effect on cell adhesion
to VCAM or fibronectin. However, direct binding experiments with
isolated
and VCAM suggest that Asp-489 and Asp-698
are not critical for ligand recognition. We found that the Asp-698
residue in
resides in a sequence resembling the
cation/ligand binding DxxxxD-S-Sx motif found in
integrins and the I-domain of
integrins, implicating
Asp-698 in cation-ligand interactions. Therefore, Asp-489 and Asp-698
may regulate cell adhesion through a novel and/or cation-dependent
mechanism.
, transfected
cells with mutated
s were analyzed for disruption of
anti-
epitopes, loss of
/
chain association or
cleavage, and impairment
of signal transduction. As shown in Fig. 2and Fig. 6,
LEV,
D489E,
D698E,
and
D811E were all recognized by a panel of mAbs
against various distinct epitopes of
. The glutamic
acid substitutions at Asp-489, Asp-698, and Asp-811 of
also did not affect the ability of
to associate
with the
subunit or the in vivo cleavage of
(Fig. 3). Therefore, the decreased cell
adhesion by K-D489E and K-D698E is unlikely to be caused by disruption
of
secondary structure.
molecules also maintained the flexibility required for undergoing
certain conformational changes during signaling. Integrins mediate
outside-in and inside-out cell signaling. VLA-4-mediated cell adhesion
can be rapidly and dramatically augmented by phorbol
esters(26) . PMA activation (inside-out signaling) does not
change the level of VLA-4 expression, and it is not dependent on de
novo protein synthesis(26, 27) . We have shown
that the adhesion of mutant
transfectants to ligand
increases in response to PMA treatment (Fig. 5A). A
number of anti-
antibodies, when binding to
integrin on the cell surface, cause
homotypic cell adhesion (outside-in signaling, (31) ).
Anti-
antibody also triggered homotypic aggregation of
the mutant
transfectants (data not shown). These
observations are consistent with the view that the Asp
Glu
substitutions did not change the global secondary structure of
.
to recognize ligand,
direct ligand binding studies with
isolated from
K-LEV, K-D489E, and K-D698E and purified VCAM were carried out (Fig. 8). The ability of
Asp-489 or
Asp-698 to bind VCAM argues that Asp-489 and Asp-698
are not critical for ligand recognition. Therefore, the D489E and D698E
substitutions may affect adhesion and the function of the
integrin in vivo by another
mechanism.
-helix/turn/
-sheet structure, whereas Asp-811 is in the
middle of a
-sheet according to the Chou-Fasman
prediction(32) . The turn regions where Asp-489 and Asp-698
reside have fairly high surface probability and flexibility, whereas
Asp-811 has low surface probability. This suggests that Asp-489 and
Asp-698 may be exposed on
and have the ability to
interact with other proteins.
function. Mutations at each
of the three divalent cation binding sites resulted in significant
inhibition of
-mediated cell adhesion to VCAM and
fibronectin. It has been reported recently that certain aspartic acid
residues in the I-domains of
and
are required for cell adhesion(18, 19) . In both
cases, the essential aspartic acid residue resides in the
DxxxxxD-S-Sx sequence (Fig. 9) as proposed by
Bajt and Loftus(14) . Furthermore, Michishita et al. (19) have also demonstrated that the same aspartic acid residue
in
is required for cation binding. In
and
subunits, the aspartic acid residues in
this motif have also been proven to be critical for ligand
binding(37, 38) . A point mutation in the
subunit of the
integrin has
been identified in the Cam variant of Glanzmann's
thrombasthenia(33) . The substitution of the aspartic acid
residue within this motif in
to tyrosine resulted in
a perturbed interaction with divalent cations and the inability of
to recognize ligand. We have
compared the Asp-698 flanking sequence with those of relevant sequences
from different
and
subunits (Fig. 9). The sequence
around Asp-698 of
resembles the divalent cation
binding motif mentioned above and thus implicates Asp-698 in divalent
cation binding. Our finding that Asp-698 is critical for the
interaction of
-transfected cells with VCAM and
fibronectin supports this hypothesis. Therefore, Asp-698 may be part of
a novel cation binding motif in
, which is required
for
function.
, critical for
-mediated cell adhesion. We found
that the sequences flanking Asp-698 resemble the cation binding motif
DxxxxxD-S-Sx, recently identified in the ligand
binding domain (I-domain) of
integrins. Since
does not contain an I-domain, this raises the possibility that
the region of
containing the
DxxxxxD-S-Sx motif is functionally similar to that of
the I-domain. The DxxxxxD-S-Sx motif in the I-domain
of
integrins appears not to be required for ligand recognition as
has been shown in studies with
(34) and
(39) . Our present findings are consistent
with these results. Asp-689 may affect cell adhesion by increasing
ligand accessibility or maintaining a binding pocket with high ligand
affinity through binding of divalent cation. Alternatively, given that
cell adhesion is a complex process, one or both aspartate residues
(Asp-489, Asp-698) may be critical for efficient cell adhesion through
an interaction with an as yet unknown cofactor.
We would like to thank Maura Macaro for flow
cytometric analysis and cell sorting.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.