(Received for publication, December 19, 1996, and in revised form, March 5, 1997)
From the Program on Cell Adhesion and the Extracellular Matrix, The amino-terminal domain of each integrin Integrins are All integrins require divalent cations to bind their ligands. An
important clue to the structural basis for ion binding was revealed by
the crystal structures of the I domains from the integrin Interestingly, the DXSXS sequence is also present
in the integrin To examine the hypothesized ion binding site within Amino acid
sequences of The structures of The Human embryonic kidney 293 cells were obtained from ATCC and maintained in Dulbecco's modified
Eagle's medium (BioWhittaker) supplemented with 10% fetal calf serum
(Irvine Scientific). Mutated constructs of human The anti- Fab-9
is an RGD-containing, synthetically engineered antibody that has been
optimized through phage display to bind to To measure the apparent affinity of integrin for Mg2+,
Fab-9 binding was measured as a function of Mg2+
concentration. The apparent affinity for cation was taken as the
concentration of cation that supported half-maximal ligand binding.
Ligand binding to cells expressing To measure the apparent affinity of integrin for divalent cation, the
binding of 125I-Vn was measured across a range of metal ion
concentration. The apparent affinity for cation was determined as the
concentration of ion at which half-maximal ligand binding was
observed.
Cell adhesion assays were performed as
described previously (23). Briefly, Fab-9 (50 nM) or
vitronectin (6 nM) were coated on 96-well plates by an
overnight incubation at 4 °C. The plates were then blocked with 1%
bovine serum albumin. Cells (1 × 105 cells/well) were
harvested with EDTA/PBS and resuspended in Binding Buffer containing
the appropriate cations as described for soluble ligand binding. Cells
were allowed to adhere at 37 °C for 45-60 min. Non-adherent cells
were removed by gentle washing, and adherent cells were detected by a
colorimetric assay for lysosomal acid phosphatase (24). Color
absorbance was detected at A405 nm.
The binding of mAb
AP5 to As a first step toward characterizing the putative metal
binding site in To examine further the homology between the I domain and the integrin
As a second step in assessing the
similarity between the metal binding site in the
To probe the structure of the ion binding site within each
The ligand binding function of each mutated form of
Table I.
The apparent affinities of
subunit is hypothesized to contain an ion binding site that is key to
cell adhesion. A new hypothesis regarding the structure of this site is
suggested by the crystallization of the I domains of the integrin
L and
M subunits (Lee, J.-O., Rieu,
P., Arnaout, M. A., and Liddington, R. (1995) Cell 80, 631-638; Qu, A., and Leahy, D. J. (1995) Proc. Natl. Acad. Sci.
U. S. A. 92, 10277-10281). In those proteins, an essential
metal ion is bound by a metal ion-dependent adhesion site
(MIDAS). The MIDAS is presented at the apex of a larger protein module
called an I domain. The metal ligands in the MIDAS can be separated
into three distantly spaced clusters of oxygenated residues. These
three coordination sites also appear to exist in the integrin
3 and
5 subunits. Here, we examined the putative metal binding site within
3 and
5 using site-directed mutagenesis and ligand binding
studies. We also investigated the fold of the domain containing the
putative metal binding site using the PHD structural algorithm. The
results of the study point to the similarity between the integrin
subunits and the MIDAS motif at two of three key coordination points.
Importantly though, the study failed to identify a residue in either
subunit that corresponds to the second metal coordination group in
the MIDAS. Moreover, structural algorithms indicate that the fold of
the
subunits is considerably different than the I domains. Thus,
the integrin
subunits appear to present a MIDAS-like motif in the
context of a protein module that is structurally distinct from known I
domains.
heterodimers that mediate cell adhesion (1,
2). Integrins participate in development and tissue remodeling and are
linked to several diseases. The integrins bind to many adhesive and
extracellular matrix proteins. The focal points of this study are the
v
3 and
v
5 integrins, both of which recognize the
Arg-Gly-Asp (RGD)1 tripeptide motif. The
v
3
integrin binds to at least nine adhesive proteins and
has two important biological functions. First,
v
3 mediates the
adhesion of osteoclasts to the bone surface (3), an event often
considered to be the first step in bone resorption (4). Second, the
v
3 integrin is expressed on the surface of angiogenic endothelial
cells, where it is required for cell survival and further vessel
development (5-7). It has been suggested that inhibitors of the
v
3 integrin could be applied as antagonists of osteoporosis and
tumor angiogenesis. The biological function of the
v
5 integrin is
less clear. This integrin can mediate cell adhesion to vitronectin. The
v
5 integrin is also required for the internalization of
adenovirus (8, 9), and it may be associated with angiogenesis (7).
L and
M subunits (10, 11). Each I domain
spans approximately 200 residues and is homologous to an "inserted"
domain in a number of other proteins including von Willebrand factor
(12). In
L and
M, the I domain is
necessary and sufficient for ligand contact. These I domains contain a
metal binding site called a MIDAS (metal ion-dependent
adhesion site). This ion binding site consists of five liganding
residues that can be separated into three groups. Each group of
coordinating residues is located at separate positions within the
primary amino acid sequence (10, 11). The first coordination group
consist of the DXSXS sequence, where D is
aspartate, X is any amino acid, and S is serine. The
aspartate and both serines coordinate with metal ion. The second group,
or coordination point, is a single threonine located 69 amino acids
from the DXSXS. The third group is comprised of a
single aspartate 102 residues from the DXSXS.
subunits (13), suggesting that they may also
contain the MIDAS (10). If correct, this would provide a common
structural basis for the regulation of all integrins by divalent metal
ions. It would also imply that all integrins are regulated in a similar manner by metal ion. Despite this hypothesized similarity, integrins behave differently with respect to metal ions. For example, we recently
demonstrated that the type of divalent ion present in the culture media
regulates the way that the
v
3 and
v
5 integrins are
organized on the cell surface (14). In fact, the same ion can direct
the two integrins to completely different locations on the cell. This
distinction suggests that the metal binding site within the
subunits is likely to have subtle but important structural differences
that have an impact on receptor function.
3 and
5, the
putative metal coordinating residues within each subunit were mutated
to alanine. Results presented here are the first to show that Asp-119
and Asp-217 within
3 are important for the binding of soluble ligand
to
v
3. The homologous aspartic acids within
5 are also key to
soluble ligand binding. Interestingly though, mutations at these
aspartic acids do not completely abrogate cell adhesion. Their mutation
decreases cell adhesion and reduces the apparent affinity of the
integrin for metal ion. This finding indicates that each aspartic acid
is likely to be part of a metal ion binding site that controls ligand
contact. The similarity in spacing and function of these aspartates to
the metal ligands in
L and
M indicate a
similarity between the integrin
subunits and the MIDAS motif found
in the I domains. However, evidence is presented here which argues that
the fold of the
subunits is distinct from that of the I domains.
The PHD structural algorithm predicts that the
3 and
5 subunits
have little structural similarity with the I domains.
Sequence Alignment and Structural Predictions
3 (residues 107-292) and
5 (residues 109-296) were
each aligned with the I domain sequences of
L (residues
125-310) and
M (residues 128-318) using the multiple sequence alignment program, ClustalW (15). Without further
manipulation, the putative metal ligands were identified in each
subunit by comparison to the known metal coordination sites within the
MIDAS motifs of
L and
M.
3,
5, and the I domains of
L and
M were analyzed using the PHD algorithm (16, 17). The
algorithm compares the environment of a single residue within a data
base of known crystal and NMR structures and then assigns the
probability that a residue is in a helix or a sheet. The algorithm also
assigns a reliability index from 0 to 9 to assist in distinguishing
residues which could be present in either structure. In this analysis, structural predictions for individual residues were only accepted when
the reliability index was greater than five. The amino acid sequences
encompassing residues 107-292 in
3, residues 109-296 in
5,
residues 125-310 in
L, and residues 128-318 in
M were subjected to this analysis. Individual structural
elements in a predicted protein were considered to be a match (compared
with elements in the known crystal structure of
L or
M) only if at least 50% of the residues in the element
were predicted to be in the correct position and of the correct
structure (helix or sheet).
3 and
5 cDNAs were cloned using
the polymerase chain reaction. The
3 or
5 cDNA in the
Bluescript II SK(+) plasmid (Stratagene) was used as the parental
plasmid for mutagenesis. Point mutations were introduced using the
Chameleon Double-stranded, Site-directed Mutagenesis Kit (Stratagene).
5
- Phosphorylated oligonucleotide primers were produced by Integrated
DNA Technologies. The oligonucleotide sequences used to produce each of
the
3 mutants are as follows: D119A,
TACTACTTGATGGCCCTGTTTAC; T182A, ATGATATGAAGGCCACCTCCTTGCC; T183A,
ATGATATGAAGACCGCCTCCTTGCC; D217A,
CACGGAACCGAGCTGCCCCAGAGGG; E220A,
GAGATGCCCCAGCGGGTGGCTTTG. The oligonucleotide sequences used
to introduce each of the
5 mutations are as follows: D121A,
CTGTACTACCTGATGGCCCTCTCCCTGTCC; N186A,
GTTGTTTCCAGCTTGCGTCCCCT; S190A,
GCGTCCCCGCCTTTGGGTTCCGCC; D220A,
CGGAACCGAGCTGCCCCTGAGGGG; E223A,
CCTGCGGGGGGCTTTGATGCAGTA. The point mutations are
indicated in bold lettering. All mutations were confirmed by dideoxy
sequencing, and the cDNAs were subsequently subcloned into the
mammalian expression vector pcDNA3 (Invitrogen).
3 or
5 cDNA
in plasmid pcDNA3 were transfected into 293 cells using DOTAP
transfection reagent (Boehringer Mannheim). Following selection in 500 µg/ml geneticin (Life Technologies, Inc.) for approximately 3 weeks,
the top 5% of the positive fluorescent population of cells was
obtained by sterile FACS using either the anti-
v
3 monoclonal
antibody (mAb) LM609 or the anti-
v
5 mAb P1F6. Cells expanded from
the sorted population were continuously monitored for high expression
of transfected integrin throughout the duration of the study. FACS
analysis was performed using standard protocols. Cells were incubated
with mouse primary antibody against
v
3 or
v
5 (5 µg/ml),
washed, and then incubated with fluorescein isothiocyanate
(FITC)-conjugated goat anti-mouse secondary antibody (Caltag). All
cells used for binding studies exhibited stable integration of the
subunit cDNA into the genome.
v
3 mAb
LM609 was purchased from Chemicon. Nonspecific mouse IgG was obtained
from Calbiochem. Monoclonal antibody P1F6 (anti-
v
5) was purchased
from Becton Dickinson. Synthetic peptides with sequences GRGDSP and
SPGDRG were obtained from Coast Scientific.
v
3 Expressed on 293 Cells
3-integrins (18, 19).
Fab-9 has at least a thousand-fold lower affinity for
v
5 (18) and
does not bind specifically to 293 cells transfected with
5. 293 cells expressing the
3 mutants were harvested from tissue culture
flasks with 0.2 mM EDTA in phosphate-buffered saline (EDTA/PBS) and washed with cold Binding Buffer (Hanks' balanced salt
solution lacking MgCl2, CaCl2, and
MnCl2 (Life Technologies, Inc.), 50 mM HEPES,
pH 7.4, 3 mg/ml bovine serum albumin) supplemented with 0.5 mM MgCl2 and 0.05 mM
MnCl2. These cation conditions were optimized for Fab-9
binding. Fab-9 was labeled with Na125I (Amersham Corp.)
using IODO-GEN (Pierce) to approximately 40,000 cpm/ng. Cells (3 × 106/ml) were incubated with increasing concentrations of
125I-Fab-9 at 14 °C for 70 min. Nonspecific binding was
measured by including 20 mM EDTA in the incubations (18),
although prior study in this lab has shown that competition with an
excess of unlabeled Fab-9 yields nearly identical values. Following
incubation, free 125I-Fab-9 was separated from cell-bound
ligand by centrifugation through 20% sucrose, 50 mM
Tris-buffered saline, pH 7.4, at 14,000 rpm for 3 min in disposable
microcentrifuge tubes (Fisher). The bottoms of the tubes were cut off
and counted in a gamma counter. All data represent the average of
triplicate measurements. All assays were repeated at least twice
yielding identical results. The affinity of
v
3 for Fab-9 was
calculated by Scatchard analysis (20).
v
5
v
5 and mutant
forms of
5 was measured with 125I-vitronectin (Vn) using
a tracer format (21). For that purpose, vitronectin was purified from
human serum as described previously (22) and labeled to high specific
activity (150,000 cpm/ng) with Na125I. Cells expressing the
mutants of
5 were harvested with EDTA/PBS and washed with Binding
Buffer containing 0.5 mM MgCl2 and 0.02 mM CaCl2. Cells (107/ml) were
incubated with 0.5 nM 125I-Vn and increasing
concentrations of unlabeled Vn in cation-supplemented Binding Buffer at
14 °C for 70 min. Specific binding was determined by subtracting the
EDTA-sensitive binding from total binding. The affinity of
v
5 for
vitronectin was derived using Scatchard analysis (20).
v
3 on 293 cells was measured as described previously (25).
Briefly, cells were incubated with 50 µg/ml FITC-conjugated AP5 in
the presence of varying concentrations of Ca2+ and analyzed
by FACS. Mean fluorescence intensity was determined per 10,000 cells.
Sequence and Structural Comparison between the I Domain of
M and the Amino-terminal Domain of
3 and
5
3 and
5, their sequences were compared with the I
domain of
M. The sequence of
3 (residues 107-292)
was aligned with the I domain of
M using the multiple
sequence alignment program ClustalW (15). The sequence of
5 was
incorporated into this alignment using the extensive identity between
3 and
5. The DXSXS motif and an aspartic
acid residue representing the third coordination group in the MIDAS
align well in all three proteins. The
subunits diverge from the
MIDAS motif at the middle metal coordinating position (Thr-209 in
M). The
subunits contain a small disulfide-bonded
loop in this region, a structure absent in the I domains. Within this
disulfide-bonded loop,
3 contains two threonine residues (Thr-182
and Thr-183) that could be homologues of the coordinating threonine in
M. Within the same disulfide-bonded loop of
5,
Asn-186 and Ser-190 could potentially ligand with metal ion.
3 and
5 subunits, structural predictions were made using the PHD
algorithm under strict conditions (16, 17). As shown in Fig.
1, the algorithm correctly predicted 11 of 13 structural
elements in
M and 10 of 13 elements in the I domain of
L (not shown). These findings validate the fidelity of
the algorithm. The algorithm predicted that
3 contains only 3 of the
13 structural elements in the I domains and that
5 may contain up to
2 of the I domain elements. Consequently, the overall fold of this
domain in the
subunits is likely to be significantly different than
that of the known I domains.
Fig. 1.
Sequence alignment and structural prediction
of the M I domain with
3 and
5. The I domains
of
L and
M and the corresponding regions
in
3 (residues 107-292) and in
5 residues (109-295) were
aligned using the program ClustalW (15). The positioning of the MIDAS
residues in
L and
M are identical, so
only
M is shown. The amino acid residues that coordinate
metal ion in
M and the predicted coordinating residues
in
3 and
5 are boxed. These same regions were
subjected to structural prediction using the PHD algorithm (16, 17).
The predicted structures were compared with the actual structure of
M as determined by crystallographic data (10).
strands are represented as hatched rectangles;
helixes
are shown as shaded rectangles, and turns/random coils are
left open. Secondary structural elements according to the
M crystal structure are labeled as
sheet strands
A-F and
helixes 1-7 (10). Crystal structure
is abbreviated as X and predicted structure as P.
The position and length of each element in the figure is shown to
scale.
[View Larger Version of this Image (64K GIF file)]
v
3 and
v
5 Is Similar to the
I Domain of
M
subunits and the
MIDAS, the ion preference of
v
3 and
v
5 was compared with
that of
M (26). The I domain of
M will
bind ligand in a series of metals, although Ca2+ and
Ba2+ are largely ineffective in supporting binding.
Therefore, we tested the same panel of metals for the ability to
support ligand binding to
v
3 and
v
5. This analysis was done
by measuring the binding of ligand to each
v-integrin as a function
of the type of divalent metal ion present in the binding buffer. All metals were tested at a concentration of 1 mM to be
consistent with the prior study of the I domain of
M
(26). As shown in Fig. 2, most metal ions tested
support the binding of vitronectin to
v
5. However,
Ca2+ and Ba2+ supported only minimal binding to
v
5. A similar metal preference was observed for the binding of
ligand to
v
3 (not shown). The only significant difference between
the two
v-integrins was the inability of Cd2+ to support
ligand binding to
v
3 (not shown). These data show that ligand
binding to
v
3,
v
5, and the I domain is supported by similar
metals.
Fig. 2.
Integrin v
5 demonstrates a cation
preference similar to an I domain. A panel of metal ions was
tested for the ability to support 125I-vitronectin binding
to
v
5 expressed on kidney 293 cells. Each ion was included at a concentration of 1 mM. Binding is
expressed as a percentage of maximum specific binding which was
achieved in the presence of Co2+. The data represent the
average of two experiments that yielded nearly identical results.
[View Larger Version of this Image (22K GIF file)]
3 and
5 in 293 Cells
subunit, we mutated putative metal liganding residues to alanine. In
3 these are Asp-119, which represents the
DXSXS sequence; Thr-182 and Thr-183, which are
hypothesized to make up the second coordination group; and Asp-217,
which is thought to comprise the third coordinating group. Within
5
the putative coordinating residues are Asp-121, Asn-186, Ser-190, and
Asp-220. Because of their proximity to the last putative coordination
residue, we also mutated Glu-220 within
3 and Glu-223 within
5.
Each cDNA construct was used to transfect 293 cells. Following
antibiotic selection and FACS sorting, transfected cells were found to
express nearly equivalent levels of each of the mutated integrins on
the cell surface (Fig. 3). One mutant,
3 E220A, was
not expressed on the cell surface, even though repeated attempts were
made to transfect this mutant. To confirm proper heterodimer formation, the mutant integrins were immunoprecipitated from lysates of
125I-labeled cells using antibodies against
v
3
(LM609) and
v
5 (P1F6). Each antibody immunoprecipitated
v and the relevant
subunit in an approximate 1:1
stoichiometry, confirming that the mutated subunits complex with
v (data not shown).
Fig. 3.
3 and
5 mutants are expressed on the
cell surface with
v. Human kidney 293 cells were transfected
with
5 (top row) or
3 (bottom row) cDNA
containing point mutations at putative metal ion-coordinating residues
(indicated in figure). Cells were maintained in antibiotic selection
and FACS-sorted to obtain a population that expressed high levels of
integrin. Here, FACS was used to analyze the expression level of the
v
3 or
v
5 heterodimer on each sorted cell population. Cells
were incubated with a nonspecific mouse IgG (clear peak) or
an antibody (shaded peak) that binds to the
v
3 (mAb
LM609) or
v
5 (mAb P1F6) complex. The binding of primary antibody
was detected with secondary antibody conjugated to FITC. Kidney 293 cells mock-transfected with the pcDNA3 expression vector did not
exhibit any shift in fluorescence (not shown).
[View Larger Version of this Image (20K GIF file)]
3
v
3
was measured using the model ligand Fab-9 which has been characterized previously (18, 19, 27). This ligand was chosen because the binding
affinity of soluble vitronectin for
v
3 on
293 cells was too low to yield reproducible binding data. In these
binding studies, the metal concentration was set to an optimal level. Wild-type
v
3 bound to
125I-Fab-9 with an affinity of 9 ± 3 nM
(n = 10). Within the detectable range of binding, the
mutation of
3 residues D119A and D217A abolished binding of soluble
Fab-9 to the cell surface (Fig. 4). Surprisingly, cells
expressing the T182A and T183A mutations bound to
125I-Fab-9 with an affinity identical to that of wild-type
v
3. To determine whether the mutations T182A and T183A had a more subtle effect on cation-dependent ligand binding, we
measured their apparent affinities for Mg2+. The apparent
affinities of T182A or T183A for Mg2+, as reported by Fab-9
binding, were identical to wild-type
v
3 (Table I).
Thus, unlike the corresponding threonines within
L and
M, neither of the candidate threonines within
3
appear to be crucial metal ligands.
Fig. 4.
Mutation of acidic residues in 3
eliminates binding of soluble ligand. Human 293 cells expressing
mutant
v
3 were incubated in solution with 125I-Fab-9
at 14 °C. Specific binding (
) was calculated as the difference between total (
) and nonspecific binding (
), which was measured in the presence of EDTA. In nearly identical experiments we found the
level of nonspecific binding to be the same when excess Fab-9 was used
as a competitor. Each point is the average of triplicate data points.
Scatchard plots (bound (B) versus bound/free
(B/F)) and calculated affinities (Kd) are
shown in the insets. The correlation coefficients of each
Scatchard plot are as follows: wild-type (WT),
r2 = 0.97; T182A, r2 = 0.92; and T183A, r2 = 0.85. No specific binding
was detected for D119A and D217A. Each figure is
representative of at least two experiments in which identical results
were obtained. Ligand binding to cells expressing mutant
v
3 was
always done in parallel with cells expressing WT
v
3.
[View Larger Version of this Image (29K GIF file)]
3 mutants T182A and T183A for
magnesium (Mg2+)
v
3 or
mutants T182A and T183A were allowed to bind 125I-Fab-9 as a
function of increasing Mg2+ concentrations. The apparent
affinity of integrin for Mg2+ was determined as the
concentration of Mg2+ at which half-maximal ligand binding
occurred.
Human 293 cells expressing wild-type (WT)
v
3 or
mutants T182A and T183A were allowed to bind 125I-Fab-9 as a
function of increasing Mg2+ concentrations. The apparent
affinity of integrin for Mg2+ was determined as the
concentration of Mg2+ at which half-maximal ligand binding
occurred.
Cell line
Apparent affinity for
Mg2+
n
nM
WT
0.5
4
T182A
0.5,
0.7
2
T183A
0.5, 0.7
2
Cell adhesion is a multimeric interaction between clustered integrins
and a non-diffusable matrix. Therefore, it can often proceed even when
the affinity between integrin and ligand is very low. Consequently, we
measured the effect of mutations within the 3 subunits on cell
adhesion to immobilized Fab-9. Surprisingly, cells expressing
3
mutated at Asp-119 and Asp-217 adhered to Fab-9 (Fig.
5), even though they failed to bind soluble ligand. The
mutated forms of
3 supported adhesion to a level that usually reached approximately 40% that of wild-type
3. More importantly, both mutations also exhibited a shift in the apparent affinity for
metal ion. This was measured by determining the level of metal ion that
supported half-maximal adhesion. The study was done with Mn2+ because it has the highest affinity for the integrin.
The D119A mutation exhibited an apparent affinity for ion that was
approximately 6-10-fold lower than that of wild-type
3. The
mutation at Asp-217 was even more deleterious, exhibiting an apparent
affinity for metal that was 15-20-fold lower than wild-type
3.
These are the first data to demonstrate that mutations at putative
metal-coordinating residues within an integrin
subunit shift the
ion response curve. This can be interpreted to indicate that Asp-119
and Asp-217 contribute to metal binding affinity.
Assessing the Ligand Binding Function of Mutant Forms of the
The binding of vitronectin to wild-type v
5 on 293 cells was initially characterized in conditions containing 500 µM Mg2+ and 20 µM
Ca2+. Under these cation concentrations, the binding of
vitronectin was specific and saturable with a Kd of
9 nM. Vitronectin binding to cells expressing wild-type
v
5 could be completely inhibited with function-blocking mAb P1F6
or GRGDSP peptide (data not shown). Each mutant of
v
5 was evaluated for its ability to bind
soluble vitronectin. The binding of vitronectin was assayed as a
function of the concentrations of Mg2+ or Mn2+
(Fig. 6). The titration of Mn2+ was carried
out to only 5 mM because artifactual binding of vitronectin was detected above this concentration. In this experiment, the data are
expressed as a percentage of maximal binding to wild-type
v
5
which was always measured in parallel. Cells expressing alanine mutations at Asp-121 and Asp-220 failed to bind to soluble Vn in either
Mg2+ or Mn2+. In the radioligand binding assay
that was employed, we were only able to detect vitronectin binding to
integrin when the Kd was below 500 nM. Since
wild-type
v
5 has a Kd of 9 nM for
soluble vitronectin, we conclude that mutations at Asp-121 and Asp-220
cause at least a 55-fold reduction in the affinity of the integrin for
vitronectin. These data are consistent with the role of each aspartate
in metal coordination and with nearly identical data obtained for
3
(see above). The mutation of Asn-186 and Ser-190 to alanine had no
effect on ligand binding. Thus, we were unable to identify a residue in
5 that corresponds to the metal coordinating threonine (Group 2) in
L and
M. It is also interesting to note
that the mutation of Glu-223 to alanine eliminated the ability of
v
5 to bind soluble vitronectin. Although this residue is not
homologous to any of the metal ligands in the MIDAS, our data indicate
that it has a role in ligand binding function. It may, in fact,
substitute for the missing second metal ligand (see
"Discussion").
The ability of each mutant form of v
5 to mediate adhesion to
immobilized vitronectin was also measured (Table II).
The substitution of alanine at Asp-121 and Glu-223 of
5 resulted in
complete abrogation of cell adhesion, whereas alanine substitutions at
5 residues Asn-186 and Ser-190 had no effect on the ability of the
cells to adhere to vitronectin. In contrast,
5 containing D220A
mediated cell adhesion, although the absolute level of adhesion at
saturation was lower than wild-type
v
5. The apparent affinity of
this mutant form of
v
5 for metal ion was 5-50-fold lower than
that exhibited by wild-type
v
5 (45-62 versus 1-10
µM). This observation is consistent with a role for
Asp-220 in coordinating metal ion and is also consistent with the fact
that the homologous residue in
3 (Asp-217) contributes to metal
binding affinity.
|
Integrins contain two classes of ion binding sites, one that
promotes ligand binding, called a Ligand Competent site, and another
that inhibits ligand binding, called an Inhibitory site (27-30). The
monoclonal antibody AP5 binds to the amino-terminal domain of the 3
subunit and reports the occupation of the Inhibitory Ca2+-binding site (25). As an extension of the present
study, we measured the effect of each point mutation within
3 on the
sensitivity of the binding of the AP5 antibody to Ca2+. As
shown in Fig. 7, the binding of AP5 to wild-type
v
3 and to both
3 D119A and
3 D217A was blocked by
Ca2+. Thus, Asp-119 and Asp-217 are not part of the
Inhibitory ion binding site.
The primary objectives of this study were to examine the
possibility that the amino-terminal portion of the integrin subunit contains a MIDAS-like metal binding site and to assess whether this
motif in the integrin
subunit is positioned at the apex of an I
domain structure. The simplest step in this analysis involved a
comparison of the two structures. The I domains and the amino-terminal portion of the integrin
subunits have similar hydropathy profiles (10) and also exhibit some sequence homology, particularly at residues
known to ligand with metal. Both observations suggest the potential for
a common fold. Here, we examined this possibility in more detail using
the PHD algorithm, which generates a predicted structure based on the
propensity of individual residues within a given local environment to
exist in a helix, a sheet, or a disordered loop. Importantly, the
algorithm relies on known crystal and NMR structures to predict
tertiary structure from the primary sequence. It is reported to have a
success rate of approximately 70% (17). The PHD algorithm correctly
predicted 10 of 13 structural elements within the I domains of
L and
M, attesting to its ability to identify the major elements within an I domain. In contrast, the algorithm predicted that only 2-3 of the 13 I domain elements are
present in the corresponding positions of
3 and
5. Although the
subunits appear to have some sequence similarity with the I
domains, an in-depth analysis using a sophisticated algorithm suggests
that the three-dimensional structure of the integrin
subunits is
likely to be significantly different from that of the I domains. Based
on this analysis it does not appear that the integrin
subunits
contain an I domain-like region. This does not exclude the possibility
that a metal-binding MIDAS motif could be presented in the context of a
different backbone structure.
Therefore, a series of biochemical studies were performed to further
assess metal and ligand binding to the v
3 and
v
5 integrins.
As a first step, the ion specificity of the ligand binding event was
tested. Indeed, both integrins have an ion preference that is
remarkably similar to that reported for the I domain of
M (26). Although some minor differences exist between
v
3 and
v
5, transition state metal ions like
Co2+ and Mn2+, as well as the cation
Mg2+, support ligand binding. Divalent ions like
Ca2+ and Ba2+ were far less effective. Although
we know the regulation of ligand binding to
v-integrins to be
complex and that it can involve regulation by two classes of ion
binding sites (27, 28, 30, 32), this simple test shows that the ligand
binding event for
v-integrins has an ion specificity that is more
similar to that of an I domain (26) than to an EF-hand (33).
A more detailed analysis of metal binding involved the mutation of the
putative metal coordinating residues within 3 and
5. This
approach identified two distantly spaced aspartic acid residues that
greatly influence receptor function. These are Asp-119 and Asp-217 in
3 and Asp-121 and Asp-220 in
5. By sequence alignment, each
aspartate appears to be a homologue of metal ligands in the MIDAS
motifs of
L and
M. Substitution of any of
these aspartates with alanine reduces the affinity of the
v-integrins for soluble ligands by at least 50-fold. Interestingly,
mutation of Asp-119 and Asp-217 within
3 and Asp-220 in
5 did not
completely abrogate receptor function because integrins with these
mutations could still mediate cell adhesion. Despite the inability of
each mutated integrin to bind soluble ligand, the ability of mutants at
3 residues Asp-119 and Asp-217 and
5 Asp-220 to mediate cell
adhesion proves that these aspartates are not absolutely essential for ligand contact. It is important to emphasize that cell adhesion to an
immobilized substratum is the summation of multivalent receptor-ligand contacts brought about by integrin clustering. In addition the ligand
is immobilized and cannot freely diffuse; therefore, cell adhesion can
often be observed even when the affinity between ligand and integrin is
too low to measure in soluble ligand binding assays. Thus, another
interpretation of this result is that each aspartate contributes to
ligand binding affinity. We believe this to be a reasonable inference
especially since FACS analysis indicates that each mutant is expressed
on the cell surface at a level equivalent to the wild-type integrin.
However, because the mutant integrins fail to interact with soluble
ligand, we are unable to provide a quantitative measure of the
difference in ligand binding affinity.
It is also key to assess whether the mutations at putative metal
coordination sites alter the affinity of the integrin for metal ion.
Unfortunately, the inability to generate milligrams of recombinant
integrin, and the relatively low affinity of the integrin for ion,
makes a direct measure of this parameter nearly impossible. We were,
however, able to assess metal binding affinity indirectly by measuring
the apparent affinity of the integrin for metal ion as reported by
ligand binding. This was accomplished by measuring cell adhesion across
a range of metal ion. From this analysis it is evident that the
mutation of Asp-119 and Asp-217 in 3, and Asp-220 in
5, reduces
the apparent affinity of each integrin for metal ion. Mutation of each
aspartic acid lowered the apparent affinity for either Mn2+
or Mg2+ by 10-20-fold. This is the first evidence we are
aware of in which an aspartic acid within an integrin
subunit has
been shown to influence metal ion affinity. The simplest interpretation
of this finding is that each of these aspartic acid residues is part of
a metal ion binding site. Without a direct measure of metal binding
affinity to each mutant, and without a three-dimensional structure,
these aspartates cannot be unequivocally assigned as metal ligands.
Yet, because each of the aspartate residues in question aligns well
with metal ligands in the MIDAS motif, this finding strongly implies a
similarity in the way the two protein modules ligand with ion at the
first and third coordination groups.
This study also identifies an important distinction between the metal
ligands in the MIDAS and in the subunits. In the MIDAS motif, the
second coordination group is a single threonine that coordinates with
bound metal. Based on the alignment presented in Fig. 1, we
hypothesized that the analogous threonine in
3 was at residue
Thr-182 or Thr-183. Interestingly, the
5 subunit lacks this
threonine, and we originally hypothesized that this difference in
sequence at a metal ligand was key to the way in which
3 and
5
differentially organize on the cell surface in response to metal ions
(14). However, the data presented here indicate neither Thr-182 nor
Thr-183 within
3 makes a significant contribution to
metal-dependent ligand binding, nor do Asn-186 and Ser-190
within
5. Thus, the distinct organization patterns of
v
3 and
v
5 on the cell surface do not appear to be related to a
difference in metal coordination in this region of the
subunit. The
inability to identify a metal ligand within the
subunits that is
analogous to the second coordination group in the MIDAS is also a clear
distinction in the way the two ion binding sites are structured.
Each integrin subunit contains the sequence DXPE. The
aspartate in this motif corresponds to Asp-217 in
3 and Asp-220 in
5. Here, we present evidence that the glutamic acid in this motif is
important for integrin function. Transfection of 293 cells with a
cDNA in which
3 Glu-220 is mutated to alanine failed to yield a
cell line in which the
v
3 heterodimer was expressed. The simplest
interpretation of this observation is that
3 Glu-220 is required for
proper folding or for assembly of a heterodimer with
v. In contrast,
the mutation of Glu-223 of
5 to alanine allows association with
v
and expression on the cell surface but eliminates ligand binding
function. Other reports in the literature also point to the region
surrounding the DXPE motif as an important domain that may
be part of the ligand binding cleft. Two independent studies showed
that synthetic peptides encompassing
3 residues 211-222 and
217-231 could block ligand binding to the
IIb
3 integrin (31,
34).
In conjunction with the present study, these data suggest a hypothesis
regarding the RGD binding site. Collectively the two lines of data
indicate that the DXPE sequence may come together with the
DXSXS motif to form a metal binding site that is
also part of the RGD binding cleft. In this respect, the integrin subunits appear to contain a site that is similar to the MIDAS motif,
where metal-coordinating residues are distantly spaced in the primary
sequence but come together in the tertiary structure of the protein to
make contact with a metal ion. This similarity must be interpreted in
the context of several key differences between the
subunits and the
I domains. Structural algorithms indicate that the
subunits lack
similarity to the I domains, so the
subunits are likely to contain
a MIDAS within a different backbone. Within the putative metal binding
domain, the
subunits contain two disulfide bonds, whereas the I
domains do not. The two domains function differently as well. The
v
3 and
v
5 integrins bind the RGD motif and require the
association of both subunits for this function.
A final objective of the present study was to classify the putative ion
binding site within the subunits. There are two classes of metal
ion binding sites on
v
3 and on
5
1 (25, 27, 30). These two
cation binding sites have opposing effects on ligand binding. One class
of site(s), called Ligand Competent sites, must be occupied for ligand
to bind (23, 27, 30). The second class of sites are called Inhibitory
sites because, when occupied with Ca2+, these sites
interfere with ligand binding. The Inhibitory sites are allosteric to
the ligand binding site and act by increasing the ligand dissociation
rate (27). The data presented here provide the first mutational
evidence that the Ligand Competent and Inhibitory sites are separate.
The elimination of the Inhibitory cation binding site by mutation would
be expected to decrease the ligand dissociation rate, thereby
increasing overall ligand affinity. Inactivation of the Ligand
Competent cation binding site would prevent ligand binding. Mutations
at
3 Asp-119 and Asp-217 clearly belong to the class of sites called
Ligand Competent sites. Importantly, the Inhibitory cation binding site
appears to remain functional in both mutated integrins because the
binding of the reporter antibody AP5 remains sensitive to
Ca2+. Thus, mutations at Asp-119 and Asp-217 within
3
disrupt the Ligand Competent site without affecting the activity of the
Inhibitory cation binding site.
While this paper was in review, reports on the same
topic were published (Puzon-McLaughlin, W., and Takada, Y. (1996)
J. Biol. Chem. 271, 20438-20443; Tozer, E. C.,
Liddington, R. C., Sutcliffe, M. J., Smeeton, A. H., and Loftus, J. C. (1996) J. Biol. Chem. 271, 21978-21984; Goodman,
T. G., and Bajt, M. L. (1996) J. Biol. Chem. 271,
23729-23736). In each report, similar mutations were made in other
integrins, yielding similar data. It should be noted, however, that the
interpretation of the data are somewhat different. Based on the data
presented here, we are reluctant to classify the amino-terminal regions of 3 and
5 as "I domains." As discussed above, we believe
there is sufficient reason to suspect that the
3 and
5 subunits
bind to metal using a MIDAS-like motif but that the backbone of the domain containing the MIDAS is structurally distinct from the known
conformation of the I domains.