(Received for publication, July 7, 1995; and in revised form, October 11, 1995)
From the
The integrin subunits play a major role in the regulation
of ligand binding specificity. To gain further insight into the regions
of the
subunits that regulate ligand specificity, we have
utilized
/
chimeras to identify
regions of
that when substituted for the homologous
regions of
switched the ligand binding phenotype of
to that of
. We report that the ligand
recognition specificity of
integrins is regulated by
the amino-terminal one-third of the
subunit. Substitution of the
amino-terminal portion of
with the corresponding 334
residues of
reconstituted reactivity with both
-specific activation-dependent
(PAC1) and -independent (OPG2) ligand mimetic antibodies in addition to
small highly specific activation-independent ligands. In contrast,
substitution of the amino-terminal portion alone or the divalent cation
repeats alone were not sufficient to change ligand binding specificity.
These data in combination with previous studies demonstrate that
integrin ligand recognition requires cooperation between elements in
both the
and
subunits and indicate that the ligand binding
pocket is a structure assembled from elements of both the
and
subunits.
Integrins are heterodimeric adhesion receptors composed of
noncovalently associated and
subunits. The integrin
superfamily consists of at least 20 members that are composed of
different combinations of nine
and more than 15
subunits.
The different combinations of
and
subunits produce
receptors that often possess a distinct ligand recognition specificity.
With regard to integrin ligands, a number of discrete sites recognized
by integrins have been identified and high resolution structures have
been obtained for a number of integrin
ligands(1, 2, 3) . An emerging general theme
from these structural studies is that integrins recognize protein
ligands through interaction with short peptide sequences often
presented on extended
loops(1, 2, 3, 4, 5) .
There is much less precise information concerning the sites within
integrins that recognize ligands. A number of potential ligand
interactive sites have been identified in the integrin subunits.
Chemical cross-linking, site-directed mutagenesis, and immunological
approaches have implicated a highly conserved sequence in the
subunit in the ligand binding
function(6, 7, 8, 9, 10, 11, 12, 13, 14) .
A second site in the same region has also been reported to be involved
in ligand binding (15, 16) . Six of the integrin
subunits contain an additional
200-residue inserted (I) (
)domain, and compelling evidence supports a role for the I
domain in ligand binding (17, 18, 19, 20) . Mutational
evidence and sequence alignment indicates that the I domain and
integrin
subunits might utilize a similar mechanism for ligand
recognition(10, 18, 21) . These data have led
to the hypothesis that the I domain and the conserved
subunit
ligand recognition site are structurally related and may define a novel
motif essential for integrin receptor
function(10, 21) . A high resolution structure of a
recombinant I domain (22) supports this hypothesis.
A
combination of approaches have been utilized to investigate potential
ligand binding sites in subunits that do not contain an I domain;
however, the results have been inconsistent. Cross-linking studies have
demonstrated that bound ligand was proximal to the four divalent cation
binding sites in
and
(23, 24) . Synthetic peptides (25) as well as a recombinant fragment (26) from this
region of
have been reported to bind ligand. A
homology scanning approach mapped the epitopes of antibodies that block
ligand binding to
to the NH
terminus, but
not to the cation binding motifs (27) . Finally, the minimal
ligand binding fragments of
lack
the COOH-terminal portions of the receptor, but contain more than half
of the entire
subunit(28, 29) .
Thus, the structures critical for ligand recognition by integrin
subunits that lack an I domain remain to be elucidated.
A major
difficulty in determining the role of integrin subunits in the
regulation of ligand binding specificity is that the binding of most
macromolecular ligands is activation-dependent, i.e. the
binding of these ligands is highly regulated by the conformational
state of the receptor(30, 31) . In contrast, the
binding of small peptide ligand mimetics is often
activation-independent(32, 33) . A limitation of
previous studies aimed at identification of ligand binding sites was
that a spectrum of both activation-dependent and -independent ligands
were not analyzed. To gain further insight into the structures in the
subunits that regulate ligand recognition specificity, we
exploited the unique tools available for the integrins
and
. These two integrins share the
common
subunit, and the two
subunits are 36%
identical in primary sequence(34) . They recognize a number of
common ligands as well as small peptides containing the Arg-Gly-Asp
(RGD) sequence(35) . In addition, there exist highly specific
small activation-independent
ligands(36, 37, 38) . Moreover, true ligand
mimetic monoclonal antibodies, PAC1 (39) and OPG2(40) ,
have been prepared against
. The
ligand mimetic property of both mAbs is linked to the tripeptide
sequence RYD within the third complementarity-determining region that
appears to mimic the RGD recognition sequence(4, 5) .
The binding of both antibodies to
is blocked by adhesive protein and small competitive peptide
ligands(39, 40) . Neither antibody binds to ligand
binding defective mutants of
(10) . However, these two antibodies differ in that the
binding of PAC1 is activation-dependent while the binding of OPG2 does
not require prior receptor activation. Finally, ligand binding to these
receptors can be assessed indirectly by the conformational changes
reported by the exposure of LIBS epitopes(41) . Utilizing this
integrin pair, we have defined the region of the
subunit that
regulates recognition specificity for both activation-dependent and
-independent ligands. We report here that neither the cation binding
repeats or the NH
terminus alone is sufficient to control
the ligand recognition specificity of this integrin pair. Ligand
specificity requires both regions. A minimal sequence encompassing the
amino-terminal one third of the
subunit was required to transfer
ligand recognition specificity.
Figure 1:
Schematic representation of the
/
chimeric
subunits. Each
chimera consists of the backbone of
(solid
line) from which the indicated portions have been removed and
replaced with the homologous region of
(shaded
boxes). The location of the three silent restriction sites
introduced into the wild type
sequence to facilitate
exchanges and the endogenous SphI site are indicated. Solid black rectangles indicate the position of the four
divalent cation binding repeats present in each
subunit. The
position of
residues that delineate chimeras are
indicated.
Figure 2:
Flow cytometric analysis of stably
transfected cell lines expressing /
chimeric
subunits. CHO cells co-transfected with wild type
and the indicated
subunit and were examined for
receptor expression by flow cytometry. Cells transfected with wild type
or the indicated
/
chimeric
subunit were stained by indirect
immunofluorescence with the anti-
mAb LM142. Cells
transfected with
were stained with the
-specific mAb D57. Results are
depicted as histograms with the log of the fluorescence intensity on
the abscissa and the cell number on the ordinate.
Since several of the substitutions resulted
in reactivity with -specific mAbs,
the binding of the ligand mimetic mAb PAC1 was examined utilizing flow
cytometry. The binding of mAb PAC1 is
activation-dependent(39) . Therefore, while resting
exhibited low reactivity with mAb
PAC1, activation with the mAb anti-LIBS2 significantly increased the
binding of mAb PAC1 (Fig. 3). mAb anti-LIBS2 acts directly upon
, provoking high affinity ligand
binding function(30) . The binding of mAb PAC1 was specific
since it was completely blocked by GRGDSP peptide. Similarly, cells
expressing the chimeras
2b(L1-Q459) or
2b(L1-P334) specifically bound mAb PAC1 in the
presence of activating mAb anti-LIBS2. In contrast, cells expressing
wild type
,
2b(L1-F223)
, or
2b(1-4C)
failed to bind
mAb PAC1 after activation with the mAb anti-LIBS2. The lack of mAb PAC1
binding to these chimeras was not due to the failure of anti-LIBS2 to
bind to the chimeric receptor as the epitope was present on each of
these receptors as assayed by flow cytometry (data not shown). These
data suggest that the chimeras
2b(L1-Q459) and
2b(L1-P334) have a ligand binding pocket very similar
to
.
Figure 3:
Binding of mAb PAC1 to cells stably
transfected with ,
, or chimeric
/
receptors. The
binding of the
activation-specific
mAb PAC1 to CHO cells stably transfected with
and the
indicated
subunit was examined by flow cytometry. Results are
depicted as histograms of cell number versus fluorescence
intensity. Transfected cells were incubated (activated) in the presence
of 8 µM purified IgG anti-LIBS2 for 30 min followed by the
addition of mAb PAC1 (IgM). Cells were washed, stained with
fluorescein-conjugated goat anti-mouse IgM for 30 min, and analyzed.
The binding of mAb PAC1 was analyzed in the presence (open
histogram) or absence (solid histogram) of 1 mM GRGDSP peptide.
Interaction of these
/
chimeras with another ligand
mimetic mAb, OPG2, was also examined by flow cytometry (Fig. 4).
OPG2 inhibits the binding of adhesive proteins to
and its binding is blocked by RGD
peptides(40) . However, unlike mAb PAC1, the binding of mAb
OPG2 to
is activation-independent.
Cells expressing wild type
stained
brightly with mAb OPG2. Consistent with the results obtained with mAb
PAC1, mAb OPG2 bound to cells expressing
2b(L1-Q459)
or
2b(L1-P334)
. No specific mAb
OPG2 staining was observed with cells expressing wild type
,
2b(L1-F223)
, or
2b(1-4C)
. The fact that
neither mAb bound to the chimera
2b(L1-F223) indicates
that the NH
-terminal region alone does not control ligand
binding specificity.
Figure 4:
Expression of the OPG2 epitope on
recombinant wild type ,
, or chimeric
/
receptors. The
binding of the
complex-specific
mAb OPG2 to CHO cells stably transfected with
and the
indicated
subunit was examined by flow cytometry. Results are
depicted as fluorescence-activated cell sorting histograms. In each
panel, the binding of mAb OPG2 (solid histogram) is
superimposed on the binding of the anti-
mAb 142 (open histogram).
To determine the capacity of the chimeras
containing substitutions of the divalent cation repeats to bind small
activation-independent ligands specific for
, we examined the capacity of the
-selective peptidomimetic Ro
43-5054 (38, 51) to increase the binding of mAb
anti-LIBS1 by flow cytometry. Since the mAb anti-LIBS1 binds
preferentially to the occupied conformation of the
receptor(41) , increased binding of mAb LIBS1 is evidence of
receptor-ligand interaction. In the presence of Ro 43-5054, there was
an increase in the binding of anti-LIBS1 to cells expressing
but not to cells expressing
(Fig. 5). Similarly, Ro
43-5054 failed to stimulate the binding of mAb anti-LIBS1 to cells
expressing the chimeras
2b(2+3C) or
2b(3+4C), indicating lack of binding to the
receptor. Unexpectedly, mAb anti-LIBS1 bound maximally to cells
expressing the chimeras
2b(1+2C) or
2b(1-4C) even in the absence of ligand. This
result suggested that these two chimeras possessed a structure that is
slightly altered from that of the wild type receptors. Although the
anti-LIBS1 epitope was exposed on these two chimeras, additional data
(see below) indicate that their ability to bind ligand was not
impaired.
Figure 5:
Flow cytometric analysis of the capacity
of transfected cells to bind an
-specific peptidomimetic. The
binding of the
-selective
peptidomimetic Ro 43-5054 (38) to cells stably transfected with
and the indicated
subunits was examined with
the mAb anti-LIBS1. For LIBS1 binding analysis, cells were incubated
with or without Ro 43-5054 (5 µM) and LIBS1 mAb (0.1
µM) for 30 min on ice. Cells were washed and incubated
with fluorescein-conjugated goat anti-mouse Ig. Results are expressed
as histograms of cell number versus fluorescence intensity. In
each panel, the binding of LIBS1 in the presence of Ro 43-5054 (solid histogram) is overlaid on the binding of LIBS1 in the
absence of Ro 43-5054 (open
histogram).
To test whether the chimeric receptors containing
substitutions of the cation binding repeats possessed an intact RGD
ligand recognition function and to test their capacity to distinguish
between the RGD and fibrinogen chain sequence, the ligand binding
function of the recombinant receptors was analyzed by affinity
chromatography (Fig. 6). Detergent lysates of radiolabeled,
transfected cells were applied to an RGD affinity column and eluted
with the fibrinogen
chain peptide K16, followed by elution with
EDTA. The eluted fractions were then immunoprecipitated with an
anti-
mAb. Eluted fractions of the control
-expressing cells were
immunoprecipitated with anti-
antiserum(30) . Precipitated proteins were then resolved
by SDS-polyacrylamide gel electrophoresis. Consistent with previous
reports(48) , wild type
was
poorly eluted by the K16 peptide (data not shown). While 64% of the
bound wild type
was eluted from
the affinity matrix by the
chain peptide K16, the chimeras
2b(1-4C) (8.4%),
2b(1+2C)
(3.4%),
2b(2+3C) (7.5%), or
2b(3+4C) (13%) were poorly eluted by the K16
peptide from the RGD affinity matrix (Fig. 6). Each of these
receptors bound to the RGD matrix and was readily eluted from the
matrix by EDTA. Both wild type
and
receptors and all the chimeras were
readily eluted from the affinity matrix by RGD peptide (data not
shown). The fact that the chimeras
2b(1-4C) and
2b(1+2C) bound to the RGD affinity matrix and
were specifically eluted by EDTA or RGD peptide indicates that the
alteration in structure reported by anti-LIBS1 did not affect the
ligand binding function of these receptors. These data show that all
chimeras containing substitutions of the cation binding motifs can
recognize the RGD sequence, but that substitution of the
divalent cation binding regions with the corresponding regions
from
was not sufficient to change the ligand
binding specificity of
to that of
.
Figure 6:
Fibrinogen chain peptide K16 does
not displace
or the
/
divalent cation binding repeat
chimeras from a RGD affinity matrix. CHO cells stably expressing the
wild type
or chimeric
/
receptors were radioiodinated and
lysed, and the extract was applied an GRGDSPK-Sepharose 4B column.
After incubation and washing, the bound proteins were sequentially
eluted with 1.5 mM K16, followed by 5 mM EDTA. The
eluted fractions were immunoprecipitated with the anti-
mAb LM142. The immunoprecipitated proteins were resolved by
SDS-polyacrylamide gel electrophoresis on 7% nonreducing acrylamide
gels and detected by autoradiography. Lanes 1,
immunoprecipitate of K16-eluted material; lanes 2,
immunoprecipitate of subsequent EDTA-eluted
material.
Figure 7:
Direct binding of an
-selective peptidomimetic. The
binding of the
-specific
peptidomimetic SC52012 (37) to stably transfected cell lines
expressing
,
, or the indicated chimeric
/
receptor
was determined by incubating transfected cells with
[
H]SC52012 (500 nM) at room temperature.
After 40 min, bound ligand was separated from free ligand by
centrifugation through 20% sucrose. The pellet associated counts were
determined by liquid scintillation spectrometry. Background binding was
measured in the presence of 5 mM EDTA. Shown are
representative results of three separate assays. Results shown are mean
± S.D. of triplicates.
The major findings of the present study are as follows. 1)
Ligand recognition specificity of integrins is
regulated by the amino-terminal one-third of the
subunit.
Substitution of the amino-terminal portion of
with
the corresponding 334 amino acid residues of
switched the ligand recognition specificity of
to that of
. This change in ligand specificity
was observed with an activation-dependent ligand mimetic antibody, an
activation-independent ligand mimetic antibody, and small
activation-independent ligands. 2) Neither the amino-terminal region or
the cation binding repeats alone is sufficient to control ligand
specificity. Chimeras that omit the amino-terminal 140 residues or
first two divalent cation binding repeats of
fail
to change ligand specificity. Thus, the ligand binding pocket of
is a structure that contains
elements of both the
and
subunits.
Previous studies have
suggested that the regions that control ligand binding to
reside in the amino-terminal
portion of
, but the minimal
structures identified in these studies encompassed more than one half
of constituent subunits(28, 29) . In the present
study, we have mapped the regions that regulate ligand specificity to a
smaller region of
. The chimera designated
2b(L1-Q459) contained the amino-terminal portion and
all four divalent cation repeats of
and reacted
with several
complex-specific
mAbs. In addition, this chimera specifically bound small
activation-independent
-specific
peptidomimetics and both activation-dependent (PAC1) and
activation-independent (OPG2) ligand mimetic mAbs. The chimera
2b(L1-P334) retains the amino-terminal portion of
but contains only the first two divalent cation
repeats of
. This chimera also exhibited a ligand
binding phenotype consistent with that of
in that it bound specific
peptidomimetics, the ligand mimetic mAbs PAC1 and OPG2, and several
-specific mAbs. These results
indicate that the ligand specificity of
can be reconstituted with the first 334 amino acid residues of
and does not require the third or fourth divalent
cation repeats of
.
Chimeras that omit the 140
amino-terminal residues or the first two divalent cation motifs of
fail to change the ligand specificity of
to that of
. The chimera
2b(1-4C) contains a substitution of the entire
divalent cation repeat region of
with the
corresponding region of
. This chimera was expressed
on the cell surface and could bind ligand as demonstrated by its
ability to bind to an RGD affinity matrix. However, this chimera was
poorly displaced from the matrix by a fibrinogen
chain peptide
and did not bind the ligand mimetic mAbs PAC1 and OPG2 or an
-specific peptidomimetic. These
data indicate that substitution of the divalent cation repeats alone is
not sufficient to change the ligand binding specificity. Similarly, the
chimera
2b(R140-P334) did not bind the ligand mimetic
mAbs PAC1 and OPG2 or the
-specific
mimetic peptidomimetic. This chimera contains the first two divalent
cation repeats of
but is missing the first 140
amino-terminal residues of mature
. This result
suggests a requirement for residues near the amino terminus and
indicates that an extended portion of the receptor is required for
ligand specificity.
The approach of homolog-scanning mutagenesis (52, 53) is of general use for the identification of
functional domains. A recent report used this technique to localize the
putative ligand binding domains of by mapping the
epitopes for function blocking antibodies to the amino-terminal
portion, but not the divalent cation repeats of
(19) . In the present study, we demonstrated
that the mAb AP2, which blocks ligand function(42) , binds
strongly to the chimera
2b(L1-F223). However, a ligand
binding domain cannot be ascribed to this region since this chimera did
not bind the ligand mimetic mAbs PAC1 and OPG2 and did not bind an
-specific peptidomimetic. Thus, our
results based on the interaction of true ligand mimetics demonstrates
the inherent limitations of relying solely on the localization of the
epitopes of function blocking mAbs to map ligand binding sites.
Previous studies have clearly demonstrated a role for the
subunit in ligand binding to
.
Single amino acid substitutions in a highly conserved region of
completely block the ligand binding function of
(9, 10) . This
loss of ligand binding is not due to an effect on the activation state
of the receptor as the mutations also block the activation-independent
binding of mAb OPG2 and small ligand mimetics. However, our present
results demonstrate that the specificity for the binding of PAC1, OPG2,
and specific peptidomimetics to
is
controlled by the first 334 amino acid residues of the
subunit.
Together, these results indicate that ligand recognition requires
cooperation between elements in both the
and
subunits and
indicates that the ligand binding pocket is a topographical structure
that is assembled from regions of both the
and
subunits.