(Received for publication, May 31, 1995)
From the
The I domains of the leukocyte integrins have
been shown to be essential for ligand recognition. Amino acid
substitutions of Asp
and Ser
, which reside
in a conserved cluster of oxygenated residues, abrogate divalent cation
ligand binding function of
.
Presently, we evaluated the role of two I domain regions in
ligand recognition: 1) the conserved
cluster of oxygenated residues (Asp
, Asp
,
Ser
, and Ser
) and 2) a 7-amino acid region
(Phe
-Tyr
), conserved in
and
but absent in
of the
integrins. Recombinant
was expressed on COS-7 cells, and function was assessed by iC3b
recognition. Alanine substitution at position Asp
,
Asp
/Ser
, Ser
, or
Ser
produced a complete loss in the capacity of
to recognize iC3b and attenuated the
binding of a divalent cation-dependent epitope recognized by monoclonal
antibody 24. Moreover, alanine substitution at Asp
or
Tyr
or deletion of Phe
-Tyr
abolished iC3b ligand recognition as well as the binding of a
blocking antibody. In contrast, these mutations did not affect the
binding of the cation-dependent epitope. These data implicate a second
region within the I domain important for
ligand binding function and suggest that this region does not
affect a divalent cation-dependent conformation of
.
The leukocyte integrins comprise a group of
closely related adhesion receptors that mediate critical events during
normal and inflammatory immune
responses(1, 2, 3) . The
integrins consist of three glycoproteins,
(CD11b/CD18, MAC-1),
(CD11a/CD18, LFA-1), and
(CD11c/CD18, p150,95), containing a
common
subunit (
) noncovalently linked to three
distinct but homologous
subunits (
,
, and
)(4) . All three
subunits contain an insertion of approximately 200 amino acids termed
the ``I'' domain (5, 6, 7, 8, 9) . The I
domain is absent in all other known integrin subunits except for
of
(10) ,
of
(11), and
of
(12) .
is the major leukocyte integrin
expressed on neutrophils and mediates phagocytosis of opsonized
particles(13) , adherence to the
endothelium(14, 15) , neutrophil homotypic
aggregation, and chemotaxis(16) . Like other integrins,
is promiscuous and recognizes a
multiplicity of protein ligands, including complement C3 fragment
iC3b(13, 17) , ICAM-1(18) ,
fibrinogen(19) , and Factor X(20) . The identification
of regions of
that contribute to
multifunctional ligand recognition have not been completely
characterized. The current state of our knowledge indicates the
presence of multiple ligand contact points in
. We have previously identified a
cluster of oxygenated residues within
that are
essential for ligand binding of
and
(21) . In addition, the I
domain has been shown to be essential for
ligand binding by the localization of blocking antibody epitopes
and by direct ligand binding to isolated I
domain(22, 23, 24) . The
I
domain also binds cations(25) . Mutations of the highly
conserved residues Asp
, Ser
, and
Asp
in
I domain, which reside in a
cluster of oxygenated residues, abolish divalent-cation binding and
ligand recognition of
iC3b(25) . To date, the location and structure of additional
ligand binding sites within the
I domain is entirely
speculative. In the present study, we have identified a novel
I domain ligand binding site
(Phe
-Tyr
), which is conserved in
and
but absent in
(see Fig. 1). In addition, we have further characterized a
cluster of highly conserved oxygenated residues (Asp
,
Asp
, Ser
, and Ser
) in the
I domain. Our results further implicate Asp
and Ser
and designate analogous importance to
Ser
, Asp
, and Tyr
in the
ligand binding function of
.
Furthermore, these two identified ligand binding domains differentially
alter the interaction of bound divalent cations to
.
Figure 1:
Amino acid alignment of the
I domain of with other integrin
subunit I domains. The deduced sequence of
(CD11b, (5, 6, 7) ) was aligned with
human
(CD11c, (8) ),
(CD11a, (9) ),
(10) ,
(11) , and
(12) using single-letter code. Gaps were introduced to
maximize alignment (dots). Boxed residues were
mutated to alanine. Asterisks indicate previously identified
residues in
critical for ligand
recognition(25) .
COS-7 cells (a monkey kidney fibroblastoid cell line from the
ATCC) were maintained in Dulbecco's modified Eagle's medium
(Irvine Scientific, Santa Ana, CA) supplemented with 10% fetal calf
serum (Hyclone Laboratories, Logan, UT), 1% glutamine (Irvine
Scientific), 1% penicillin and streptomycin (Irvine Scientific), and 1%
nonessential amino acids (Sigma). COS-7 cells were co-transfected by
electroporation with wild-type (32) subcloned
into pCDNA1/neo (Invitrogen) and either wild-type or mutant
constructs. Mock transfected cells were transfected with pCDM8
vector without insert. Cells were evaluated for surface expression and
function 48 h after electroporation.
Forty-eight hours
post-transfection, COS-7 cells were harvested with tissue culture
trypsin/EDTA solution (Sigma), and 2 10
cells were
seeded in duplicate onto 6-well tissue culture plates in supplemented
Dulbecco's modified Eagle's medium as described above.
Cells were allowed to adhere for 3 h at 37 °C. Adherent monolayers
were washed once with HBSS buffer and incubated for 30 min at 37 °C
in 0.5 ml of HBSS in the presence or absence of blocking
anti-
mAb 8H1 (50 µg/ml). 100-µl aliquots of
iC3b-E (2
10
cells/ml) were added, and the plates
were further incubated for 1 h at 37 °C. Adherent monolayers were
gently washed with HBSS buffer to remove nonadherent erythrocytes.
Rosettes (>10 erythrocytes/COS-7 cell, >50 cells examined) were
examined by light microscopy at 200
magnification.
COS-7 cells were transiently co-transfected with the
wild-type or mutant together with the wild-type
. Cell surface expression and heterodimer association
of the integrins were evaluated by immunoprecipitation of
detergent-lysed surface-labeled cells (Fig. 2). Anti-
specific antibody (TS1/18) immunoprecipitated both the
and
subunits from cells co-transfected with
and the
wild-type
or any of the mutant
,
except for
(D134A), indicating that both subunits were
associated on the cell surface. Similar results were obtained utilizing
anti-
antibody (LM2/1) (data not shown). In contrast,
alanine substitution of
at D134A was not expressed
when co-transfected with
. Anti-
specific antibody did not precipitate
heterodimers.
Moreover, immunoprecipitation of surface-labeled
(
246-252)
consistently
showed moderate cell surface expression in comparison with the
wild-type or other mutant
receptors.
Figure 2:
SDS-polyacrylamide gel electrophoresis of
wild-type or mutant (s)
transiently expressed on COS-7 cells. COS-7 cells co-transfected with
or
mutants were surface-iodinated, lysed, and immunoprecipitated
with mAb TS1/18 (anti-
). The precipitated proteins
were resolved by electrophoresis on 7.5% SDS-polyacrylamide gels under
reduced conditions and detected by autoradiography. The co-transfected
wild-type or mutant
(s) in each cell line are listed above each lane. The position of the
and
subunits are shown on the right, and molecular mass standards are
indicated on the left in kDa.
Figure 3:
Expression of 3H5 epitope on recombinant
wild-type or representative
mutant
receptors as
determined by flow cytometry. The binding of
-specific
antibodies LPM19c (a) and 3H5 (b) to cells bearing
the wild-type
or
mutants was examined by flow
cytometry. Results are depicted as histograms with the log of the
fluorescence intensity on the abscissa and the cell number on the
ordinate. Transfected cells were incubated in the presence of mAbs
LPM19c or 3H5 for 30 min at room temperature. Cells were washed,
stained with fluorescein-conjugated goat anti-mouse F(ab`)
for 30 min, and analyzed on a FACSan.
Figure 4:
Adhesion of cells expressing wild-type
or mutant receptors to
immobilized iC3b. Fluorescently labeled cells were allowed to attach to
microtiter wells coated with iC3b in the absence (open bars)
or presence (hatched bars) of blocking mAb 8H1
(anti-
) for 30 min at 37 °C. Unbound cells were
removed, and adherent cells were quantitated by florescence using a
Pandex fluorescence concentration analyzer. The data are expressed as
the percentage bound where 100% equals the total number of cells that
bound to the mAb 8H1-coated wells to correct for the levels of integrin
expressed by the different transfectants. Results are representative of
three separate experiments. Bars represent the mean ±
S.E. of three determinations.
To further substantiate the importance of these
I domain residues in iC3b ligand recognition, we
tested the ability of the adherent COS-7 cell transfectants to bind to
iC3b-coated erythrocytes (iC3b-E) (Table 2, Fig. 5). COS-7
cells expressing the wild-type
or
those expressing
mutations F246A, G247A, P249A,
L250A, or N224A were able to rosette iC3b-E, and binding was blocked by
anti-
antibody, 8H1. In contrast, cells expressing
recombinant
with
mutations D140A, S142A, D140GS/A140GA, S144A, D248A, Y252A, or
246-248 were unable to rosette iC3b-E. None of the COS-7
transfectants were able to bind erythrocytes coated with IgM (results
not shown). These results further implicate Asp
and
Ser
as playing an essential part in the ligand binding
function of
and designate analogous
importance to Ser
, Asp
, and Tyr
in ligand recognition.
Figure 5:
Binding of iC3b-E by cells expressing
wild-type or representative mutant receptors. COS-7 cells transiently transfected with wild-type
(A and B),
(L250A)
, or
(D140A)
were detached and replated on
6-well tissue culture plates for 3 h at 37 °C. Cells were then
incubated in the absence (A, C, and D) or
presence (B) of blocking mAb 8H1 (anti-
) for
30 min at 37 °C. iC3b-E were added with transfectants for 60 min at
37 °C. Unbound erythrocytes were removed by washing, and rosettes
were examined by light microscopy.
Figure 6:
Expression of mAb 24 epitope on
recombinant wild-type or mutant
receptors as determined by flow cytometry. The binding of
cation-dependent antibody 24 to COS-7 cells transfected with wild-type
or mutant
receptors was examined by
flow cytometry. Transfected cells were incubated with mAb 24 in the
presence of 0.5 mM MnCl
or 10 mM EDTA for
30 min at 37 °C. Cells were washed, stained with
fluorescein-conjugated goat F(ab`)
anti-mouse
immunoglobulins for 30 min, and analyzed. The binding of mAb 24 is
expressed as the percent of mAb 24 binding in the presence of 0.5
mM MnCl
(mean linear fluorescence) to the total
number of receptors/cell line as determined by the binding of
anti-
antibody (mean linear fluorescence). Results
represent the mean ± S.E. of three separate
experiments.
These studies establish the following. 1) Individual alanine
substitutions of I domain residues did not affect
heterodimer formation or surface expression when co-transfected with
in COS-7 cells with the exception of residue
Asp
, which was not expressed. 2) Alanine substitution of
the clustered oxygenated residues at position Asp
,
Ser
, D140GS/A140GA, or Ser
resulted in the
complete loss of the capacity of
to
recognize iC3b. 3) Alanine substitution of Asp
,
Tyr
, or deletion of residues
Phe
-Tyr
(
246-252)
abolished the binding of
to iC3b as
well as the recognition of the function blocking anti-
antibody 3H5. 4) Substitution of residues Asp
,
Ser
, D140GS/A140GA, or Ser
with alanine
attenuated the binding of the divalent cation-dependent antibody mAb
24. 5) Alanine substitution at Asp
, Tyr
, or
246-252 did not affect cation-dependent mAb 24 binding
function. These results further implicate Asp
and
Ser
and designate analogous importance to
Ser
, Asp
, and Tyr
in the
ligand binding function of
.
Furthermore, these two identified ligand binding domains differentially
alter the interaction of bound divalent cations to
.
Since the disclosures of the
contribution of the I domain in ligand recognition, the location and structure of the ligand
binding sites within the I domain has been a subject of intense
investigation. Double alanine substitutions of the highly conserved
oxygenated residues Asp
and Ser
(D140GS/A140GA), which reside in a cluster of oxygenated residues of
the
I domain, have been reported to abolish divalent
cation-dependent binding of
to
iC3b(25) . Our data are in agreement with these previous
observations and specifically extend the functional role of this highly
conserved cluster of oxygenated residues in this domain in ligand
recognition. The present work suggests that Ser
in
addition to Asp
and Ser
of
I domain appear to be critical for ligand binding function of
. This conclusion is based on the
failure of cells expressing
(D140A),
(S142A),
(D140GS/A140GA), or
(S144) cotransfected with
to adhere
to immobilized iC3b or to bind iC3b-E.
In addition to the effect on
ligand binding, our results support a direct interaction between ligand
binding and a divalent cation-dependent conformation. This conclusion
is based on the attenuated binding of mAb 24 in the presence of
Mn to cells expressing
(D140)
,
(S142)
, or
(D140GS/
A140GA)
and to a lesser extent to cells expressing
(S144A)
. Expression of mAb 24 epitope
is associated with the Mg
or Mn
bound form of the
integrins(27) .
Therefore, the present work suggests that these I domain mutations
perturb the interaction between bound divalent cation and
. This is further supported by the
identification of these residues as part of the three-dimensional metal
coordination sites within the
I domain by high
resolution crystal structure: Ser
, Ser
, and
Thr
as well as secondary coordination sites between
Asp
to Ser
and Asp
to
Ser
(37) . Furthermore, alanine substitution of
Asp
and a synthetic peptide containing this residue
inhibit iC3b binding to
(23) .
We have previously
identified a conserved amino acid alignment of these I domain residues with a similar divalent cation-dependent ligand
binding motif, DXSXS, in
and
subunits that contribute to ligand binding of
,
,
and
, respectively (21, 38) . In addition, substitution of Asp
in
disturbs binding of divalent
cations(39) , and substitutions in the corresponding homologous
residues in
(40) and
(41) abolish the ligand binding function. Therefore, we
hypothesized that this common binding motif participates in all
integrin functions through the interaction of ligand with divalent
cations occupying a divalent cation binding site in the
integrins(21, 38) . This is further supported by the
finding that alanine substitution of the corresponding residues in the
I domain are essential for the ligand binding
function of
(42) . Alignment
of these
subunit sequences with the I domain has led to the
proposal of an integrin
subunit I domain(37) . Previous
studies identifying natural
mutations (leukocyte
adhesion deficiency) suggest that this region of
(residues 128-361) represents critical contact sites required for
heterodimer formation(43) . Interestingly, while
alanine substitution of Asp
, Ser
,
D140GS/A140GA, or Ser
did not affect the capacity of
to efficiently associate with
on
COS cells, alanine substitution at Asp
resulted in loss
of
surface expression based on
immunoprecipitation with anti-
antibody. These results
suggest a role for the I domain not only in ligand recognition but in
heterodimer formation. It is particularly noteworthy that
residue Asp
aligns with the
corresponding residue Asp
of
, which we
previously identified as essential for surface expression of
and
(21) .
These I domain
sequences may participate directly in a complex between cation, ligand,
and receptor as has been reported for the identified homologous ligand
binding domain in the subunit(44) . It was
proposed that residues 118-131 of
bind both
divalent cation and ligand, resulting in the displacement of cation
from this region of
and subsequently exposing
secondary binding sites. In the present study we have identified a
second region within
I domain, which is important in
ligand recognition of iC3b. This
conclusion is based on the inability of the cells expressing I domain
mutations at Asp
, Tyr
or deletion of
residues 246-252 to adhere to immobilized iC3b or to rosette
iC3b-E. However, in contrast to mutations at Asp
,
Ser
, or Ser
, these mutations do not perturb
the expression of the Mn
-induced mAb 24 epitope.
Taken together, these results demonstrate that the two distinct I
domain regions differentially alter the interaction of a
cation-dependent conformation of
.
This suggests that alanine substitution at Asp
,
Ser
, or Ser
perturb the interaction between
bound cation and the integrin, while substitution at Asp
,
Tyr
, or
246-252 do not. Therefore, an
alternative hypothesis is that the binding of divalent cations to
Asp
, Ser
, and Ser
may
maintain an integrin conformational structure that allows access to
distinct binding sites within the receptor. This is supported by the
localization of the epitope recognized by receptor blocking mAb 3H5 to
Asp
and Tyr
, if we assume that function
blocking mAbs bind close to the ligand binding site. In contrast,
substitution at Asp
, Ser
, or Ser
did not affect the binding of any of the blocking
anti-
antibodies tested.
In conclusion, we have
further characterized a cation ligand-interactive region in the
I domain that further supports the proposal of a
common ligand binding mechanism (21, 38) essential
for all integrin receptor function. In addition, we have identified a
second unique ligand binding domain in the
I domain
that does not affect cation-dependent conformation of
. The mechanisms by which the
multiple ligand binding domains in the
and
subunits
participate in ligand specificity is speculative. Differences in ligand
recognition and specificity may be controlled by small sequence
differences in specific regions such as residues
Phe
-Tyr
of the
I
domain. This region is unique to
and
but absent in
of the
integrins.
and
share a common ligand, iC3b, that is
not recognized by
(1, 2, 3) .
We are presently addressing this issue.