(Received for publication, July 18, 1994; and in revised form, October 11, 1994)
From the
The ``I'' domains of the (CD18)
leukocyte integrins are implicated in ligand binding function.
Moreover, rather than recognizing linear peptide sequences, this class
of integrins generally recognizes multiple discontinuous sites on
immunoglobulin superfamily adhesion receptors. A conserved cluster of
oxygenated residues is involved in ligand recognition by
and
integrins. In the present study, we
evaluated the role of this region in the I domain-containing
integrins. Recombinant
(LFA-1, CD11a/CD18) and
(MAC-1, CD11b/CD18) were expressed on COS cells, and function was
assessed by adhesion to ICAM-1 or iC3b, respectively. Alanine
substitution at position Asp
or Ser
in
produced a complete loss in the capacity of both
and
to support cell adhesion. In contrast, substitution at
Asp
or Ser
resulted in loss of
surface expression when co-transfected with
(CD11a) or
(CD11b). These data provide the
first evidence for involvement of the
subunit in
ligand binding to I domain integrins.
The integrin subfamily mediates leukocyte
interactions during normal immune and inflammatory
responses(1, 2, 3) . The leukocyte integrins,
(LFA-1, CD11a/CD18),
(MAC-1, CD11b/CD18), and
(p150, 95, CD11c/CD18), share a
common
subunit (CD18) that associates with three unique but
homologous
subunits(4) . Unique to the three
subunits of the
integrins and
and
subunits of the
integrins is a
200-amino acid inserted or ``I''
domain(5, 6, 7, 8, 9, 10) .
Each of the integrins contributes to leukocyte
adhesive interaction by recognition of a multiplicity of protein
ligands.
recognizes three members of
the immunoglobulin superfamily: ICAM-1(11) ,
ICAM-2(12) , and ICAM-3 (13) .
binds the complement protein
iC3b(14, 15) , ICAM-1(16) ,
fibrinogen(17) , and factor X(18) . Little
information about the structural features involved in the binding of
the various adhesive proteins to the
integrins is
known. Perhaps the best characterized domain implicated in ligand
recognition is a discrete region of the
(GPIIIa)
subunit of the platelet integrin
(GPIIb/IIIa)(19) . Based on mutational analysis, a highly
conserved cluster of oxygenated residues within this region of
has been implicated in the ligand recognition
function of
(20, 21) . Further, substitution of the
corresponding residue in
integrin abolished ligand
recognition of
(22) . The
remarkable conservation of this region among the known integrin
subunits suggests that this region may also be involved in ligand
recognition in all other integrins (Fig. 1). However, unlike the
and
integrins, the
leukocyte integrins do not appear to utilize small linear peptide
sequences to bind but apparently recognize multiple discontinuous sites
on their adhesive proteins(32, 33, 34) . In
addition, epitope mapping of functional blocking monoclonal antibodies
implicate the I domains as a ligand binding site in
,
, and
(35, 36, 37, 38, 39) .
Thus, I domain integrins may utilize a distinct ligand recognition
mechanism from other integrins.
Figure 1:
Alignment of the conserved cluster of
oxygenated residues of the human integrin subunits. The amino
acid sequences of human
(23) ,
(24) ,
(25) ,
(26, 27) ,
(28) ,
(29) ,
(30) , and
(31) are
shown using the single letter code. Squaredresidues were mutated to alanine. Asterisks indicate previously
identified residues in
and
critical
for ligand recognition (20, 21, 22) .
To test this idea, we evaluated the
role of the integrin subunit in ligand recognition by
the I domain integrins
and
. Recombinant
and
were expressed on COS cells, and function was assessed by the
adhesion to ICAM-1 and iC3b, respectively. Alanine substitution at the
conserved Asp
or Ser
of
resulted in a complete loss of the capacity of both
and
to support cell adhesion. These data implicate the
subunits
of I domain integrins in ligand binding and implicate Asp
and Ser
as part of a ligand binding domain in
and
.
COS-7 cells were transfected by electroporation with
the wild-type or the mutant
constructs together with the wild-type
subunit (9) or
subunit (47) cDNAs subcloned
into the expression vector pCDM8 (Invitrogen Corp.). Cells were
evaluated for surface expression and adhesion 48 h after
electroporation. Flow cytometric and immunoprecipitation analyses were
carried out as previously described (48) .
Figure 2:
SDS-polyacrylamide gel electrophoresis
analysis of wild-type or mutant and
receptors transiently expressed on
COS cells. A, COS cells co-transfected with
or
mutants were surface-iodinated, lysed, and immunoprecipitated
with mAb 38 (anti-
, lanesa) or mAb
TS1/18 (anti-
, lanesb). B,
COS cells co-transfected with
or
mutants were surface-iodinated,
lysed, and immunoprecipitated with mAb 3H5 (anti-
, lanesa) or mAb TS1/18 (anti-
, lanesb). Immune complexes were isolated, and reduced
samples were resolved by SDS-polyacrylamide gel electrophoresis on 7.5%
acrylamide gels and detected by autoradiography. Molecular mass markers
are indicated on the left in kDa.
Figure 3:
FACS histograms of wild-type or mutant
and
receptors transiently expressed on COS cells. A, COS
cells co-transfected with
or
mutants were incubated in the
presence of mAb 38 (anti-
) (a) or mAb TS1/18
(anti-
) (b). B, COS cells
co-transfected with
or
mutants were incubated in the
presence of mAb 3H5 (anti-
) (a) or mAb TS1/18
(anti-
) (b). After 30 min, cells were washed,
further incubated with fluorescein isothiocyanate-conjugated goat
anti-mouse F(ab`)
fragments for 30 min, and analyzed on a
FACScan. Abscissa, log of the fluorescence; ordinate,
cell number.
Figure 4:
Adhesion of cells expressing wild-type or
mutant and
receptors to purified ICAM-1 and
iC3b, respectively. Fluorescently labeled cells were allowed to attach
to microtiter wells coated with immunoaffinity-purified ICAM-1 (A) or iC3b (B) in the absence or presence of the
indicated monoclonal antibodies for 30 min at 37 °C. Unbound cells
were removed, and adherent cells were quantitated by fluorescence using
a Pandex fluorescence concentration analyzer. The data are expressed as
the percentage bound where 100% equals the total number of cells
adhered to the mAb TS1/18 (anti-
)-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 determine whether these residues
of also affected
ligand recognition, the ability of the transfected cells
expressing this integrin to adhere to microtiter wells coated with iC3b
was examined (Fig. 4B). Cells bearing the recombinant
attached to iC3b and were blocked by
anti-
(mAb 3H5)-specific antibody. In contrast, cells
bearing
(D134A) or
(S136A) failed to adhere to
iC3b-coated microtiter wells. Consequently, Asp
along
with Ser
play an integral role in the ligand binding
function of both
and
.
The major findings in this work are as follows. 1) Alanine
substitution of two of the conserved oxygenated residues in
, Asp
, or Ser
did not
affect heterodimer formation or surface expression when co-transfected
with
or
in COS cells. 2)
Substitution of these residues to alanine resulted in deficits in both
and
ligand binding function. 3) Alanine substitution of Asp
or Ser
resulted in loss of
surface expression when co-transfected with
or
. These data demonstrate the role of the
subunit
in ligand recognition by the I domain-containing leukocyte integrins.
Two of the individual alanine substitutions, Asp and
Ser
, impede heterodimer formation of
with the co-transfected
or
.
The lack of surface expression may be due to faulty folding and rapid
intracellular degradation. However, previous studies identifying
mutations from patients with genetic deficiencies of
(leukocyte adhesion deficiency) suggest that mutations within
this critical region of
(residues 128-361)
represent critical contact sites between the
and
chain
precursors. These contact sites are presumably required for
precursor association and biosynthesis (reviewed in (51) ). In
addition, extensive investigation of bovine leukocyte adhesion
deficiency, prevalent among Holstein cattle, led to the identification
of two
mutations, one of which results in the
substitution of Asp for Gly at position 128 (D128G)(52) .
Therefore, the present results further implicate that this region is
critical in
heterodimer surface expression in the leukocyte
integrins.
In contrast, alanine substitution at residue Asp or Ser
did not affect the capacity of the mutant
to form heterodimers with co-transfected
or
. This is based on the finding that
anti-
or anti-
coprecipitated
or
, respectively. Furthermore, surface expression was
confirmed by reactivity with a panel of anti-
or
antibodies, suggesting that the amino acid substitutions did not
affect receptor structure. The present work suggests that these
residues of
appear to be critical for ligand binding
function of the leukocyte integrins. This conclusion is based on the
failure of cells expressing
(D134A) or
(S136A) co-transfected with
or
to adhere to immobilized ICAM-1 or iC3b,
respectively. Considered together, these data suggest that this region
of
is critical to the common ligand binding
structural characteristics of
integrins
and
.
These residues
(Asp
and Ser
) are highly conserved among
the
subunits and play a role in ligand recognition in
(corresponding residues Asp
and
Ser
)(20, 21) . Substitution of
Asp
to tyrosine in
results in a
complete loss of RGD-dependent ligand binding function of
. In addition, amino acid
substitution of the corresponding residue in
(Asp
) (22) abrogates the RGD-dependent
binding of
to the 110-kDa cell
binding fragment of fibronectin. However, the
integrins, unlike certain
and
integrins, do not utilize RGD-like sequences to interact with
their ligands. The present data suggest that the
,
, and
integrins utilize these same
conserved residues to interact with their respective ligands despite
the very dissimilar nature of their ligands. This suggests the
possibility of a common mechanism of ligand recognition by integrins.
The linear spacing of the conserved cluster of oxygenated residues
approximates that of the calcium binding residues in EF-hand proteins (53) . Given the absolute requirement of divalent cations for
the function of all integrins and the high degree of conservation of
this cluster of oxygenated residues, it has been hypothesized that a
common feature of ligand binding to integrins is the interaction of
ligand with divalent cations occupying a divalent cation binding site
in the integrins(20, 54) . In this working hypothesis,
oxygenated residues in the ligand, such as the Asp in RGD, would
contribute to the coordination of cation binding. This is further
supported by the finding that substitution of Asp to
tyrosine in
also resulted in disruption of divalent
cation binding of
as determined by
the binding of a divalent cation-dependent antibody,
PMI-1(20) .
Since the I domain integrins
do not utilize RGD-like sequences in ligand recognition, we hypothesize
that an unidentified oxygenated residue(s) in their ligands contributes
to the coordination of cation binding similarly to the Asp in
RGD-containing ligands for
and
integrins. This is further supported by the finding that the
substitution of Asp
to alanine in
also
results in a loss of ligand binding of
to the non-RGD-containing ligand, invasin(22) . In
addition, other small peptide ligands that interact with
and
integrins, such as LGGAKQAGDV (55) and EILDVPST(46) , contain oxygenated residues
that may be capable of coordinating divalent cations.
In addition,
results of previous studies implicate the I domain of the integrins as an integral part of the ligand binding function of
,
,
and
(35, 37, 39) . Inhibitory anti-
antibodies have been mapped to the I domains of the
,
, and
. Moreover, alanine
substitution of certain conserved oxygenated residues in the
I domain (D140GS/AGA) abolished divalent
cation-dependent binding of
to
iC3b(36) . Alignment of these critical residues of the I
domains with the presently described region of the
subunits
demonstrates a remarkable conservation of these oxygenated residues
between the I domain and the
subunits, suggesting that these
sequences may comprise a ligand binding motif essential for all
integrin receptor function(21) . This is further supported by
the finding that alanine mutagenesis of one of these conserved residues
in the I domain of
(Asp
) blocked the
binding of
to collagen(38) .
However, further mutational analysis is in progress to determine
whether other conserved and nonconserved
residues are
essential for either ligand binding or surface expression.
In
summary, we have verified a ligand-interactive region in the
subunit of the leukocyte integrins
and
. This is the first evidence for a
ligand binding site in the
subunit of the
``I'' domain integrins. Moreover, that Asp
along with Ser
play a role in the ligand
recognition of both
and
further supports the proposal of a
common ligand binding mechanism(20, 21) , which is
essential for integrin receptor function irrespective of the presence
of the RGD sequence in the ligand. Since the I domain as well as
domains V and VI of
(56) have been implicated
in ligand binding function, it is likely that multiple sites in
integrins cooperate in recognition of ligands.