From the
Integrins mediate signal transduction through interactions with
multiple cellular or extracellular matrix ligands. Evidence is
accumulating that the I (or A) domain, a
Integrins are a supergene family of cell adhesion receptor
heterodimers, which recognize multiple ligands and mediate cell-cell
and cell-extracellular matrix
interactions
(1, 2, 3, 4, 5, 6) .
Integrins
Some
integrin
Ueda et al.(29) recently reported that C3bi
directly bound to recombinant
After this study was submitted, a report describing the
crystal structure of the I domain of
We thank to D. Altieri for antibodies; D. Dottavio for
recombinant ICAM-1; D. Hickstein for cDNAs; and Drs. M. Schwartz, S.
Shattil, and J. Smith for critical reading of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
200-residue inserted
sequence in some integrin
subunits, mediates ligand binding. We
have previously shown that Thr-221 of the putative ligand binding sites
within
2 I domain of
2
1 is critical for binding to
collagen (Kamata, T., and Takada, Y. (1994) J. Biol. Chem. 269, 26006-26010). Here we report that the mutation of Thr-206 of
L blocks intercellular adhesion molecule 1 (ICAM-1) binding to
L
2 and mutation of Thr-209 of
M blocks ICAM-1 and C3bi
binding to
M
2. The data indicate the Thr residues of
M
and
L corresponding to Thr-221 of
2 are critically involved
in the ligand interaction with
2 integrins. The mutations of the
Asp-137 and Asp-239 of
L also block ICAM-1 binding to
L
2, as do the corresponding Asp residues of
2 or
M
in collagen/
2
1 or C3bi/
M
2 interactions,
respectively. These data suggest that these Thr and Asp residues,
conserved among I domains, are critical for interaction with
structurally distinct ligands (e.g. ICAMs, C3bi, and
collagen).
L
2 and
M
2, which share a common
2
(CD18) subunit (M
95,000), are broadly distributed
on all leukocytes of lymphoid and myelomonocytic lineage and mediate
crucial adhesive recognition through their ability to bind multiple
ligands (intercellular adhesion molecules (ICAMs),
(
)
fibrinogen, Factor X)
(2, 7) .
subunits have an I (or A) domain that consists of about
200 amino acids and is located close to the three metal binding
sites
(8, 9, 10, 11, 12, 13, 14, 15) .
The I domain is present in the integrin
L (8),
M
(9, 10, 11) , and
X subunits
(12) of the
2 subfamily, in the
1
(13, 14) and
2
(15) subunits of the
1 subfamily,
and in the
E subunit
(16) of the
7 subfamily. The I
domains are also present in other proteins, including von Willebrand
factor
(17) , cartilage matrix protein
(18) , complement
factors C2 and B
(19, 20) , and collagen type
VI
(21, 22) . There is growing evidence that the I
domains are important in ligand binding and receptor activation. A
number of function-blocking or activation-dependent antibodies map to
the I domains of
L
2
(23, 24) ,
M
2
(25, 26) ,
2
1
(27) , and
1
1
(28) (collagen/laminin receptors). The recombinant
M I domain fusion protein binds to fibrinogen, ICAM-1
(26) ,
C3bi
(29) , or hookworm neutrophil inhibitory
factor
(30, 31) . Also, an
L I domain fusion protein
binds to ICAM-1
(24) and an
2 I domain fusion protein to
collagen
(32) . Asp-140 and Asp-242 within the
M I domain
are critical for the binding of divalent cations to the I domain, and
for binding of
M
2 to C3bi
(33) (Fig. 1). Asp-151
and Asp-254 in the
2 I domain (corresponding to Asp-140 and
Asp-242 of the
M I domain) were shown to be critical for
collagen/
2
1 interaction as well
(27) . The Asp-253 of
the
1 I domain (corresponding to Asp-242 of
M) has been shown
to be critical in collagen IV/
1
1 interactions
(28) .
Since Asp-151 or Asp-254 of
2 are not required for the recombinant
2 I domain/collagen interaction
(27) , it is likely that the
Asp residues are indirectly involved in the collagen binding to
2
1 probably through divalent cation binding to the I domain
(Fig. 1).
Figure 1:
Conserved Asp and Thr residues among I
domains potentially important in ligand binding. The putative ligand
binding sites of the 2 I domain were recognized by
function-blocking anti-
2 mAbs (27). Thr-221 of
2 has been
identified as critical in collagen binding by alanine scanning
mutagenesis of the
2 I domain (32). Asp-140 and Asp-242 were
critical in divalent-cation binding to the
M I domain and in
binding to C3bi (33). Asp-151 and Asp-254 of
2 were critical in
binding to collagen (27). Asp-137, Asp-239, and Thr-206 of
L, and
Thr-209 of
M were tested for ligand interactions in this
study.
Recently we have shown that function-blocking
anti-2 mAbs recognize a small region (residues 173-259) of
the
2 I domain, suggesting that the region may be involved in
ligand binding to
2
1 (putative ligand binding
sites)
(27) . Notably, we identified Thr-221 of the putative
ligand binding site as critical residue for binding of collagens to
both the recombinant
2 I domain and intact
2
1 (32),
suggesting that Thr-221 is directly involved in collagen binding. In
the present study we introduced mutations of the conserved Thr and Asp
residues to study their role in
2 integrin/ICAM-1 interactions.
The data suggest that the conserved Thr and Asp residues are equally
critical in
2 integrin/ICAM-1 or C3bi interactions, and that there
may be similar ligand recognition mechanisms among different I domains
that recognize structurally distinct ligands (e.g. C3bi,
collagens, ICAM-1).
Materials
Monoclonal antibodies TS1/22 (L)
(34) and TS1/18 (
2)
(34) were obtained from ATCC.
mAbs OKM1 (
M)
(35) and 60.3 (
2)
(36) are gifts
of D. Altieri (Yale University, New Haven).
M and
2
(37) cDNAs were obtained from D. D. Hickstein (University of
Washington, Seattle).
L cDNA was obtained by amplifying partial
cDNAs from a human peripheral leukocyte cDNA library by a polymerase
chain reaction using synthetic oligonucleotides from the published
sequence
(8) and ligating the cDNA fragments. ICAM-1/mouse
C
fusion protein was obtained from D. Dottavio (Sandoz Research
Institute, East Hanover, NJ). C3bi was purified as described
elsewhere
(38) .
Adhesion Assay
Wells of 96-well Immulon-2 plates
were coated with goat anti-mouse C chain polyclonal antibody
(Caltag, South San Francisco, CA) (0.2 µg/well in 100 µl of
PBS) overnight at 4 °C. After washing the wells with PBS, wells
were incubated with ICAM-1/mouse C
fusion protein (0.4
µg/well) at room temperature for 1 h. Unoccupied protein binding
sites were blocked by incubating the wells with 1% heat-denatured
bovine serum albumin (BSA) for 1 h at room temperature. After washing
with PBS, cells (10
/well) were added and incubated for 1 h
at 37 °C in 100 µl of Tyrode, 5 mM Hepes buffer, pH
7.4, in the presence of 0.1% BSA and 0.1 mM MnCl
.
After rinsing the wells to remove unbound cells, bound cells were
quantified by assaying endogenous phosphatase activity
(39) .
Adhesion of cells to C3bi was measured as described above except that
purified C3bi (1 µg/well in 100 µl of PBS) was directly coated
onto wells.
Other Methods
Site-directed mutagenesis was
carried out using unique restriction site elimination
(40) . The
presence of mutations was confirmed by DNA sequencing. M,
L,
and
2 cDNAs were subcloned into pBJ-1
(41) . Transfection
into CHO cells by electroporation, selection of the transfected cells
with G418, immunoprecipitation, and flow cytometric analysis were
carried out as previously described
(42) .
Expression of Wild-type (wt) and Mutant
We introduced T206A (Thr-206 to Ala),
T208A, D137A, or D239A mutations into the I domain of the 2
Integrins on CHO Cells
L
subunit. wt and mutant
L cDNA in pBJ-1 expression vector were used
to transfect CHO cells together with wt
2 cDNA. Forty-eight h
after transfection, CHO cells transiently expressing wt and mutant
L
2 were surface
I-labeled and analyzed by
immunoprecipitation with anti-
L and anti-
2 antibodies.
Fig. 2a demonstrates that wt and mutant
L
(M
175,000) and
2 (M
95,000) were co-precipitated in each case. The data indicate that
wt and mutant
L are expressed on the surface of CHO cells in
association with wt
2, suggesting that the mutations do not affect
the subunit association of
L
2. We cloned CHO cell lines
stably expressing wt or mutant
L
2 using cell sorting to
obtain cells expressing high levels of wt or mutant
L
2
(Fig. 2b). While the transient expression of T208A
mutant was low, we obtained clonal CHO cells stably expressing the
mutant.
Figure 2:
Surface expression of wt and mutant
L
2 on CHO cells. a, immunoprecipitation of
L
and
2 subunits from CHO cells transiently expressing wt or mutant
L
2. Cells were surface
I-labeled and lysates
were immunoprecipitated with anti-
L (TS1/22) or anti-
2 (60.3)
mAbs. Immunoprecipitated samples were analyzed by SDS-polyacrylamide
gel electrophoresis (7% gel) under nonreducing conditions. Dried gels
were exposed for 3 days. Expression level of T208A
L mutant was
low as compared to other mutants. b, CHO cells stably
expressing wt or mutant
L
2 were cloned using cell sorting.
Clones were stained with anti-
L (TS1/22) or anti-
2 (TS1/18)
mAbs and analyzed in a cell sorter. Levels of
L
2 expression
were similar in each case, except for CHO cells expressing T208A
L
mutant/
2, which show a significantly lower expression
level.
We also introduced the T209A and T211A mutations into M
cDNA to examine the effects of the mutations on
M
2/ICAM-1
interaction. CHO cell lines expressing wt and mutant
M in
association with wt
2 were developed using the same strategy
described above. Upon immunoprecipitation, wt and mutant
M were
shown to be expressed on CHO cells in association with wt
2
(Fig. 3). The cloned CHO cells homogeneouslyexpressed high level
M
2 assayed by flow cytometry (data not shown).
Figure 3:
Surface expression of wt and mutant
M
2 on CHO cells. CHO cells stably expressing wt or mutant
M
2 were cloned using cell sorter. Cells were surface
I-labeled and lysates were immunoprecipitated with
anti-
M (OKM1) or anti-
2 (60.3) mAbs. Immunoprecipitated
samples were analyzed by SDS-polyacrylamide gel electrophoresis (7%
gel) under nonreducing conditions. Dried gels were exposed for 3
days.
The Effects of Mutations within the I Domain of
We examined the adhesion
of CHO cells expressing wt or mutant L or
M on the Interaction with ICAM-1
L or
M (with wt
2).
All the CHO cell lines examined adhered very well to fibronectin via
endogenous
5
1 (43), but not to heat-denatured BSA. The
ICAM-1/C
fusion protein concentration at approximately 0.2
µg/well gives adhesion of 50% of added CHO cells expressing wt
L (Fig. 4a) and the concentration at 0.4
µg/well was used throughout the experiments. Mn
was required to induce the adhesion of wild-type
L
transfectants. CHO cells expressing wt
L/wt
2 or T208A mutant
L/wt
2 bound to the ICAM-1, but the CHO cells expressing
T206A mutant
L/wt
2 did not (Fig. 4b). T206A
mutant
L did not respond to the increase in coating concentration
of ICAM-1 up to 1.6 µg/well (Fig. 4a). Also, CHO
cells expressing T211A mutant
M/wt
2 adhered to ICAM-1, but
those expressing T209A mutant
M/wt
2 did not (Fig. 5,
a and b). Cells expressing the T209A mutant
M
did not respond to the increase in ICAM-1 coating concentration up to
1.6 µg/well (Fig. 5a). The T209A mutant
M did
not show binding to C3bi, but T211A had no effect on the binding
(Fig. 5c). These data clearly show that Thr-206 of
L or Thr-209 of
M that correspond to Thr-221 of
2, are
critical for adhesion to ligands. By contrast, mutation of nearby
residues Thr-208 of
L or Thr-211 of
M have no effect.
Figure 4:
Effects
of mutations within L I domain on ICAM-1/
L
2
interactions. a, cells (10
/well) were added to
wells of 96-well Immulon-2 plates coated with ICAM-1/mouse C
fusion protein (0-2.0 µg/well in 100 ml of PBS) and incubated
for 1 h at 37 °C in 100 µl of Tyrode, 5 mM Hepes
buffer, pH 7.4, in the presence of 0.1% BSA and 0.1 mM
MnCl
. After rinsing the wells to remove unbound cells,
bound cells were quantified as described under ``Experimental
Procedures.'' b, cell adhesion assay was performed with
saturating concentrations of ICAM-1/mouse C
fusion protein (0. 4
µg/well) as described above. Most (91.8-98.9%) of added cells
of all cell lines bound to fibronectin (coating concentration at 1
µg/well in 100 µl of PBS). Expression (%) and mean fluorescent
intensity of
L on CHO cell lines with mAb TS1/22: parent CHO cells
(1.8%, 2.8), wt
L (94.3%, 458), T206A
L (94.6%, 428), T208A
L (99.0%, 135), D137A
L (99.8%, 532), and D239A
L (98.3%,
437). The data indicate that mutation of Thr-206, Asp-137, and Asp-239
blocked the ICAM-1/
L
2 only or C3bi interaction but that
mutation of Thr-208 did not.
Figure 5:
Effects of Thr-209 and Thr-211 mutations
of M on the interaction of
M
2 with ICAM-1 or C3bi. Cell
adhesion to ICAM-1 (a, b) or to C3bi (c)
were determined as described above except that C3bi (1 µg/well) was
directly coated onto wells. Most (93.4-99.3%) added cells of all
cell lines bound to fibronectin. Expressions (%) and mean fluorescent
intensity of
M on CHO cell lines with mAb OKM1: parent CHO cells
(1.4%, 2.1), wt
M (97.4%, 133), T209A
M (93.4%, 152), T211A
M (99.3%, 334). The data indicate that mutation of Thr-209 in
M blocked the ICAM-1 or C3bi/
M
2 interactions but that
mutation of Thr-211 did not.
CHO
cells expressing D137A or Asp-239 mutant L/wt
2 did not show
significant binding to ICAM-1 (Fig. 4b). These data
suggest the conserved Asp-137 and Asp-239 of
L are critical for
ICAM-1/
L
2 interaction, as Asp-151 and Asp-254 of
2 are
for collagen/
2
1
(27) or Asp-140 and Asp-242 of
M
are required for C3bi/
M
2
(33) interactions.
Potential Roles of the Conserved Asp Residues in Ligand/I
Domain Interactions
While mutation of Asp-151 or Asp-254 of
2 completely block the binding of
2
1 to collagen,
Asp-151 (and probably Asp-254 as well) are not critical in collagen
binding to the recombinant
2 I domain fusion
protein
(27, 32) . We demonstrated that recombinant
2 I domain/collagen interaction is not divalent cation dependent,
while
2
1/collagen interaction requires cations
(32) .
This suggests that Asp-151 and Asp-254 are involved in the regulation
of
2
1 conformation rather than in the actual binding to
collagen. Such regulation could occur through effects on accessibility
of ligands to the binding sites or maintenance of active conformation
of the ligand binding site. These effects are likely to be mediated
through binding of divalent cations to the I domain. In this study we
established that the Asp residues at position 137 and 239 of
L I
domain are also critical for binding to ICAM-1. These data indicate the
possibility that there is a regulatory mechanism of ligand binding
through divalent cation binding common to I domain-containing
integrins.
Critical Roles of the Conserved Thr Residues in Ligand/I
Domain Interaction
We have reported that the T221A mutation of
2 almost completely blocks binding of collagen to both intact
2
1 and recombinant
2 I domain
(32) . By contrast,
D151A or D254A mutations block binding of
2
1 but not
recombinant I domain
(27) . The present study establishes that
the corresponding Thr residues in
L and
M at positions 206
and 209, respectively, are critical for the interaction with ICAM-1.
The present data suggest that there may be a common mechanism of
interaction (requiring the conserved Thr residues) between I domains
and ligands as structurally distinct as the IgG superfamily protein
ICAM-1, C3bi, and collagen type I. The Thr-206 of
L are well
conserved among integrin I domains (Fig. 6), whereas sequences
around these Thr residues are not. It will be interesting to examine if
the conserved Thr residues are equally involved in ligand interactions
of other I domain-containing integrins,
1
1,
X
2, and
E
7, or in the interactions between
2 integrins and the
other ligands (e.g. ICAM-2, fibrinogen, neutrophil inhibitory
factor).
Figure 6:
The critical Thr residues are conserved
among I domains. The Thr residues (marked by *) critical in ligand
binding in 2,
L, and
M are conserved among I domains,
but amino acid sequences around the Thr residues are not.
L (8),
M (9-11),
X (12),
1 (13, 14),
2 (15),
E
(16), von Willebrand factor (vWf) (17), cartilage matrix
protein (CMP) (18), complement factors C2 and B (19, 20), and
collagen type VI (21, 22).
L
2 and
M
2 integrins play important
roles in the inflammatory response such as arthritis,
glomerulonephritis, and tissue injury induced by ischemia-reperfusion
and therefore the efforts have been made toward modulating their in
vivo functions using mAbs to
2 and
ligands
(1, 2, 7) . The present data may be
useful to focus the study for designing specific inhibitors of
ICAM-1/
2 integrin interactions on a region of the I domain.
M I domain and a linear peptide A7
(residue 232-245) of the I domain blocked the binding. The
peptide A7 includes Asp-242, but not Thr-209
(29) . The peptide
A5 that contains Thr-209 had no effect on C3bi/
M I domain
interaction
(29) . These findings are not necessarily
contradictory to the data in the present study if more than one
potential binding site is involved in ligand/I domain interactions.
Also, linear peptides are likely to have random conformation and might
not be inhibitory if conformation of the corresponding protein sequence
is critical for ligand recognition. More structural and mutagenesis
studies will be required for detailed understanding of the ligand/I
domain interactions, especially the roles of the critical Thr and Asp
residues.
M appeared
(44) . The
domain adopts a classic
/
``Rossmann'' fold and
contains an unusual Mg
coordination site at its
surface. Interestingly, Asp-140, Asp-242, and Thr-209 are all involved
in the coordination of Mg
on the surface,
underscoring the importance of these residues in ligand binding.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.