From the Center for Blood Research and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115
Received for publication, January 22, 2001
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ABSTRACT |
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The adhesiveness of integrins is regulated
through a process termed "inside-out" signaling. To
understand the molecular mechanism of integrin inside-out signaling, we
generated K562 stable cell lines that expressed LFA-1
( Integrins are heterodimeric adhesion molecules that mediate
important cell-cell and cell-extracellular matrix interactions. To
date, 25 different integrin The adhesiveness of leukocyte integrins is dynamically regulated in
cells by cytoplasmic signals, a process termed inside-out signaling.
For example, T-cell receptor cross-linking or activation of leukocytes
with phorbol esters rapidly increases adhesiveness through LFA-1
(6-8). The enhanced adhesion is transient, and by 30 min after
stimulation, cells lose their ability to bind to ICAM-1. This may
provide a mechanism for regulating T cell adhesion and de-adhesion with
antigen-presenting and target cells. In addition to activation by
intracellular signals, divalent cations can directly modulate the
ligand-binding function of leukocyte integrins (9-11). Activation can
also be mimicked by certain antibodies that bind to the extracellular
domain of the leukocyte integrins (12-15).
A key question on integrins is how signals are transduced from the
cytoplasm to the ligand-binding site in the extracellular domain. The
integrin The importance of integrin Here we test the hypothesis that association between the membrane
proximal regions of the integrin Monoclonal Antibodies--
The murine mAbs TS1/22, TS2/4
to Construction of Mutant
The
The Transient and Stable Transfection--
Transient
transfection of 293T cells was described previously (45). Stable
transfection of K562 cells and maintenance of stable cell lines were as
described previously (27, 45).
Immunofluoresence Flow Cytometry--
Flow cytometry of cells
was described previously (27). Briefly, cells (105) were
incubated with primary antibody in 100 µl of L15/fetal bovine serum
on ice for 30 min, except for KIM127 and m24. Incubation with mAbs
KIM127 and m24 was carried out at 37 °C for 30 min (9, 13). mAbs,
except for X63, were used as purified IgG at 10 µg/ml. The nonbinding
IgG X63 was used at 1:20 dilution of hybridoma supernatant. Cells were
then washed twice with L15/fetal bovine serum and incubated with
fluorescein isothiocyanate-conjugated goat anti-mouse IgG (heavy and
light chain, Zymed Laboratories Inc. Laboratories, San
Francisco, CA) for 30 min on ice. After washing, cells were resuspended
in cold phosphate-buffered saline and analyzed on a FACScan (Becton
Dickinson, San Jose, CA).
Metabolic Labeling and Immunoprecipitation--
Metabolic
labeling and immunoprecipitation was described previously (27).
Briefly, 2 × 107 cells in 4 ml of labeling medium
(methionine and cysteine-free RPMI 1640 containing 15% dialyzed fetal
bovine serum) were labeled with 0.4 mCi of
[35S]methionine and cysteine (ICN Biochemicals) overnight
in a 37 °C incubator. Labeled cells were lysed, and the lysate was
incubated with antibody-coupled-Sepharose beads overnight at 4 °C.
The immunoprecipitates were subjected to 7.5% SDS-polyacrylamide gel
electrophoresis and fluorography.
Cell Adhesion--
ICAM-1 was purified from human tonsil, and
coated on 96-well plates as described previously (27). Human complement
component iC3b was purchased from CalBiochem and coated at 10 µg/ml.
Cell adhesion to immobilized ligand was described previously (27, 45).
Bound cells were expressed as a percentage of total input cells.
The Membrane Proximal Region of the Complete Truncation of the
The
Thus, complete truncation of the Activating
mAb CBRM1/5 recognizes an activation epitope in the Replacing
To test whether the acidic and basic peptide cytoplasmic domains indeed
formed an
To test the hypothesis that cytoplasmic association between
The adhesive function of
Constitutive ligand binding by We have demonstrated that the membrane proximal region of eight
residues in the It has been shown previously that partial truncations of the
The mechanism by which membrane proximal cytoplasmic domain segments
regulate integrin activity and efficient association of the Direct association between the membrane proximal GFFKR sequence of the
The activation state of an integrin is dependent on the cell type in
which it is expressed. For example, We have demonstrated that activating cytoplasmic domain mutations
induce or enhance expression of activation epitopes in
We propose the following model for integrin activation (Fig.
1B). The membrane proximal regions of the L
2) or Mac-1
(
M
2) with mutations in the cytoplasmic
domain. Complete truncation of the
2 cytoplasmic domain,
but not a truncation that retained the membrane proximal eight
residues, resulted in constitutive activation of
L
2 and
M
2,
demonstrating the importance of this membrane proximal region in the
regulation of integrin adhesive function. Furthermore, replacement of
the
L and
2 cytoplasmic domains with
acidic and basic peptides that form an
-helical coiled coil caused
inactivation of
L
2. Association of these
artificial cytoplasmic domains was directly demonstrated. By contrast,
replacement of the
L and
2 cytoplasmic
domains with two basic peptides that do not form an
-helical coiled
coil activated
L
2. Induction of ligand
binding by the activating cytoplasmic domain mutations correlated with the induction of activation epitopes in the extracellular domain. Our data demonstrate that cytoplasmic, membrane proximal
association between integrin
and
subunits, constrains an
integrin in the inactive conformation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
heterodimers have been reported (1).
The leukocyte integrin subfamily consists of four members that share
the common
2 subunit (CD18) but have distinct
subunits,
L (CD11a),
M (CD11b),
X (CD11c), and
D for LFA-1, Mac-1,
p150,95 and
D/
2, respectively (2-4).
LFA-1 is expressed on all leukocytes and is the receptor for three Ig
superfamily members, intercellular adhesion molecule-1, -2, and -3 (ICAM1-1, -2 and -3). Mac-1
and p150,95 are primarily expressed on myeloid lineage cells and bind
ligands including ICAM-1, the complement component iC3b, and
fibrinogen. The leukocyte integrins mediate a wide range of adhesive
interactions that are essential for normal immune and inflammatory
responses (5). Patients with lymphocyte adhesion deficiency have
defective expression of leukocyte integrins on the cell surface because
of mutations in the common
2 subunit. This disease
causes an inability of phagocytic cells to bind to and migrate across
the endothelium at sites of inflammation, resulting in severe bacterial
and fungal infections (3).
and
subunits are both type I transmembrane glycoproteins. Electron microscopy of integrins reveals an overall structure with a globular headpiece connected to the plasma membrane by
two long stalks each about 16 nm long (16). The two stalks correspond
to the C-terminal portions of the
and
subunits. The headpiece
binds ligand and contains the more N-terminal domains, including a
predicted
-propeller domain and I domain in the
subunit and an
I-like domain in the
subunit. Both conformational change (affinity
regulation) and receptor clustering in the membrane (avidity
regulation) have been proposed as mechanisms for the enhancement of
integrin adhesiveness through inside-out signaling (17-20).
Conformational change in integrin I domains has been demonstrated in
structural studies (21-23) and found to regulate ligand binding affinity (23-25).
and
subunit cytoplasmic domains in
inside-out signaling has been demonstrated by mutagenesis studies.
Whereas partial deletions of the
L cytoplasmic domain have no effect on binding to ICAM-1, complete truncation of the cytoplasmic domain or internal deletion of the conserved membrane proximal GFFKR sequence constitutively activates LFA-1 (26, 27).
Truncation of the
IIb cytoplasmic domain before, but not after the conserved GFFKR sequence renders the
IIb
3 integrin constitutively active (28,
29). These findings demonstrate the importance of the membrane proximal
subunit GFFKR sequence in the regulation of integrin adhesiveness.
Partial truncations of the
3 cytoplasmic domain maintain
IIb
3 in a low affinity state, but
complete truncation or deletion of the membrane proximal seven residues
causes constitutive ligand binding by
IIb
3, indicating that this membrane
proximal region of the
3 subunit is required to maintain
IIb
3 in a low affinity state (30). It has
been suggested that interactions between the
and
subunit
cytoplasmic/transmembrane domains that include complementary negatively
and positively charged residues restrain integrins in an inactive state
(29, 31). Several proteins that associate with integrin cytoplasmic
domains have been identified (32-36); however, how these
integrin-associated proteins function in physiological activation of
integrins remains unclear.
and
cytoplasmic domains constrains an integrin in the inactive state. We demonstrate that the
membrane proximal region of the
2 cytoplasmic domain
plays an important role in the formation of cell surface
heterodimers and maintenance of
L
2 and
M
2 in an inactive state. Replacement of
the
L and
2 cytoplasmic domains with
acidic and basic peptides that form an
-helical coiled coil renders
the integrin inactive in cell types in which wild type
L
2 is either basally active or inactive,
whereas replacement of the cytoplasmic domain with two basic peptides
that do not form a heterodimer renders the integrin active in cell
types in which wild type
L
2 is either basally active or inactive. Our findings directly demonstrate that
association between the membrane proximal segments of the
and
cytoplasmic domains regulates ligand binding by integrin extracellular domains.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
L (CD11a), CBR LFA-1/7 and CBR LFA-1/2 to
2 (CD18), CBRM1/33, CBRM1/20 and CBRM1/5 to
M (CD11b), and the nonbinding IgG X63 were described
previously (15, 37-39). KIM127 (13) was kindly provided by M. Robinson (Celltech Limited, England). mAb m24 (9) was a kind gift from N. Hogg
(Imperial Cancer Fund, England). mAb 2H11 (40) was generously provided
by H-C Chang (Dana-Farber Institute, Boston).
L and
2
Subunits--
2 truncation mutants
2710*
and
2702* were generated by introducing a stop codon at
710 and 702 in the
2 subunit, respectively (the 22 amino
acid signal sequence was not included in
2 numbering; Fig. 1A). A NotI restriction site was designed in
the downstream PCR primer after the stop codon. The PCR upstream primer
corresponded to
2 cDNA sequences from 1651-1672.
The wild-type
2 in plasmid AprM8 (41) was
used as template for PCR reaction. The PCR product was cut with
BstBI at nucleotide 1977 and NotI, and swapped
with the corresponding BstB1-NotI fragment from
wild-type
2 in AprM8.
Lacid and
Lbase constructs were
generated by fusing a 30-amino acidic peptide or a 30-amino basic
peptide (42) to the extracellular and transmembrane domains of the
L subunit following residue Tyr-1087 (Fig.
1A). The constructs were made by overlap extension PCR (43,
44) using the nucleotide sequences of the acidic and basic peptides
(40). A stop codon was designed at the end of the peptide, and
following the stop codon was an SphI site. The outer left
PCR primer was 5' to the BstXI site at nucleotide 2963 of
the
L cDNA. Two rounds of overlap PCR were performed. The final PCR product was cut with BstXI and
SphI, and the BstXI-SphI fragment was
swapped into the same sites of wild-type
L cDNA in
plasmid AprM8. The unique NheI site in the
acidic and basic peptide nucleotide sequences was used for mutant identification.
2base construct was made by fusing the basic peptide
(42) to residue Trp-701 at the end of the putative
2
transmembrane domain with overlap extension PCR (Fig. 1A).
The PCR strategy was similar to that for making the
Lacid and
Lbase constructs. The final PCR
product was digested with BstBI and NotI, and the BstBI-NotI fragment was swapped into the same
sites in wild-type
2 cDNA contained in plasmid
AprM8. All mutations were verified by DNA sequencing.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2 Cytoplasmic
Domain Regulates Integrin
Heterodimer Formation on the Cell
Surface--
To examine the role of the membrane proximal region of
the
2 cytoplasmic domain in the regulation of
heterodimer formation and ligand binding activity, we generated
2 cytoplasmic domain truncation mutants
2710* and
2702*.
2710*
retained the membrane proximal sequence KALIHLSD, whereas
2702* contained a complete truncation of the
2 cytoplasmic domain (Fig.
1A). The
2
truncation mutants were transiently coexpressed with wild-type
L in 293T cells. Cell surface expression of
heterodimeric
L
2 was determined by
indirect immunofluorescence staining with mAbs TS1/22 to
L, CBR LFA-1/7 to
2, and TS2/4 to
L in the
L
2 complex (Table I). The level of cell surface
heterodimeric
L
2710* was comparable with
that of wild-type
L
2. However, the level
of
L
2702* was greatly reduced (Table I).
Complete truncation of the
2 cytoplasmic domain also
greatly reduced cell surface expression of the
M
2702* heterodimer (data not shown).
Thus, the membrane proximal sequence KALIHLSD plays a role in the
formation of cell surface
heterodimer. Complete truncation of
the
L cytoplasmic doman in mutant
L1090* reduced surface expression as previously described (27). The reduction
in expression of
L1090*
2 was similar to
that seen with
L
2702* (Table I).
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Fig. 1.
Schematic diagram of
L and
2 subunit mutants. A,
the sequences of the mutants are shown in the membrane proximal
regions. Numbers to the right (+16 and +28) indicate the number of
C-terminal residues not shown. B, scheme for regulation of
integrin adhesiveness by cytoplasmic domain association/dissociation
with the acid and base peptide fusions. The acid and base
peptides are shown as (
) and (+ + +), respectively.
Flow cytometric measurement of L
2 expression on
the surface of 293T transfectants
2 was transiently coexpressed with
wild-type
L in 293T cells. Included for comparison is the
L cytoplasmic domain truncation mutant
L1090*
(27) coexpressed with wild-type
2. Cell surface expression
of heterodimeric
L
2 was determined by flow
cytometry using mAb TS1/22 to
L, mAb TS2/4 to
L
in the
L
2 complex, and mAb CBR LFA-1/7 to
2. Mean fluorescence intensity shown was subtracted by that
of the nonbinding IgG X63. Data are mean ± difference from the
mean of two independent experiments.
2 Cytoplasmic Domain
Results in Constitutive Ligand Binding by
L
2 and
M
2--
To examine the effect of
2 cytoplasmic domain truncations on ligand binding by
L
2, the
2 truncation
mutants
2710* and
2702* were stably
coexpressed with wild-type
L in K562 cells. Clones of
wild-type
L
2,
L
2710*, and
L
2702* transfectants that expressed
similar levels of surface
L
2, as
determined by flow cytometry (Fig.
2A), were selected and tested
for their ability to bind to purified ICAM-1. Transfectants that
expressed wild-type
L
2 and
L
2710* showed low basal binding to
ICAM-1; however, binding was greatly increased by the activating mAb
CBR LFA-1/2 to the
2 subunit (Fig.
3A). By contrast,
L
2702* showed strong constitutive binding
to ICAM-1, and the level of binding was comparable with that of the
constitutively active
L truncation mutation
L1090*. CBR LFA-1/2 did not further enhance ligand
binding by
L
2702* and
L1090*
2, suggesting that these mutants are fully active without activation. All binding was specific, as shown with
inhibition by mAb TS1/22 to the
L I domain and by
comparison to mock transfectants.
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Fig. 2.
Expression of truncation mutants on the
surface of K562 cell transfectants. Wild-type
L (A) or
M (B) was
coexpressed with wild-type (WT)
2, truncation
mutant
2710* or
2702* in K562 cells, or
cells were transfected with vector alone (mock). Cell
surface expression of the
L
2
(A) and
M
2 (B)
complexes was determined by immunofluorescence flow cytometry.
Numbers in parentheses after the transfectant names are
clone numbers. mAbs and their specificity are indicated on the
left. X63 is a nonbinding myeloma IgG
control.
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Fig. 3.
Ligand binding activity of
L
2
and
M
2
mutants. A, binding to ICAM-1 of K562 stable
transfectants expressing wild-type
L
2 or
L
2 with truncated
2
(
2710* and
2702*) or truncated
L (
L1090*). ICAM-1 was immobilized in
wells of a 96-well plate, and binding of K562 transfectants was
determined in the absence (control) or presence of the
blocking mAb TS1/22 or the activating mAb CBR LFA-1/2 (10 µg/ml).
B, binding to immobilized iC3b of K562 stable transfectants
that express wild-type
M
2 or
M
2 with truncated
2. iC3b
was immobilized, and the binding of K562 transfectants was determined
in the absence (control) or presence of the blocking mAb
CBRM1/33 or the activating mAb CBR LFA-1/2 (10 µg/ml). Results are
mean ± S.D. of triplicate samples and are representative of three
independent experiments. The expression level of cell surface
L
2 and
M
2
is shown in Fig. 2.
2 cytoplasmic domain truncation mutants
2710* and
2702* were also stably
coexpressed with the wild-type
M subunit in K562 cells.
Clones of K562 transfectants that expressed similar levels of
cell surface
M
2 heterodimer, as
determined by flow cytometry (Fig. 2B), were tested for
binding to immobilized iC3b. K562 transfectants that expressed
wild-type
M
2 or
M
2710* did not bind to iC3b in the
absence of the activating mAb CBR LFA-1/2. By contrast,
M
2702* bound strongly to iC3b without
activation. Binding was specific, because it was inhibited by mAb
CBRM1/33 to the
M I domain.
2 cytoplasmic domain
constitutively activates ligand binding by
L
2 and
M
2,
whereas partial truncation that retains the membrane proximal eight
residues does not. These results suggest that the membrane proximal
region in the
2 cytoplasmic domain constrains
2 integrins in the inactive state.
2 Truncation Mutations Expose
Activation-dependent Epitopes in
L
2 and
M
2--
mAb m24 has been used as a
reporter for
L
2 activation (10, 27, 46).
Recently, mAb m24 has been mapped to the I-like domain of the
2 subunit (47). mAb KIM127 recognizes an epitope in the
2 stalk region that becomes exposed upon receptor
activation (48). We therefore tested expression of the m24 and KIM127
epitopes by
L
2 containing
2 truncation mutations. There was little expression of
the m24 epitope by wild-type
L
2 or
L
2710* (Table
II). However, the truncation mutation
2702* greatly induced the m24 epitope. Basal expression
of the KIM127 epitope on wild-type
L
2 was
higher than that of the m24 epitope, and there appeared to be a
moderate increase in the
2710* mutant. However,
expression of the KIM127 epitope was greatly increased by the
2702* mutation (Table II). Expression of the KIM127
epitope on
L
2702* was nearly maximal; i.e. comparable with constitutively expressed epitopes such
as TS2/4.
Expression of activation epitopes by L
2 mutants
L
2 were stably expressed in
K562 cells, and reactivity of transfectants with mAb m24 and KIM127 was
determined by immunofluorescence flow cytometry. Mean fluorescence
staining of each antibody after subtraction of the mean fluorescence of
the control IgG X63 is expressed as the % mean fluorescence with mAb
TS2/4. mAb TS2/4 recognizes the
L subunit in the
L
2 complex and reacts with wild-type and mutant
L
2 equally well as shown by comparison to many
other mAb specific for the
L and
2 subunits. Data
are mean ± difference from the mean of two independent
experiments.
M
I-domain near the metal ion-dependent adhesion site (MIDAS)
motif (39, 45). Expression of the CBRM1/5 epitope was greatly enhanced by the activating
2 truncation mutation
2702*, whereas the
2710* mutation did not
significantly increase CBRM1/5 binding compared with wild type (Table
III). The
2702* mutation
also greatly increased mAb KIM127 binding to
M
2 (Table III). Thus, constitutively
strong ligand binding by
L
2 and
M
2 containing the truncation mutation
2702* correlates with exposure of activation epitopes in
the extracellular domain.
Expression of activation epitopes by M
2 mutants
M
2 with wild-type or truncated
2 was determined by immunofluorescence flow cytometry. Mean
fluorescence staining of each antibody was subtracted by the mean
fluorescence of the control IgG X63, and is expressed as % mean
fluorescence staining of mAb CBRM1/20, which binds to
M in
the
M
2 complex. mAb CBRM1/20 reacts with
wild-type and mutant
M
2 equally well. Data are
mean ± difference from the mean of two independent experiments.
L and
2 Cytoplasmic Domains
with an
-Helical Coiled Coil Constrains
L
2 in the Inactive State--
The above
results suggest that the membrane proximal region of the
2 subunit cytoplasmic domains play an important role in the regulation of
2 integrin function and formation of
the
heterodimer. The membrane proximal GFFKR sequence in the
L cytoplasmic domain regulates
and
heterodimerization and ligand binding by
L
2 (27). We hypothesized, therefore, that
the membrane proximal regions of the
and
cytoplasmic domains
associate, and such association constrains the integrin in an inactive
conformation. To test this hypothesis, we replaced the cytoplasmic
domains of
L and
2 with a heterodimeric
coiled coil. Peptides termed "acid" and "base" were fused to
L and
2. These peptides preferentially form heterodimeric as opposed to homodimeric
-helical coiled coils
(42). These fusions were termed
Lacid and
2base, respectively (Fig. 1). As a control, both
L and
2 cytoplasmic domains were replaced
by the basic peptide (
Lbase and
2base).
Dimerization of the two basic peptides is disfavored because of
interhelical electrostatic repulsion (42). K562 cell clones were
selected that stably expressed
Lacid
2base
and
Lbase
2base at similar levels on the
cell surface (Fig. 4).
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Fig. 4.
Expression of
L
2
coiled coil fusion mutants on the surface of K562 stable
transfectants. K562 cells were stably co-transfected with
wild-type
L and
2,
Lacid
and
2base, or
Lbase and
2base. Clones of the stable K562 transfectants were
stained with the indicated mAbs (shown on the left) and
analyzed by flow cytometry. Clone numbers are indicated in
parentheses. X63, nonbinding IgG.
-helical coiled coil, we examined reactivity with mAb
2H11, which specifically recognizes the acidic and basic peptide
heterodimer, and not monomer or homodimer (40). mAb 2H11
immunoprecipitated the
Lacid
2base
complex, but not wild-type
L
2 or
Lbase
2base (Fig.
5). By contrast, mAb TS2/4 to the
L subunit immunoprecipitated all three types of
L
2 heterodimers (Fig. 5). These results
demonstrate that in
Lacid
2base, the cytoplasmic peptides noncovalently associate to form an
-helical coiled coil.
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Fig. 5.
The acid and base peptides of
Lacid
2base
associate in an
-helical coiled coil.
Mock-transfected K562 cells or K562 transfectants that stably express
wild-type
L
2,
Lacid
2base or
Lbase
2base were metabolically labeled
with [35S]methionine and cysteine. Lysates of the labeled
cells were immunoprecipitated with mAb TS2/4 specific for
L in the
L
2 complex or mAb
2H11 specific for the
-helical coiled coil formed by the acidic and
basic peptides. Lysates from equal numbers of labeled cells were
subjected to immunoprecipitation, SDS 7.5% polyacrylamide gel
electrophoresis, and fluorography. The
L subunit with
the acidic peptide or basic peptide and the
subunit with the basic
peptide migrated slightly faster than wild-type
L and
2, respectively.
L and
2 regulates ligand binding, ligand
binding activity was compared of
Lacid
2base,
Lbase
2base, and wild-type
L
2. Clones of K562 stable transfectants
that expressed similar levels of surface
L
2 (Fig. 4) were tested for binding to
immobilized ICAM-1. Cells that expressed wild-type
L
2 or
Lacid
2base did not bind to ICAM-1 without
activation (Fig. 6A). The
activating mAb CBR LFA-1/2 or Mn2+ greatly increased
binding of both wild-type
L
2 and
Lacid
2base to ICAM-1. By contrast, cells
expressing
Lbase
2base strongly bound to
ICAM-1 in the absence of activation, and mAb CBR LFA-1/2 or
Mn2+ did not further increase binding by
Lbase
2base (Fig. 6A).
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Fig. 6.
Binding of
L
2
coiled coil fusion mutants to purified ICAM-1. A,
binding of stable K562 transfectants to ICAM-1. Binding of cells to
ICAM-1 was performed in the absence (control) or presence of
10 µg/ml of the inhibitory mAb TS1/22 or the activating mAb CBR
LFA-1/2 in L15 medium that contains Mg2+ and
Ca2+. For the Mn2+ experiment, the binding
assay was conducted in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl supplemented with 1 mM
MnCl2. Cell surface expression of
L
2 is shown in Fig. 4. The results are
mean ± S.D. of triplicate samples and are representative of three
independent experiments. B, binding of 293T transient
transfectants to ICAM-1. 293T cells were transiently transfected, and
binding of the transfectants was determined in the absence
(control) or presence of the inhibitory mAb TS1/22 or the
activating mAb CBR LFA-1/2 at 10 µg/ml. The percent of cells bound to
ICAM-1 was divided by the fraction of transiently transfected cells
expressing
L
2. The percent of cells
expressing
L
2 was 90, 44, and 18% for
wild-type
L
2,
Lacid
2base, and
Lbase
2base transfectants, respectively
(determined by flow cytometry with mAb TS2/4 to
L in the
L
2 complex). The data are mean ± S.D. of triplicate samples and are representative of two independent
experiments.
Lacid
2base
and
Lbase
2base was further examined in
293T transfectants, in which wild-type
L
2 is basally active (Fig. 6B). Wild-type
L
2 and
Lbase
2base in 293T transfectants
constitutively bound to ICAM-1, and binding was specific as shown by
inhibition with mAb TS1/22 to the
L I domain. By
contrast,
Lacid
2base showed little
binding. However, mAb CBR LFA-1/2 increased ligand binding by
Lacid
2base to a level comparable with
wild-type
L
2 and
Lbase
2base, indicating that lack of
ligand binding by
Lacid
2base was not
because of loss of function (Fig. 6B).
Lbase
2base
correlated with the expression of activation epitopes in the
extracellular domain (Table II). Binding of
activation-dependent mAbs m24 and KIM127 to
Lbase
2base in K562 cells was greatly
increased compared with wild-type
L
2,
whereas the level of m24 and KIM127 binding to
Lacid
2base was similar to wild-type
L
2 (Table II).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2 cytoplasmic domain is required for
efficient expression of integrin
L
2 and
M
2 heterodimers on the cell surface and
for maintenance of these integrins in the inactive state. Furthermore,
we have provided evidence that membrane proximal cytoplasmic
association between the
and
subunits constrains an integrin in
the inactive state, whereas a lack of association between membrane
proximal regions activates an integrin.
2 subunit cytoplasmic domain, or mutation of the three
contiguous threonines at amino acid residues 736-738 or the
phenylalanine at residue 744, abolishes LFA-1 activation by phorbol
ester, suggesting that the membrane distal region is involved in the
inducible activation of LFA-1 (26, 49). A more recent study showed that
complete truncation of the
2 cytoplasmic domain
activated binding through
L
2 of K562
transfectants to ICAM-1 (50). However, the previous studies did not
localize the region in the
2 cytoplasmic domain that
maintains LFA-1 in the default inactive state. We have shown that the
function of restraining LFA-1 in an inactive state can be localized to
a membrane proximal segment with the sequence KALIHLSD. Complete
truncation of the
2 cytoplasmic domain, but not
truncation after the KALIHLSD sequence, constitutively activated ligand
binding by
L
2. Furthermore, we generalized this observation to
another
2 integrin,
M
2.
Moreover, we demonstrated that the KALIHLSD sequence is required for
efficient formation of cell surface
L
2
and
M
2 heterodimers. The membrane
proximal sequence in the cytoplasmic domain is conserved among integrin
subunits (50). Deletion of the membrane proximal seven residues in
the
3 cytoplasmic domain results in a constitutively
active
IIb
3 integrin (30). Similarly, in
integrin
subunits, the conserved membrane proximal GFFKR sequence
has been shown to be important for regulating integrin
heterodimer assembly and adhesive function (27, 29, 31). Thus, the
conserved membrane proximal regions of both integrin
and
cytoplasmic domains control integrin subunit association and ligand
binding activity.
and
subunits has been unclear. Our data strongly support the hypothesis
that association between these segments regulates ligand binding
activity. We replaced the cytoplasmic domains in
L
2 with peptides that either favor or
disfavor association to form an
-helical coiled coil (42).
Association of the membrane proximal segments was confirmed by
reactivity with mAb 2H11 that recognizes the acid and base peptides
only when they are associated with one another in a coiled coil
heterodimer (40). We tested the integrin coiled coil fusions in two
different cellular contexts, 293T cells and K562 cells, in which
wild-type
L
2 is active and inactive,
respectively. 293T transfectants that expressed
Lacid
2base showed little binding to
ICAM-1, whereas cells expressing wild-type
L
2 strongly bound to ICAM-1. The
activating mAb CBR LFA-1/2 to the
2 subunit or Mn2+
could activate ligand binding by
Lacid
2base, showing that its extracellular domain is competent for activation. Furthermore,
Lbase
2base, in which association of the
cytoplasmic basic peptides is disfavored, was constitutively active
when expressed in K562 cells in which wild-type
L
2 has little basal activity. These findings provide strong evidence that association of membrane proximal
cytoplasmic domains renders an integrin inactive, and that lack of
association renders an integrin active (Fig. 1B).
IIb subunit and the KLLITIHD sequence of the
3 subunit has been proposed previously based on
mutational studies (31). Mutation of Arg-995 in the
IIb
GFFKR sequence or Asp-723 in the
3 KLLITIHD sequence
resulted in 39-70% activation of
IIb
3, whereas the complementary mutations
IIb Arg-995
Asp
and
3 Asp-723
Arg resulted in a significantly lower
activation of 12% (31). However, no direct evidence for association
between the
IIb and
3 cytoplasmic domains
was presented. Moreover, we cannot generalize this result to
2 integrins, because mutation of the corresponding
Asp-709 in the
2 cytoplasmic domain did not activate
ligand binding by
L
2 and
M
2 (3 and 2% of maximal binding,
respectively; data not shown). Thus, mechanisms other than a proposed
salt bridge between integrin
and
cytoplasmic domains may
regulate integrin adhesiveness. Indeed, despite extensive evidence that
mutations of the membrane proximal segments of the integrin cytoplasmic domains are activating, there has to date been no direct evidence that
association between these segments, either direct or mediated by
integrin-associated proteins, regulates adhesiveness. We have demonstrated for the first time that close spatial proximity between membrane proximal
and
subunit segments maintains integrins in
an inactive state and that a lack of association results in activation.
2 integrins in K562 cells require activation to bind to ligands, whereas
2
integrins are constitutively active in 293T cells, as shown here and
previously (25). Presumably, this is due to differential expression of proteins or other factors that modulate integrin function in different cell types. We examined the effect of the
L
2 coiled coil fusions in both types of
cellular environments. Interestingly, the effect of the coiled coil
mutations was independent of cellular environment. Thus,
Lacid
2base was inactive in both K562 and
293T cells, whereas
Lbase
2base was active
in both cell types. The dominance of the peptides over the cellular
environments suggests that the factors that modulate differential
integrin activity exert their effect by binding to the cytoplasmic
domains of the integrins. Activation of these factors by cytoplasmic
signals may regulate binding to integrin cytoplasmic domains and hence
the transition between inactive and active wild-type integrins (Fig.
1B).
L
2 and
M
2.
For
L
2, complete truncation of the
2 cytoplasmic domain or replacement of the
L and
2 cytoplasmic domains with the
basic peptides exposed the m24 epitope and enhanced KIM127 epitope
expression. Both epitopes localize to the
2 subunit. The
m24 epitope localizes to loops in the I-like domain that are predicted
to be near its metal ion-dependent adhesion-like site (47).
The KIM127 epitope localizes within cysteine-rich repeat 2, to residues
504, 506, and 508 in the C-terminal region (48). For
M
2, complete truncation of the
2 cytoplasmic domain greatly increased expression of the
CBRM1/5 epitope in the
M I domain, as well as the KIM127
epitope in
2. CBRM1/5 binds to the
M I domain very
close to the ligand binding site (39, 45). Thus, the activating
cytoplasmic domain mutations cause conformational changes in diverse
extracellular domains of
L
2 and
M
2. It has previously been shown that
complete truncation of the
2 cytoplasmic domain alters
the localization of LFA-1 into clusters, and hence increases cell
adhesion (50). Our results suggest that conformational change (affinity
regulation), as well as receptor clustering (avidity regulation) play a
role in the enhanced adhesiveness of
L
2
and
M
2 induced by activating cytoplasmic
domain mutations.
and
subunit cytoplasmic domains can associate either directly or
indirectly. Under physiological conditions, there is an equilibrium
between the association and dissociation of the membrane proximal
segments of the cytoplasmic domains that is dynamically regulated.
Association constrains the integrin in the inactive state, and
dissociation results in activation. Inside-out signaling and integrin
binding proteins regulate this equilibrium. Separation of the two
membrane proximal regions results in activation. Truncation of the
or
subunit cytoplasmic domain or deletion of the membrane proximal
region disrupts this constraint, and activates the integrin. The
inactive and active states of an integrin can be mimicked by replacing the integrin cytoplasmic domains with peptides that favor or disfavor, respectively, noncovalent association into a coiled coil. Thus, association of membrane proximal cytoplasmic segments is sufficient to
regulate integrin activation and conformational change.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Hsiu-Ching Chang, Nancy Hogg, and Martyn Robinson for providing mAbs.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants CA31798 and CA31799.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a fellowship from the Cancer Research Institute.
Present address: Millennium Pharmaceuticals, 75 Sidney St., Cambridge,
MA 02139.
§ To whom correspondence should be addressed: Tel.: 617-278-3200; Fax: 617-278-3232.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M100600200
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ABBREVIATIONS |
---|
The abbreviations used are: ICAM, intercellular adhesion molecule; mAb, monoclonal antibody; PCR, polymerase chain reaction; FACS, fluorescent-activated cell sorter.
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