From the Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom
Received for publication, December 23, 2002, and in revised form, February 27, 2003
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ABSTRACT |
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The ligand-binding head region of integrin Integrins mediate a wide variety of essential cell-matrix and
cell-cell interactions and also participate in many common disease processes (1, 2). Integrins are heterodimers containing non-covalently
associated The molecular basis of integrin function has been powerfully elucidated
by the recent x-ray crystal structures of the extracellular domains of
Recently, evidence (16, 17) has been presented that the bent form of
the integrin is in the inactive state and that this may undergo a
switchblade-like straightening to attain the active conformation.
Nevertheless, precisely how this straightening is linked to activation
of ligand binding in the head domain is uncertain. A key regulator of
integrin activity is known to be the conformation of the Here we provide evidence that changes in the expression of activation
epitopes in the hybrid domain are linked to shape-shifting in the Monoclonal Antibodies--
Rat mAbs 16 and 13 recognizing the
human Expression Vector Construction and Mutagenesis--
C-terminally
truncated human Transfection--
Chinese hamster ovary cells L761h variant (24)
were maintained in Dulbecco's modified Eagle's medium supplemented
with 10% fetal calf serum, 2 mM glutamine, and 1%
non-essential amino acids (growth medium). Cells were detached using
0.05% (w/v) trypsin, 0.02% (w/v) EDTA in phosphate-buffered saline,
and plated overnight into 6-well culture plates (Costar). Approximately
1 µg of wild-type or mutant
For comparison of purified wild-type heterodimers with heterodimers
containing the L358A or S359A mutations in Proteins--
A recombinant fragment of fibronectin containing
type III repeats 6-10 (III6-10) was produced and purified
as described previously (12). A mutant fragment in which the RGD
integrin-binding sequence is replaced by the inactive sequence KGE
(III6-10KGE, see Ref. 26) was produced and purified in the
same manner. III6-10 was biotinylated as before (8) using
sulfo-LC-NHS biotin (Perbio, Chester, UK). Fab fragments of N29,
TS2/16, and 12G10 were prepared by ficin cleavage of purified IgG,
followed by removal of Fc-containing fragments using protein
A-Sepharose, according to the manufacturer's instructions (Perbio).
None of the Fab fragments showed any reactivity with goat anti-mouse
IgG (Fc-specific) peroxidase conjugate (Sigma).
Effect of Divalent Cations on 15/7 and HUTS-4
Binding--
96-Well plates (Costar 1/2-area EIA/RIA, Corning
Science Products, High Wycombe, UK) were coated with goat anti-human
For comparison of the effects of divalent cations on 15/7 and HUTS-4
binding to wild-type heterodimer and the L358A and S359A mutants,
plates were coated with anti-human Fc, blocked as described above, and
then incubated with supernatant from cells transfected with wild-type
or mutant heterodimers. mAb binding was measured as described above in
2 mM EDTA, 2 mM Mn2+, 2 mM Mg2+, or 2 mM Ca2+.
Measurements obtained were the mean ± S.D. of four replicate wells.
Effect of mAbs and Ligand on 15/7 and HUTS-4 Binding--
Plates
were coated with anti-human Fc and blocked as described above. Wells
were then incubated with supernatant from cells transfected with
wild-type or mutant heterodimers for 1-2 h at room temperature as
above. Wells were washed three times with 200 µl of 150 mM NaCl, 1 mM MnCl2, 25 mM Tris-Cl, pH 7.4, containing 1 mg/ml BSA (buffer B). 15/7
or HUTS-4 (l µg/ml in buffer B) was added to the plates (50 µl/well) either alone or in the presence of Fab fragments of N29,
TS2/16, or 12G10 (5 µg/ml), mAb 13 IgG (10 µg/ml), or
III6-10 (20 µg/ml). The plates were then incubated at
30 °C for 2 h. Unbound antibody was aspirated, and the wells
were washed three times with buffer B. Bound 15/7 or HUTS-4 was
quantitated by addition of 1:2000 dilution of goat anti-mouse IgG
(Fc-specific, precleared with rat serum proteins) peroxidase conjugate
(Sigma) in buffer B for 30 min at room temperature (50 µl/well).
Wells were then washed four times with buffer A, and color was
developed as above. Background binding of mAbs to wells incubated with
supernatant from mock-transfected cells was subtracted from all
measurements. Measurements obtained were the mean ± S.D. of four
replicate wells.
Comparison of Epitope Expression by Wild-type and Mutant
Heterodimers--
Plates were coated with anti-human Fc and blocked as
described above. The blocking solution was removed, and cell culture supernatants were added (25 µl/well) for 1-2 h. All supernatants were assayed in triplicate, and supernatant from mock-transfected cells
was used as a negative control. The plate was washed 3 times with
buffer B (200 µl/well), and anti- Effect of L358A and S359A Mutations on III6-10
Binding--
Plates were coated with anti-human Fc and blocked as
described above. Wells were then incubated with protein A-purified
heterodimers diluted to ~1 µg/ml with 150 mM NaCl, 25 mM Tris-Cl, pH 7.4 (25 µl/well), for 1-2 h at room
temperature. Wells were washed three times with 200 µl of buffer B. Biotinylated III6-10 (0.1 µg/ml) in buffer B was added
to the plate (50 µl/well) alone or in the presence of N29, TS2/16, or
12G10 (5 µg/ml). The plate was then incubated at 30 °C for 2 h. Unbound ligand was aspirated, and the wells were washed three times
with buffer B. Bound ligand was quantitated by addition of 1:500
dilution of ExtrAvidin® peroxidase conjugate (Sigma) in buffer B for
20 min at room temperature (50 µl/well). Wells were then washed four
times with buffer B, and color was developed using
2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) substrate
(50 µl/well). Background binding to BSA-coated wells was subtracted
from all measurements. Measurements obtained were the mean ± S.D.
of four replicate wells.
Mapping of the 15/7 and HUTS-4 Epitopes--
Substitution of
human residues with the corresponding residues in murine
In each assay involving a comparison between different heterodimers,
the binding of mAb 8E3 (5 µg/ml) was used to normalize any
differences between the amounts of the different heterodimers bound to
the wells. For example, normalized A405 for 15/7
binding = (AM15/7 Homology Modeling of the Head Region of
Expression of the 15/7 and HUTS-4 Epitopes Is Regulated by
Conformational Changes in the
Activation of the integrin head is known to involve conformational
changes in
Conformational changes in
Taking these data together, the active conformation of A Mutation in the The L358A Mutant Mimics the Ligand-occupied Conformation--
We
next tested whether the L358A mutant caused any other conformational
changes associated with activation or ligand binding. For this purpose
we used a panel of mAbs recognizing epitopes on both the
The L358A Mutation Causes Activation of Ligand Binding--
If the
conformation of the L358A mutant is akin to the ligand-occupied state,
it would be predicted that this mutant should be constitutively active
for ligand binding (38). Tr The Epitopes of 15/7 and HUTS-4 Map to a Region of the Hybrid
Domain Close to an Interface with the
Comparison with the crystal structures of
By using the conformation-sensitive mAbs 15/7 and HUTS-4 and
site-directed mutagenesis, we have studied conformational changes in
integrin subunits contains a von Willebrand factor type A domain (
A). Ligand
binding activity is regulated through conformational changes in
A,
and ligand recognition also causes conformational changes that are transduced from this domain. The molecular basis of signal transduction to and from
A is uncertain. The epitopes of mAbs 15/7 and
HUTS-4 lie in the
1 subunit hybrid domain,
which is connected to the lower face of
A. Changes in the expression
of these epitopes are induced by conformational changes in
A caused
by divalent cations, function perturbing mAbs, or ligand
recognition. Recombinant truncated
5
1
with a mutation L358A in the
7 helix of
A has constitutively high
expression of the 15/7 and HUTS-4 epitopes, mimics the conformation of
the ligand-occupied receptor, and has high constitutive ligand binding
activity. The epitopes of 15/7 and HUTS-4 map to a region of the hybrid
domain that lies close to an interface with the
subunit. Taken
together, these data suggest that the transduction of
conformational changes through
A involves shape shifting in
the
7 helix region, which is linked to a swing of the hybrid domain
away from the
subunit.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
subunits; each subunit has a large extracellular
domain linked to a transmembrane segment and a short cytoplasmic tail.
Integrins participate in bi-directional signaling; ligand recognition
is dynamically regulated by "inside-out" signaling, and ligand
occupancy leads to "outside-in" signals that affect cell migration,
growth, differentiation, and survival (3-5). Modulation of integrin
activity is essential in such processes as leukocyte migration to sites
of tissue injury and the aggregation of platelets to form a hemostatic
plug. Integrin activation can be mimicked in vitro by
divalent cations such as Mn2+ or Mg2+ (6).
Three major conformational states of integrins can be distinguished
using monoclonal antibodies
(mAbs)1: an inactive (resting
or low affinity) state, an active (or high affinity) state, and a
ligand-occupied state (7). The conformations of the inactive and active
states are discriminated by low and high expression, respectively, of
activation epitopes (such as those recognized by 12G10, 15/7, and 9EG7
for the
1 subunit, see Refs. 8-10). The ligand-occupied
conformer expresses high levels of ligand-induced binding site (LIBS)
epitopes (which are generally also activation epitopes) and shows
decreased expression of ligand-attenuated binding site (LABS) epitopes
(such as mAb 13 for the
1 subunit, see Ref. 11). The
conformational states are in equilibrium; therefore, antibodies that
recognize activation epitopes or LIBS tend to cause activation and
stabilize the ligand-occupied state. Conversely, antibodies that
recognize LABS appear to block ligand binding by preventing
conformational changes involved in ligand recognition (7, 11, 12).
V
3 in both an unliganded state (13) and
in complex with a small peptide ligand (14). Overall, the integrin
structure resembles that of a "head" on two "legs." The
ligand-binding head region of the integrin contains a seven-bladed
-propeller in the
subunit, the top face of which is in close
juxtaposition with a von Willebrand factor type A domain in the
subunit (
A).
A consists of seven
helices encircling a central
-sheet and is connected at its N and C termini to an
immunoglobulin-like "hybrid" domain and forms an extensive
interface with it. The key regions involved in ligand recognition are
loops on the upper surface of the
-propeller and the upper face of
the
A, which contains a metal ion-dependent adhesion
site (MIDAS) and an adjacent MIDAS cation-binding site (13-15). A
small number of subtle conformational changes between the unliganded
and liganded states were observed. The most important of these appeared
to be a shift of the
1 helix in
A, and a slight closing up of the
interface between the upper surface of the
-propeller and the upper
face of the
A. A surprising feature of the crystal structures was
that the two legs are severely bent at the "knees," such that the
head is in close contact with lower legs. Because the peptide ligand
was soaked into the crystals of unliganded
V
3, it is unclear whether the small
conformational changes observed between the unliganded and liganded
structures (13, 14) are representative of those that take place upon ligand occupancy of the native integrin. Importantly, no pathway for
the transduction of conformational changes from the head to the legs
(or from legs to head) was evident. Hence, the molecular basis of both
outside-in and inside-out signaling remains to be clarified.
A domain
(15, 18), and we have shown that a movement of the
1 helix activates
this domain (8). We hypothesized that the
1 helix could occupy two
different positions: position 1 characterized by high binding of the
mAb 12G10 (the active conformation), and position 2 characterized by
low binding of 12G10 (the inactive conformation). The inward movement
of
1 helix observed in the liganded
V
3
structure (14) supports this proposal. Hence, position 1 appears to
correspond to the "in" state and position 2 to the "out" state
of the
1 helix. About half of the integrin
subunits contain a
similar domain (
A or I), and in these domains an inward movement of
the
1 helix is linked to rearrangement of cation-coordinating
residues at the MIDAS and a dramatic downward shift of the C-terminal
7 helix and its preceding loop (19). However, no change in the
position of the
7 helix of
A was observed between the two x-ray
structures, and it was suggested that activation of
A does not
involve
7 movement (13-14, 20).
7
helix region of
A. This movement appears to participate in the
conformational changes involved in both activation and ligand binding.
Our data suggest that an outward swing of the hybrid domain is coupled
to
7 helix motion, and hence lend support to a recent model of
integrin activation (21). There are both strong similarities and some
differences between
A and
A domain activation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5 and
1 subunits, respectively,
were gifts from Dr. K. Yamada (NIDCR, National Institutes of Health,
Bethesda). Mouse anti-human
5 mAb P1D6 was a gift from
Dr. E. Wayner (Fred Hutchinson Cancer Research Center, Seattle, WA).
Mouse anti-human
5 mAb SNAKA52 and mouse anti-human
1 mAbs 12G10 and 8E3 were produced as described (22, 23). Mouse anti-human mAb TS2/16 was a gift from F. Sánchez-Madrid (Hospital de la Princesa, Madrid, Spain). Mouse
anti-human mAbs JB1A and N29 were gifts from J. Wilkins (University of
Manitoba, Winnipeg, Canada). Mouse anti-human mAb 15/7 was a gift from
T. Yednock (Elan Pharmaceuticals, South San Francisco, CA). Mouse anti-human mAbs 4B4 and HUTS-4 were purchased from Beckman Coulter (High Wycombe, UK) and Chemicon (Harrow, UK), respectively. All mAbs
were used as purified IgG.
5 and
1 constructs
encoding
5 residues 1-613 and
1 residues
1-455 fused to the hinge regions and CH2 and
CH3 domains of human IgG
1 (
5-(1-613)-Fc
and
1-(1-455)-Fc) were generated as described
previously (24). To aid the formation of heterodimers, the
CH3 domain of the
5 construct contained a
"hole" mutation, whereas the CH3 domain of the
1 constructs carried a "knob" mutation as described
(24, 25). The L358A and S359A mutations in the
1
subunit were carried out using oligonucleotide-directed PCR
mutagenesis, as described (24). Oligonucleotides were purchased from
MWG Biotech (Southampton, UK). The presence of the mutations (and
the lack of any other changes to the wild-type sequence) was verified
by DNA sequencing.
1-(1-455)-Fc or
1-(121-455)-Fc and 1 µg of wild-type
5-(1-613)-Fc DNA/well was used to transfect the cells
using LipofectAMINE Plus reagent (Invitrogen) according to the
manufacturer's instructions. After 4 days, supernatants were harvested
by centrifugation at 1000 × g for 5 min.
1,
75-cm2 flasks of sub-confluent CHOL761h cells were
transfected with 5 µg of wild-type or mutant
1-(1-455)-Fc and 5 µg of
5-(1-613)-Fc DNA as described above. After 4 days, culture supernatants were harvested by centrifugation at 1000 × g for 5 min.
Wild-type or mutant heterodimers were purified using protein
A-Sepharose essentially as described before (24).
1 Fc (Jackson Immunochemicals, Stratech Scientific, Luton, UK) at a concentration of 2.6 µg/ml in Dulbecco's phosphate-buffered saline (50 µl/well) for 16 h. Wells were then blocked for 1
3 h with 200 µl of 5% (w/v) BSA, 150 mM NaCl, 0.05% (w/v)
NaN3, 25 mM Tris-Cl, pH 7.4 (blocking buffer).
Blocking buffer was removed, and supernatant from cells transfected
with wild-type
5-(1-613) and
1-(1-455)-Fc diluted 1:1 with 150 mM NaCl,
25 mM Tris-Cl, pH 7.4 (25 µl/well), was added for 1-2 h
at room temperature. Wells were then washed three times with 200 µl
of 150 mM NaCl, 25 mM Tris-Cl, pH 7.4, containing 1 mg/ml BSA (buffer A). Buffer A was treated with Chelex beads (Bio-Rad) to remove any small contaminating amounts of endogenous Ca2+ and Mg2+ ions. mAbs (1 µg/ml) in buffer
A with varying concentrations of Mn2+, Mg2+, or
Ca2+ were added to the plate (50 µl/well). The plate was
then incubated at 30 °C for 2 h. Unbound antibody was
aspirated, and the wells were washed three times with buffer A. Bound
antibody was quantitated by addition of 1:1000 dilution of goat
anti-mouse IgG (Fc-specific) peroxidase conjugate (Jackson
Immunochemicals) in buffer A for 30 min at room temperature (50 µl/well). Wells were then washed four times with buffer A, and color
was developed using 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) substrate (50 µl/well). Absorption at 405 nm was measured using
a plate reader (Dynex Technologies). Background binding to mAbs to
wells incubated with supernatant from mock-transfected cells was
subtracted from all measurements. Measurements obtained were the
mean ± S.D. of four replicate wells.
5 or
anti-
1 mAb (5 µg/ml) was added (50 µl/well). The
plate was incubated for 2 h and then washed 3 times in buffer B. Peroxidase-conjugated anti-rat or anti-mouse secondary antibodies
(1:1000 dilution in buffer B; Jackson Immunochemicals) were added (50 µl/well) for 30 min, and the plate was washed four times in buffer B,
and color was developed as above. All steps were performed at room
temperature. Results shown are the mean ± S.D. of three separate experiments.
1 within the hybrid domain sequence 361-425 was performed using a PCR-based mutagenesis kit (Gene Tailor, Invitrogen) according to the manufacturer's instructions. CHOL761h cells were transfected with wild-type or mutant constructs and supernatants harvested as described above. Binding of 15/7, HUTS-4, and TS2/16 to
mutant heterodimers was performed as described above, relative to the
wild-type control. Background binding to mAbs to wells incubated with
supernatant from mock-transfected cells was subtracted from all
measurements. Measurements obtained were the mean ± S.D. of three
replicate wells. Results shown are mean ± S.D. of three separate experiments.
Am15/7) × ((AWT8E3
Am8E3)/(AM8E3
Am8E3)), where AM15/7 = mean absorbance of wells
coated with mutant integrin; Am15/7 = mean
absorbance of wells coated with mock supernatant; AWT8E3 = mean absorbance of 8E3 binding to wells
coated with wild-type integrin; Am8E3 = mean
absorbance of 8E3 binding to wells coated with mock supernatant, and
AM8E3 = mean absorbance of 8E3 binding to wells
coated with mutant integrin. 8E3 recognizes a non-functional epitope in
the N-terminal region of the
1 subunit (24). Essentially identical results were obtained from normalization using mAb N29 against the PSI domain (Ref. 27, data not shown). In experiments using heterodimers captured from cell culture supernatants, similar results were obtained using protein A-purified heterodimers (data not shown). Each experiment shown is representative of at least three
separate experiments.
5
1--
A model of the
5-propeller and thigh domains and
1A and
hybrid domains was built based on an alignment against the
V
3 crystal structure (13), using the same
procedures as described previously (8). The PSI domain (residues 1-60
of
1) was not included in the model. Representation of
the structure was produced using
PyMol.2
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A Domain--
To investigate the
mechanisms of integrin activation, we employed a recently described
system for expression of recombinant soluble
5
1 (24). For these particular studies, we
have used a truncated version of
5
1,
5-(1-613)
1-(1-455), fused to the Fc
region of human IgG
1 (24) (hereafter referred to as
tr
5
1-Fc). This heterodimer contains the
subunit
-propeller and thigh domain, and the
subunit A,
hybrid, and PSI domains (13), and has been shown to retain the
properties of the full-length receptor (24). This system is
particularly useful (a) because it permits the rapid
analysis of the effects of mutations, and (b) because conformational changes in the head region can be studied in isolation, i.e. in the absence of any complicating effects due to the
presence of the lower leg domains (e.g. unbending, see Refs.
16 and 17) or the cytoplasmic tails (5).
A, and since
A is connected at its N and C termini to
the hybrid domain, these changes must be transduced from and to the
hybrid. HUTS-4 and 15/7 are two previously characterized mAbs whose
epitopes lie within this region of the
1 subunit (9, 29-31). Expression of the 15/7 and HUTS-4 epitopes by
tr
5
1-Fc was found to be cation-modulated
(Fig. 1, A and B).
Binding of each mAb was promoted by Mn2+ and to a smaller
extent by Mg2+, whereas Ca2+ did not stimulate
binding. Importantly, these effects parallel the effects of each
divalent ion on the ligand-binding competence of the integrin (32), and
they also mirror a conformational change in the
A domain reported by
mAb 12G10 (8). These changes have been shown to be due to cation
binding to the MIDAS (8), and in agreement with this, the MIDAS
mutation D130A prevented the cation modulation of 15/7 and HUTS-4
binding (data not shown).
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Fig. 1.
Effect of divalent cations on the binding of
15/7 (A) and HUTS-4 (B) to
tr 5
1-Fc.
Binding of mAbs was measured in the presence of varying
concentrations of Mn2+ (
), Mg2+ (
), or
Ca2+ (
).
A can also be induced by
function-perturbing mAbs with epitopes in this domain. The epitopes of all function-altering anti-human
1 mAbs that map to the
A domain include one or more residues in the sequence
Asn207-Lys218 (33), which is predicted to form
the
2 helix region (13). The epitope of mAb 12G10 also includes two
arginyl residues that lie near the base of the
1 helix (8). TS2/16
and 12G10 are examples of activating mAbs, whereas 13 is an example of
a function-blocking mAb (11). The mAb N29, whose epitope lies in the
PSI domain, was used as a
control.3 15/7 and HUTS-4
binding to tr
5
1-Fc was increased by
TS2/16 and 12G10 but markedly decreased by mAb 13 (Fig.
2). Hence, conformational changes in the
1/
2 helix region of
A appear to modulate 15/7 and HUTS-4
binding. As ligand recognition is also known to cause shape-shifting in
A and to stabilize the active conformation of this domain (8), we
tested the effect of ligand binding on the expression of the 15/7 and
HUTS-4 epitopes. A recombinant fragment of fibronectin containing the
5
1 recognition sites (26) stimulated 15/7
and HUTS-4 binding (Fig. 2) to a similar extent as TS2/16 and
12G10.
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Fig. 2.
Effect of function-perturbing mAbs and
III6-10 fibronectin fragment on binding on 15/7 and HUTS-4
to
tr 5
1-Fc.
Binding of 15/7 or HUTS-4 was measured in the absence of other mAbs or
ligand (Con) or in the presence of Fab fragments of the
non-function-perturbing N29, or the activating TS2/16 or 12G10, or in
the presence of IgG of the function-blocking mAb 13, or in the
presence of III6-10 fragment of fibronectin
(FN). The experiment was performed in the presence of 1 mM Mn2+. In control experiments the binding of
15/7 or HUTS-4 was not affected by control rat IgG or by a
III6-10 fragment of fibronectin lacking the RGD integrin
binding sequence (data not shown).
A (stabilized
by Mn2+/Mg2+, activating mAbs, or ligand) leads
to increased expression of the 15/7 and HUTS-4 epitopes. Conversely,
the inactive conformation of
A (stabilized by Ca2+ or
function-blocking mAbs) leads to decreased expression of the 15/7 and
HUTS-4 epitopes. These effects may be linked to a motion of the
1
helix (8). Our data on the effects of cations, function-perturbing mAbs, and ligand are in broad agreement with previous characterization of 15/7 and HUTS-4 as mAbs recognizing activation/LIBS epitopes (9, 29,
31).
A Domain
7 Helix (L358A) Results in
Constitutively High Expression of the 15/7 and HUTS-4
Epitopes--
The transduction of conformational changes from
A to
the hybrid domain must take place at the interface between these two modules. At its C terminus
A is joined to the hybrid domain by the
7 helix, and mutations in this region of
subunit A domains cause
activation by favoring a downward shift of
7 (34, 35). We therefore
tested whether similar mutations in the
7 helix of
A could affect
15/7 and HUTS-4 binding. A mutation
Leu358 to Ala was found to
cause increased expression of the 15/7 and HUTS-4 epitopes, whereas the
control mutation S359A had little effect (Figs. 3 and
4). Expression of the 15/7 and HUTS-4
epitopes by the L358A mutant was constitutively high compared with the wild-type receptor, and Mn2+/Mg2+ only caused a
small enhancement of the level of binding seen in the absence of
divalent ions (Fig. 3, A and B). The high level of 15/7 binding to the L358A mutant was only slightly increased by
activating mAbs TS2/16 or 12G10 and was relatively resistant to
inhibition by mAb 13 (Fig. 4); similar results were obtained for HUTS-4
(data not shown). In contrast to the wild-type receptor, ligand binding
did not increase 15/7 epitope expression by the L358A mutant (Fig. 4).
The mutation S359A had little effect on the ability of mAbs or ligand
to modulate 15/7 binding (results similar those for wild-type
tr
5
1-Fc, see Fig. 2). These findings suggest that the L358A mutation constrains the
A domain in an active
conformation and that the
7 helix region is involved in conveying
conformational changes from
A to the hybrid domain.
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Fig. 3.
Effect of L358A and S359A mutations on
modulation of 15/7 (A) and HUTS-4 (B)
binding to
tr 5
1-Fc
by divalent cations. 15/7 or HUTS-4 binding was measured in the
presence of 2 mM EDTA (open bars), 2 mM Mn2+ (filled bars), 2 mM Mg2+ (gray bars), or 2 mM Ca2+ (diagonally shaded
bars).
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Fig. 4.
Effect of L358A and S359A mutations on
modulation of 15/7 binding to
tr 5
1-Fc
by function-perturbing mAbs and III6-10 fibronectin
fragment. Binding of 15/7 or HUTS-4 to the L358A or S359A mutants
was measured in the absence of other mAbs or ligand (Con),
or in the presence of Fab fragments of N29, TS2/16, or 12G10, or IgG of
the function-blocking mAb 13, or the III6-10 fragment of
fibronectin (FN). The experiment was performed under the
same conditions as that shown in Fig. 2.
5 and
1 subunits (Fig.
5A). The results showed that the L358A mutation increased the expression of the 12G10 epitope and
decreased the expression of the 13 and 4B4 epitopes in
A. The
mutation also attenuated the expression of the epitopes of function-blocking mAbs SNAKA52, 16, and P1D6, which lie at or near the
top of the
5
-propeller domain (23), close to the
A/propeller interface (13). Although, as shown above, the L358A mutation increased the expression of the 15/7 and HUTS-4 epitopes, it
did not alter the expression of the JB1A epitope, which maps to a
different region of the hybrid domain (36). Furthermore, the expression
of epitopes that are not affected by the activation state, such as
TS2/16 and JB1A, was not altered by the L358A mutation. The control
mutation S359A had no significant effect on the expression of any of
the epitopes tested (Fig. 5B). Taken together, these results
show that the L358A mutation specifically increases the expression of
all activation/LIBS epitopes (12G10, 15/7, and HUTS-4, see Refs. 8, 9,
and 31) and decreases the expression of all LABS epitopes
(SNAKA52,4 16, P1D6, 13, and
4B4, see Refs. 11, 12, and 37). Hence, the data suggest that L358A
mutant adopts a conformation that is similar to the ligand-occupied
state. Furthermore, a conformational change in the
7 helix region of
A caused by the mutation appears to be linked to movements in the
1/
2 helix region (the location of the 12G10, 13, and 4B4
epitopes) and the proximity of the
A/propeller interface (the
location of the SNAKA52, 16, and P1D6 epitopes).
View larger version (37K):
[in a new window]
Fig. 5.
Expression of and
subunit epitopes by L358A mutant
(A) and S359A mutant (B).
Binding of anti-
5 and anti-
1 mAbs to
tr
5
1-Fc with the mutations L358A or S359A
in
1 is expressed as a percentage of the binding to
wild-type tr
5
1-Fc. Experiments were
performed in the presence of 1 mM Mn2+. Results
are mean ± S.D. of at least three separate experiments. *,
p < 0.05 by Student's t test.
5
1-Fc has low
constitutive ligand binding activity when captured onto enzyme-linked
immunosorbent assay plates using goat anti-human Fc, but the same
protein has similar activity to recombinant integrin containing the
complete extracellular domains of
5 and
1
when stimulated with mAbs such as 12G10 (24). We compared the ligand binding activity of wild-type tr
5
1-Fc
with the L358A and S359A mutants (Fig.
6). The results showed that compared with
the wild-type receptor, the L358A mutant had high constitutive ligand
binding activity, which was only slightly enhanced by activating mAbs TS2/16 or 12G10. In contrast, the S359A mutant had constitutively low
activity, similar to wild-type levels.
View larger version (36K):
[in a new window]
Fig. 6.
Effect of L358A and S359A mutations on
binding of III6-10 fibronectin fragment. Binding of
III6-10 was measured in the absence of mAbs (open
bars) or in the presence of the non-function-perturbing mAb N29
(horizontally hatched bars), or the activating mAbs TS2/16
(diagonally hatched bars), or 12G10 (cross-hatched
bars).
Subunit--
The above
results suggest that the transduction of conformational changes from
A to the hybrid domain involves a shift of the
7 helix. To
understand these changes more fully, we fine-mapped the epitopes of
15/7 and HUTS-4. Both antibodies bind to human
1 but not
to mouse
1, and their epitopes have been shown to reside
within amino acid residues 355-425 (30, 31). These residues in human
1 show 10 differences with the equivalent sequence in
murine
1 (Table I). Mutant
proteins containing single point mutations at each of these positions
were expressed and tested for binding of 15/7 and HUTS-4, using TS2/16
as a control. The mutations E371D and K417N abrogated both 15/7 and
HUTS-4 binding, whereas the mutation S370P completely blocked 15/7
binding and strongly inhibited HUTS-4 binding. The other mutations had
either no effect or showed a partial inhibition (Table I). None of the mutations affected binding of TS2/16. Ser370 and
Glu371 map to the C-D loop, and Lys417 maps to
the neighboring E-F loop of the hybrid domain (13). The spatial
proximity of this triplet of residues is consistent with them forming
an antibody epitope.
Analysis of 15/7 and HUTS-4 reactivity with 1 hybrid domain
substitution mutants
5-(1-613)-Fc and
wild-type or mutant
1-(1-455)-Fc. Cell culture supernatants
were analyzed for reactivity with anti-
1 mAbs by sandwich
enzyme-linked immunosorbent assay. Results are expressed as a
percentage of wild-type binding and are mean ± S.D. from three
separate experiments (except for the S422T mutant, from two separate
experiments). A value of 0% indicates that mAb reactivity was
identical to, or slightly lower than, reactivity with supernatant from
mock-transfected cells. All the mutants bound well to the hybrid domain
mAb JB1A, and none of the mutations affected recognition of the
III6-10 fragment of fibronectin (data not shown).
V
3 (13, 14) shows that these epitopes map
to a region of the hybrid domain that faces the
subunit
-propeller and are very close to residues that form a small
interface with it. To estimate the antibody-accessible surface, we
rolled a 20-Å sphere over the structure (16). The results (not shown)
demonstrated that Lys417 would be accessible in this
conformational state, but Ser370 and Glu371
would not. However, Ser370 and Glu371 would be
available for antibody binding if the hybrid domain moves away from the
-propeller.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 A domain, and we investigated how these
relate to signal transduction in the integrin head region. Our results show the following: (i)
A domain activation involves a
conformational change in the region of
7 helix; (ii) this
shape-shifting results in increased exposure of the 15/7 and HUTS-4
epitopes in the hybrid domain and is also associated with other
conformational changes in
A and the top of the
subunit
-propeller; (iii) the 15/7 and HUTS-4 epitopes map to a portion of
the hybrid domain that is likely to be partly masked (in the inactive
receptor) due to its close proximity to the
-propeller. Taking these
results together with previous data showing that a movement of the
1
helix is important for activation of
A (8), we propose the model of affinity regulation shown in Fig. 7.
View larger version (54K):
[in a new window]
Fig. 7.
Model of conformational changes
involved in activation of integrin and
head domains. A model of the
5-propeller and thigh domains and
1 A and
hybrid domains was built as described under "Experimental
Procedures." For the
subunit,
strands are shown in
blue, and
helices are shown in red, except
for
7 (in yellow) and
2 (in orange, partly
hidden by
1). The divalent ion at the MIDAS site of
A is depicted
by a magenta sphere. The metal ion at the adjacent to MIDAS
site of the
A domain (13) is omitted for the sake of clarity. For
the
subunit, secondary structure elements are shown in
gray. In the model, Ser370, Glu371,
and Lys417, which form the 15/7 and HUTS-4 epitopes, are
depicted by green space fill. In the transition from the
inactive to the active form of the
A domain appears to be both an
inward movement of the
1 helix and a downward shift of the
7
helix (solid arrows). A downward motion of the
7 helix
would be coupled to an outward swing of the hybrid domain (open
arrow 1), but this movement of the hybrid domain could also push
on the lower face of
A (open arrow 2), stabilizing the in
position of the
1 helix and tilting
A toward the top face of the
-propeller (open arrow 3). The
1/
2 helix region
contains the epitopes of activating and inhibitory
anti-
1 mAbs (8, 33, 47), whereas loops at the top of the
-propeller (indicated by arrowheads) contain the epitopes
of function-blocking anti-
5 mAbs (23). The tilt of
A
toward the
-propeller could partially attenuate the
anti-
5 mAb epitopes because it closes up the
A/propeller interface.
Mechanism of A Activation--
Recent debate concerning the
mechanism of activation of
A has centered on whether or not the
7
helix moves. Three distinct models have been proposed. (i) Based on the
crystal structures of
V
3 (13, 14) in
which there is no movement of
7, and the MIDAS site is unoccupied in
the absence of ligand, it was suggested that
A is regulated by
unmasking of the MIDAS site (20, 39). (ii) Based on a separation of the
head domains observed in RGD-occupied
IIb
3 after rotary shadowing/electron
microscopy (40), and on a similarity between the quaternary structures of integrins and heterotrimeric G-proteins (13), it was hypothesized that activation involves a rotation of
A away from its contact with
the
-propeller (41). In this scenario, the position of the
7
helix is fixed but the rotation of
A resulted in the same net
movement of
7 seen in
A domains. (iii) Based on studies of
inactive (Ca2+-occupied) and active
(Mn2+-occupied)
V
3 by
negative staining/electron microscopy (17), it was suggested that
activation of the head region is regulated by an outward swing of the
hybrid domain, which is predicted to be coupled to downward shift of
the
7 helix equivalent to that in
A domains (19, 42). Model i
appears unlikely because our results (this work and see Ref. 8) and the
results of others (17, 18) show that conformational changes take place
through cation binding to the MIDAS in the absence of ligand occupancy. Model ii is improbable because
A and the
-propeller appear to move closer together, rather than farther apart, upon ligand
recognition because ligand binding requires close apposition of
residues on both subunits (14, 39). Instead, our data supply strong
support for model iii because they suggest that a movement in the
7
helix region is important for activation and that this movement is
linked to a change in the position of the hybrid domain such that it moves away from the
/
subunit interface. The existence of the swing-out motion of the hybrid is further supported by the finding that
a section of the
3 hybrid domain encompassing residues
393-423 (equivalent to residues 402-432 in
1) is
exposed in the active but not the resting form of
IIb
3 (43). This portion of the hybrid
domain encompasses part of the 15/7 and HUTS-4 epitopes (Lys417).
Why was no movement of the 7 helix seen in the crystal structure of
the liganded form of
V
3 (13)? The likely
explanation is that motion of
7 would be prevented because the
hybrid domain is paralyzed by lattice contacts and by its contacts with
the leg domains (16, 17). Some conformational changes are observed in
the liganded
A domain; these include rearrangement of the loops that
coordinate the MIDAS cation, leading to an inward movement of the
1
helix. These changes are very similar, both in direction and form, to
those seen in
A domains; however, as pointed out above, the
subsequent downward motion of the
7 helix that takes place in
A
domains is probably prohibited. Thus, the structural changes observed
in the liganded
A also favor the hypothesis that the unliganded
structure represents the inactive form of
A (44).
We found that a mutation in the 7 helix, L358A, caused activation of
the
A domain. In
A domains mutation of a highly conserved isoleucine residue at the same position favors the active state. This
is apparently because this residue fits into a hydrophobic pocket
(known as "socket for isoleucine," SILEN), and this interaction favors the inactive form (34). However, unlike the
A domains, mutation of residues that form the hydrophobic pocket surrounding the
7 helix in the
1 A domain (Leu125,
Leu149, Leu253, and Ile314) did not
affect the activation state of
tr
5
1-Fc.5
Hence, the mechanism that regulates
7 movement may differ slightly from that of
A domains. Nevertheless, mutation of Leu358
may favor the active state of
A because this residue is likely to be
more exposed in the "down" position of the
7 helix than in the
"up" position. Hence, mutation of the leucine residue to the less
hydrophobic alanine would be predicted to lower the energy of the
active state. We cannot rule out the possibility that the L358A
mutation activates the receptor by altering the
A/hybrid interface.
However, mutation of Ser359 (also at the interface) did not
cause activation, and furthermore, this paradigm would not explain how
activating anti-
A mAbs cause activation and hybrid domain movement
in a similar manner to the L358A mutation. In contrast, a linked
movement of the
1 and
7 helices as seen in
A domains can
explain the mechanism of action of the anti-
A domain mAbs (see below).
Similarities and Differences between Activation of A and
A
Domains--
In the activation of
A domains a change in cation
coordination at the MIDAS is linked to an inward movement of the
1
helix. This movement pinches the hydrophobic core, squeezing out
residues in the loop that precedes the
7 helix, and results in an
~10-Å downward motion of
7 (19). A similar link between
1 and
7 helix movement in
A is suggested by our findings. For example, occupancy of the MIDAS by Mn2+ or the binding of activating
mAbs, which stabilize the in position of the
1 helix (8),
also promotes the downward motion of the
7 helix (reported by
increased exposure of the 15/7 and HUTS-4 epitopes).
Although the overall mechanisms of A and
A activation now appear
to be closely related, there are some subtle differences. For example,
for
A domains Mn2+ and Mg2+ are equally
effective for promoting ligand binding to the MIDAS (45, 46). In
contrast, Mn2+ is much more effective than Mg2+
for promoting ligand binding to the
A MIDAS (8). This property of
Mn2+ may be due to the fact that it binds with much higher
affinity than Mg2+ to the
A MIDAS, whereas the
affinities of
A domains for these two ions are more comparable (44,
45). Similar to
A domains, movement of the
7 helix appears to
form an essential part of the activation mechanism of
A because
7
movement closely parallels the activation state. However, as noted
above, the regulation of
7 motion may be slightly different to that
in
A domains.
Allosteric Mechanism of Function-perturbing mAbs--
We have
shown previously (11, 12, 37) that most function-blocking
anti-5 and anti-
1 mAbs have an allosteric
mode of action. They recognize epitopes attenuated by ligand
recognition and appear to perturb ligand binding by preventing a
conformational change involved in the formation of the integrin-ligand
complex (7). The epitopes of function-blocking anti-
1 A
domain mAbs map to the
2 or
1 helix regions (13, 33, 47), which
lie adjacent to each other. Similarly, the epitopes of
function-blocking anti-
2 A domain mAbs have been shown
to include residues in the
1/
2 or
7 helix regions (48, 49). It
is likely that these mAbs function allosterically by stabilizing
1 in the inactive (out) location and/or
7 in the
inactive (up) position. The epitopes of function-blocking anti-
subunit mAbs map to loops on the top face of the
-propeller domain
(indicated by arrowheads in Fig. 7), and the binding of
these mAbs may prevent conformational rearrangements required for
ligand binding (14). Hence both anti-
and anti-
subunit
function-blocking mAbs appear to impede conformational changes involved
in ligand recognition, as proposed previously (7).
The epitopes of activating anti-A domain mAbs map to the same
regions as inhibitory mAbs (8, 18, 33) and are likely to function by
stabilizing
1 in the active (in) location and/or
7 in the active
(down) position. It has been suggested that 12G10 activates by
affecting the
A/hybrid domain interface (44), rather than by an
effect on the
1 helix (8). However, we consider this proposal to be
incorrect because (i) the mechanism of 12G10 action is likely to be
closely overlapping with that of other activating anti-
A mAbs (whose
epitopes are not close to the interface), and (ii) 12G10 has the same
properties in a recombinant integrin that lacks a large part of the
hybrid domain, suggesting that it directly affects the conformation of
A (24). The activating effect of the 15/7 and HUTS mAbs (9, 31) is
likely to be due to their preferential binding to the active form of
the integrin in which the hybrid domain is shifted away from the
-propeller.
Implications for Inside-out and Outside-in Signaling--
A recent
NMR study of a complex between the intracellular segments of
IIb and
3 suggests that integrin
activation is prevented by a "handshake" between
and
cytoplasmic tails, and that unclasping of this handshake by proteins
such as talin represents the first stage of activation (5). Separation
of the cytoplasmic domains may then be linked to unbending of the
integrin legs, which in turn is coupled to activation of head domains
(16, 17). In this scenario, hybrid domain movement is severely
restricted by its interface with the leg domains in the inactive, bent
form of the integrin. Unbending causes activation because it is coupled to the release of the hybrid domain from these constraints, allowing swinging of the hybrid to take place, which, in turn, would cause pulling on the
7 helix of
A. Our findings generally support this
model of activation. However, since removal of the lower leg domains
(in the truncated integrin) did not favor the active state (24), hybrid
domain movement (rather than unbending per se) appears to be
the essential requirement for activation. Hybrid domain motion provides
the conduit for the transduction of signals to and from the head
region. The attenuation of the epitopes on the
-propeller in the
L358A mutant also suggests that the outward swing of the hybrid domain
is coupled to a conformational rearrangement of the
A/propeller
interface that is involved in ligand recognition (14). Similarly,
ligand binding reinforces and stabilizes the conformational changes
associated with activation (e.g. the movements of the
1
and
7 helices and hybrid domain). This feature of ligand recognition
may explain the well known ability of integrin ligands to cause
activation that persists after dissociation of the complex (50, 51).
Outside-in signaling could result, in part, from stabilization of the
active conformation, allowing the separated cytoplasmic tails to stably
interact with cytoskeletal and signaling molecules. In addition,
integrin clustering is also a major contributor to this signaling (52,
53).
In summary, we have shown that a conformational shift in the 7 helix
region of the
A domain is involved in the regulation of integrin
activity. This movement is coupled to a swing-out of the hybrid domain
and thereby provides a pathway for signal transduction. Integrins are
important therapeutic targets in many inflammatory and cardiovascular
disorders (28), and our findings suggest a novel way in which highly
specific regulators of integrin activity could be developed
(e.g. by stabilizing the hybrid/propeller interface). A more
complete understanding of the signaling mechanisms will require further
characterization and crystallization of an integrin in defined
conformational states.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank A. Coe, P. Stephens, and M. Robinson
for the 5 and
1 constructs. We are
grateful to J. Wilkins, T. Yednock, K. Yamada, E. Wayner and F. Sánchez-Madrid for mAbs; K. Yamada and S. Aota for the
III6-10 and III6-10 KGE constructs; E. Symonds for assistance with integrin purification and cell culture; A. Arnaout for the coordinates of the
V
3
structures; and J. Bella for advice on molecular modeling.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the Wellcome Trust (to M. J. H.).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.
To whom correspondence should be addressed: Wellcome Trust Centre
for Cell-Matrix Research, School of Biological Sciences, University of
Manchester, 2.205 Stopford Building, Oxford Rd., Manchester M13 9PT,
UK. Tel.: 44-161-275-5649; Fax: 44-161-275-5082; E-mail:
paul.mould@man.ac.uk.
§ Supported by a studentship from the Biotechnology and Biological Sciences Research Council.
Published, JBC Papers in Press, March 3, 2003, DOI 10.1074/jbc.M213139200
2 W. L. Delano, The Pymol Molecular Graphics System, Delano Scientific, San Carlos, CA (www.pymol.org).
3
Although previously characterized as an
activating mAb (27), N29 did not cause any activation of
tr5
1-Fc and was used as a negative control.
4 K. Clark, R. Pankov, J. A. Askari, A. P. Mould, S. E. Craig, P. Newham, K. M. Yamada, and M. J. Humphries, manuscript in preparation.
5 A. P. Mould, J. A. Askari, S. J. Barton, and M. J. Humphries, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
mAb, monoclonal
antibody;
A,
subunit von Willebrand factor type A domain;
A,
subunit von Willebrand factor type A domain;
MIDAS, metal-ion
dependent adhesion site;
BSA, bovine serum albumin;
tr
5
1-Fc, recombinant soluble integrin
heterodimer containing C-terminally truncated
5 and
1 subunits (
5 residues 1-613 and
1 residues 1-455) fused to the Fc region of human
IgG
1;
LIBS, ligand-induced binding site;
LABS, ligand-attenuated
binding site.
![]() |
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