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INTRODUCTION |
Integrins are major metazoan adhesion receptors that play
a fundamental role in cellular organization. They mediate
cell-extracellular matrix as well as cell-cell adhesion, connect
extracellular cues to the cytoskeleton, and activate many intracellular
signaling pathways (1, 2). One unique aspect of integrins is that the
affinity of their extracellular domain for biological ligands can be
rapidly up-regulated by signals from within the cell. Rapid and precise
control of integrin activation is particularly important for leukocytes
and platelets, which circulate in the vascular system, where leukocyte
emigration and thrombus formation mediated by integrins must be
triggered only at the appropriate location. Integrins compose two
noncovalently associated type I transmembrane glycoprotein
- and
-subunits (3). A crystal structure of the extracellular domain of
the integrin
V
3 revealed a bent conformation, in which there is an acute angle between the headpiece and tailpiece (4), and an extensive headpiece-tailpiece interface (5).
Recently, we have shown that the bent conformation represents the low
affinity receptor and that activation is associated with a
switchblade-like motion of the headpiece resulting in a highly extended
conformation (5).
The integrin headpiece contains the ligand-binding site. The headpiece
contains the
-subunit
-propeller and thigh domains and the
-subunit I-like and hybrid domains (Fig. 1), corresponding approximately to the N-terminal two-thirds of the extracellular domain
of each subunit. A crystal structure with a bound ligand-mimetic peptide revealed that ligand binds to an interface formed by the
-subunit I-like domain and
-sheets 2-4 of the
-subunit
-propeller domain (6) (Fig. 1).
Many experiments support the idea that the inter-subunit association at
the cytoplasmic region maintains integrins in low affinity state
(7-10). Originally, this notion led to a "hinge hypothesis," where
association/dissociation of the cytoplasmic tails caused hinging
between the two subunits, and ultimately changed the conformation of
the ligand-binding extracellular segments (11). However, the nature of
the conformational change that regulates ligand binding by integrins
has been controversial (2, 3, 5, 12-15). Hantgan et al.
(16, 17), using rotary shadowing EM, reported that binding of RGD
peptides induced separation of the headpiece of detergent-solubilized
IIb
3. These images suggested a wide
separation in the headpiece, with no interaction remaining between the
N-terminal halves of the
- and
-subunits. These observations have
been highly influential, in part because they seemed to fit with
earlier schematics of integrin activation models where a
hinge-like motion at the transmembrane domains is transmitted through
rigid
- and
-subunit tailpiece segments to the headpiece, resulting in movement apart of the
- and
-subunits in the
headpiece (11, 12, 18). However, recent high resolution negative stain EM studies have shown that the
-subunit leg can exist in two distinct conformations with respect to the headpiece and that the
-subunit leg is highly flexible (5). Existence of multiple modular
domains (4) also disfavors rigid movement of the entire stalk region.
Because the legs are flexible, it is hard to imagine that information
can be transmitted to the headpiece as proposed in the hinge model.
Furthermore, high resolution EM studies (5), as well as many other EM
studies (19-22), have shown that ligand binding to integrins is not
accompanied by head separation.
There are other reasons for the popularity of the head separation
model. First, it has been suggested that residues that have been
implicated in ligand binding are buried in the headpiece and that
separation could expose them, resulting in higher affinity binding (2).
Second, there is the mystery of the synergy site in fibronectin
type III module 9 of fibronectin, which is distant from the RGD site in
module 10. It has been proposed that separation of the headpiece (2,
15) would facilitate simultaneous binding of the
-subunit to the
synergy site and the
-subunit to the RGD site (23). However, the
liganded
V
3 crystal structure shows that
the Arg of RGD binds to the
-subunit and the Asp of RGD binds to the
-subunit (6), strongly suggesting that headpiece separation would
disrupt binding to RGD. Third, there are structural homologies between
integrins and G proteins (4, 24, 25). By taking this analogy further,
it has been suggested that upon integrin activation, the
-propeller
and I-like domains might dissociate analogously to the G protein
-
and
-subunits (2, 15).
An alternative model for integrin activation has been proposed that is
supported by high resolution EM, physicochemical studies, ligand
binding assays, introduction of disulfide bonds that lock in the bent
conformation, and localization of epitopes that become exposed after
integrin activation (5, 26). In this model, activation is regulated by
the conformational equilibrium between three states as follows: a bent
conformation with low affinity, an extended conformation with a closed
headpiece with intermediate affinity, and an extended conformation with
an open headpiece with high affinity. Binding of RGD peptide was found
not to induce head separation but to induce a dramatic change in angle
between the
-subunit I-like and hybrid domains, leading to the
swing-out of the hybrid domain away from the
-subunit (5). The
prominence of the hybrid domain in the interface between the headpiece
and the tailpiece in the bent conformation provides a mechanism for linking the change in angle upon ligand binding to the equilibrium between the bent and extended integrin conformations (5).
Definitive experimental testing of the head separation model is
important. Therefore, we have used mutagenesis to introduce disulfide
bonds between the
-subunit
-propeller domain and the
-subunit
I-like domain, and we have tested the effect of preventing head
separation on activation of
IIb
3 and
V
3 integrins on the cell surface.
Furthermore, we test the effect of ligand-mimetic compounds on the
association between the
- and
-subunits in native integrins on
the cell surface and in soluble integrin fragments that contain only
the headpiece.
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EXPERIMENTAL PROCEDURES |
Monoclonal Antibodies--
Mouse monoclonal antibody
(mAb)1 PT25-2 recognizing
human
IIb subunit (27) was a gift from Dr. M. Handa
(Keio University, Tokyo, Japan). Mouse mAb 10E5 recognizing the human
IIb
3 complex (28) was a gift from Dr.
B. S. Coller (Rockefeller University, New York). Mouse
anti-
3 AP3 was from American Type Culture Collection. All other mAbs were obtained from the Fifth International Leukocyte Workshop (29).
Plasmid Construction, Transient Transfection, and
Immunoprecipitation--
Plasmids coding for full-length human
IIb,
V, and
3 were
subcloned into pcDNA3.1/Myc-His(+) or pEF/V5-HisA as described previously (5). Mutants were made using site-directed mutagenesis, and
DNA sequences were confirmed before being transfected into 293T cells
using calcium phosphate precipitation. Transfected cells were
metabolically labeled with [35S]cysteine/methionine as
described (5). Labeled cell lysates were immunoprecipitated with
anti-
3 AP3, eluted with 0.5% SDS, and subjected to
nonreducing or reducing SDS-7.5% PAGE and fluorography.
Two-color Ligand Binding and Flow Cytometry--
Binding of
fluorescein-labeled human fibrinogen and fibronectin were performed as
previously described (5). To test the effect of EDTA treatment on mAb
epitope expression, transiently transfected 293T cells were
preincubated in 20 mM Tris-buffered saline, pH 8.4, containing either 1 mM Ca2+, 1 mM
Mg2+, or 5 mM EDTA at 37 °C for 30 min,
followed by washing and resuspension in 20 mM Hepes, 150 mM NaCl, 5.5 mM glucose, 1% bovine serum
albumin, and 1 mM Ca2+, 1 mM
Mg2+ (HBS). Cells were then incubated with mAbs on ice for
30 min, followed by staining with FITC-conjugated anti-mouse IgG and
flow cytometry. To test the effect of RGD peptide on mAb epitope
expression, cells in HBS were incubated with 100 µM
GRGDSP peptide at room temperature for 30 min before adding mAbs and
staining as above.
Stability of the
IIb
3 Headpiece in
SDS in the Presence of an RGD-mimetic Compound--
An integrin
headpiece fragment comprising
IIb residues 1-621 and
3 residues 1-472 was produced in Chinese hamster ovary Lec 3.2.8.1 cells stably transfected with plasmids coding for each
fragment. Acid-base
-helical coiled-coil peptides were fused to the
C termini of the
- and
-subunits to increase the stability of the
heterodimer. Methods were as described previously (8). A hexahistidine
tag was also attached to the C terminus of the
-subunit to
facilitate purification by nickel chelate chromatography (8). The
purified headpiece fragment was treated with 10 µg/ml chymotrypsin
for 16 h at 25 °C to remove the C-terminal clasp, and incubated
with the high affinity RGD-mimetic compound L738,167 (gift from Dr.
G. D. Hartman, Merck) (30) at 10 µM for 30 min at
37 °C. The mixture was cooled to room temperature; an equal volume
of sample buffer containing 0.1% SDS was added, and samples were
immediately subjected to nonreducing SDS-PAGE on a 4-20% gradient gel.
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RESULTS |
Introduction of Disulfide Bonds between the
-Propeller and
I-like Domains--
To make mutant integrins that were unable to
undergo head separation, we mutationally introduced cysteine residues
at the interface between the
IIb
-propeller and
3 I-like domains (Fig. 1).
Inspection of the structure of
V
3 and a
model of
IIb
3 made from the
V
3 template identified candidate
positions for disulfide bonds where C
distances between
-propeller and I-like domain residues were within 7 Å.
Disulfide-forming efficiency was assessed by transient transfection of
293T cells followed by immunoprecipitation and nonreducing SDS-PAGE.
Preliminary experiments revealed that the efficiency varied from 20 to
100%, depending on the combination of residues chosen (data not
shown). In
V
3, the combination of the
V-M400C and
3-Q267C mutations gave
~100% formation of an intersubunit disulfide bridge as indicated by the formation of a high molecular weight band in nonreducing SDS-PAGE (Fig. 2, compare wild type in lane
1 and double mutant in lane 2), whereas the
V- and
3-subunits migrated separately in
reducing SDS-PAGE (Fig. 2, lanes 3 and 4). The
combination of
V-E311C and
3-G293C
mutations resulted in slightly less efficient disulfide formation
(~70%, data not shown). Since
IIb lacks the long loop containing M400 in
V, the mutation corresponding to
V-E311C (
IIb-E324C) was tested and shown
to form an efficient disulfide when coupled with the
3-G293C mutation (Fig. 2, compare lane 6 with
wild type in lane 5). Under reducing conditions, the
disulfide-linked complex was reduced into individual
- and
-subunits indistinguishable from the wild type subunits (Fig. 2,
lanes 7 and 8). The mutations link
-propeller
blade 5 or the connection between blades 6 and 7 to the I-like domain,
whereas the ligand-binding interface locates on the opposite side of
the interface where blades 2-4 of the
-propeller domain contact the
I-like domain (Fig. 1). The mutations should therefore not directly
affect the ligand-binding site or disrupt conformational changes in
loops near the ligand-binding site. A panel of mAbs directed against
the head regions of
V
3 and
IIb
3 bound identically to mutant and wild
type receptors (data not shown), suggesting that the mutant receptors
adopt a native fold.

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Fig. 1.
The
V 3
integrin headpiece and location of introduced disulfide bonds. The
stereo view of the V 3 headpiece is based
on the crystal structure of the V 3
extracellular domain bound to a ligand-mimetic RGD peptide (6). The
V -propeller and thigh domains are red;
the 3 I-like and hybrid domains are blue, and
engineered disulfide bonds are in gold. The bound RGD
peptide is magenta, and its Arg and Asp side chains are
shown. Backbones are shown as a worm-like trace. Note that the
disulfide bonds introduced by mutations
V-M400C/ 3-Q267C and
V-E311C/ 3-G293C (which corresponds to
IIb-E324C/ 3-G293C) are located on the
side of the -propeller and I-like domain interface opposite from the
ligand-binding site. Figure was prepared with Ribbons (44).
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Fig. 2.
Formation of intersubunit disulfide bonds in
the headpieces of V 3 and
IIb 3. Lysates were prepared from
[35S]methionine- and -cysteine-labeled 293T cells that
had been transiently transfected with wild type
V 3 (lanes 1 and 3),
V-M400C/ 3-Q267C (lanes 2 and 4), wild type IIb 3
(lanes 5 and 7), or
IIb-E324C/ 3-G293C (lanes 6 and
8), immunoprecipitated with mouse mAb AP3 to human
3, and subjected to SDS-7.5% PAGE under nonreducing
(lanes 1, 2, 5, and 6) or reducing
(lanes 3, 4, 7, and 8) conditions followed by
fluorography. Positions of molecular size markers are shown on the
left.
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High affinity binding of
V
3 and
IIb
3 transfectants to fluorescent,
soluble ligands was measured with simultaneous staining of
surface-expressed
V
3 or
IIb
3 using two-color flow cytometry (5).
As described previously, wild type
IIb
3
bound soluble fibrinogen when activated by Mn2+ and
activating mAb PT25-2 but not in the absence of activation (Fig.
3A). The
IIb-E324C/
3-G293C mutant could not bind
soluble fibrinogen in the resting state but showed full activity when treated with Mn2+ and PT25-2 (Fig. 3A). Thus,
the ligand-binding phenotype of
IIb
3 containing a disulfide-linked headpiece is identical to wild type.

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Fig. 3.
Ligand binding by disulfide-linked
receptors. 293T cells expressing
IIb 3 (A) or
V 3 (B and C)
integrins were incubated with (filled bars) or without
(open bars) 1 mM Mn2+ and the
activating mAbs PT25-5 for IIb 3 or AP5
for V 3. Binding of FITC-fibrinogen
(A and B) or FITC-fibronectin (C) was
determined and expressed as the percentage of mean fluorescence
intensity relative to immunofluorescent staining with Cy3-labeled AP3
mAb. wt, wild type.
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Similarly, the mutant
V
3 receptor was
tested for activation-dependent binding to soluble
fibrinogen (Fig. 3B) and fibronectin (Fig. 3C).
As for
IIb
3, the ligand binding
activities of wild type and mutant
V
3
receptors were indistinguishable. Both showed little or no basal ligand
binding and strong binding of both ligands upon activation by
Mn2+ and activating mAb AP5 (Fig. 3, B and
C). These data show that disulfide bond formation between
the
-propeller and I-like domains has no effect on high affinity
ligand binding by
3 integrins, strongly arguing against
the notion that head separation is required for conversion to high affinity.
Introduced Disulfide Bond Blocks EDTA-induced Subunit
Dissociation--
EDTA treatment at pH 8.4 at 37 °C is known to
induce dissociation of the integrin
IIb
3
into the
IIb- and
3-subunits on the
platelet surface (31-33). We confirmed that
IIb
3 expressed on 293T transfectants is
similarly susceptible to the EDTA-induced subunit dissociation.
Incubation of cells expressing wild type
IIb
3 with 5 mM EDTA at pH 8.4 completely abolished binding of the 
complex-specific mAbs AP2
(34) and 10E5 (34) but had no effect on binding of the
3-specific mAb AP3 (Fig.
4A). The complex-specific mAbs
require the presence of a complex between
IIb- and
3-subunits for recognition, and loss of reactivity is
thus an excellent indicator of subunit dissociation. In contrast to the
wild type receptor, binding of the mAbs AP2 and 10E5 to the mutant
IIb-E324C/
3-G293C receptor was unaffected
by EDTA treatment (Fig. 4B) showing that the disulfide bond
conferred resistance to EDTA-induced subunit dissociation. Thus,
maintenance of a covalent connection between the
-propeller and
I-like domains is sufficient to protect the epitopes of the AP2 and
10E5 mAbs, suggesting that the deleterious effect of EDTA on these
epitopes is a consequence of subunit separation, rather than a direct
stabilizing effect of divalent cations on residues within the
epitopes.

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Fig. 4.
Effect of EDTA treatment and RGD peptide on
the binding of complex-specific
IIb 3
mAbs. Transiently transfected 293T cells expressing wild type
IIb 3 (A) or mutant
IIbE324C/ 3G293C (B) were
stained with the indicated mAbs in the presence of 1 mM
Ca2+/Mg2+ (open bars), 5 mM EDTA (filled bars), or 1 mM
Ca2+/Mg2+ plus 100 µM GRGDSP
peptide (shaded bars, wild type only) as described under
"Experimental Procedures." Binding is expressed as the percentage
of positive cells after subtraction of background staining by ×63 IgG
control.
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RGD Peptide Binding Does Not Induce Head Separation--
We used
the AP2 and 10E5 mAbs to determine whether RGD-mimetic peptides, like
EDTA, could induce head separation of cell surface
IIb
3. Incubation of wild type
IIb
3 293T cell transfectants with 100 µM GRGDSP peptide resulted in full exposure of cryptic epitopes called ligand-induced binding sites (data not shown), as
described previously (35, 36), suggesting that all of the
IIb
3 on the cell surface bound GRGDSP
peptide. However, preincubation with and the continued presence of 100 µM RGD peptide had no effect on binding of AP2 and 10E5
mAbs to
IIb
3 (Fig. 4A). This
result not only establishes that RGD peptide and these mAbs bind to
independent sites on the
IIb
3 headpiece
but also strongly suggests that the two subunits stay associated in the
headpiece when RGD peptide is bound.
RGD-mimetic Compound Stabilizes Rather than Destabilizes Integrin
Headpiece Association--
High affinity ligand-mimetic compounds can
stabilize integrin 
complexes and make them resistant to
SDS-induced dissociation during gel electrophoresis (37, 38). A wide
variety of integrins can be stabilized, including
IIb
3 (17, 39, 40). In these studies, the
integrins were either native or were recombinant integrins containing
the entire extracellular domain. In the bent integrin conformation,
there are substantial interactions between the
- and
-subunits in
both the headpiece and tailpiece (4, 5). To clarify whether
ligand-mimetic peptide binding stabilizes the 
interface present
within the headpiece, we designed a truncated
IIb
3 integrin fragment composed
only of headpiece segments. It contains the
IIb
-propeller and thigh domains and the
3 plexin-semaphorin-integrin (PSI), I-like, hybrid, and the first integrin-epidermal growth factor-1 domains; the only 

interface present in this fragment is between the
-propeller and
I-like domains (Fig. 1). When the purified
IIb
3 headpiece fragment was incubated
with SDS sample buffer at room temperature and subjected to SDS-PAGE,
two distinct bands corresponding to the truncated
IIb-
and
3-subunits were found (Fig.
5, lane 1). By contrast, when
the
IIb
3 headpiece fragment was incubated
with 10 µM of the RGD-mimetic compound L738,167 prior to
SDS-PAGE, a single band with higher molecular mass was found with no
free
IIb or
3 fragments (Fig. 5,
lane 2). Thus, the
IIb
3
headpiece becomes resistant to dissociation by SDS after incubation
with a ligand-mimetic peptide. The ligand-mimetic compound strengthens
association between the
-propeller domain of
IIb and
the I-like domain of
3 probably by providing an
additional interaction between them, as seen in the crystal structure
in which an RGD-mimetic peptide binds to
V through its
Arg and
3 through its Asp (6). Stabilization by the
ligand-mimetic compound of 
headpiece association is in strong
contradiction to the notion that ligand-mimetic peptides cause
dissociation of the
- and
-subunits in the headpiece.

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Fig. 5.
Ligand-induced stabilization of intersubunit
association in the headpiece. The
IIb 3 headpiece fragment comprising
IIb residues 1-621 and 3 residues 1-472
was incubated without (lane 1) or with 10 µM
L738,167 (lane 2) and subjected to SDS 4-20% gradient PAGE
and Coomassie Blue staining. Positions of molecular size markers are
shown on the left.
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DISCUSSION |
Our study definitively establishes that head separation is not
required for activation of ligand binding by integrins, as shown with
both
IIb
3 and
V
3 by introduction of a covalent linkage
between the
-subunit
-propeller domain and the
-subunit I-like
domain. Despite the covalent linkage, binding of
IIb
3 to fibrinogen, and
V
3 to fibrinogen and fibronectin, was
fully activable. We used the AP2 and 10E5 mAbs, which bind to epitopes contained wholly within the
IIb
3
headpiece and are specific for the
IIb
3
complex, to monitor separation between
IIb and
3 in the headpiece within intact
IIb
3 on the cell surface. EDTA
dissociated the
IIb
3 headpiece as shown
by loss of both epitopes. However, the ligand-mimetic GRGDSP peptide
did not dissociate the headpiece, despite saturation binding to
IIb
3 as shown by full exposure of
ligand-induced binding site epitopes. Finally, a high affinity
RGD-mimetic compound was found to stabilize association between
IIb and
3 in a fragment containing only
the headpiece. These results completely contradict the idea that
RGD-mimetics induce headpiece separation. A model for I
domain-containing integrins, in which separation between the
-subunit
-propeller and the
-subunit I-like domain allows the
-subunit I domain to bind to the
-propeller domain in place of
the I-like domain (15), also seems highly unlikely in view of our
results, because integrins that contain and lack I domains are
activated by similar mechanisms (3).
By contrast, the results are completely consistent with an alternative
model of integrin activation, in which the integrin headpiece stays
associated and ligand binding affinity is linked to the equilibrium
between bent and extended integrin conformations, and between two
headpiece conformations that differ in the angle between the I-like and
hybrid domains (5). In contrast to the headpiece separation model, the
bent-extended model has received experimental support. Disulfide bonds
that stabilize the bent conformation inhibit integrin activation (5),
and an introduced N-glycosylation site designed to wedge
open the angle between the I-like and hybrid domains activates ligand
binding (36).
The crystal structure of
V
3 bound to an
RGD ligand-mimetic peptide was obtained by soaking the peptide into a
crystal containing the bent conformer of
V
3 (6). Some movement was seen at the
-propeller interface with the I-like domain upon ligand binding, but
its magnitude was small and compatible with the disulfide bonds we have
introduced. Thus, the distances between C
atoms before and after
ligand binding, respectively, are 6.7 and 6.8 Å for
V-M400/
3-Q267, and 5.8 and 5.8 Å for
V-E311/
3-G293. In the absence of
restraining crystal lattice contacts, ligand-mimetic peptide binding
induces adoption of the high affinity, extended conformation of
V
3 and a change in angle between the
I-like and hybrid domains (5). Our disulfide bond-linked mutants are fully competent for high affinity ligand binding. Therefore, any further change in orientation of the
-propeller domain with respect to the I-like domain between the ligand-bound bent and extended conformations must be relatively small.
Apart from complete head separation, the possibility has been raised of
integrin activation by a marked tilt of the I-like domain so that it
separates from the
-propeller domain at the ligand binding interface
but not on the opposite side of the interface, near where we have
introduced disulfide bridges (2, 15). We note that the
IIb-E324C/
3-G293C mutation involves
residues that are buried in the
-propeller I-like domain interface
and that the surrounding region is highly conserved in
V
and
IIb. Therefore, pivoting around the introduced
disulfide bond to open the ligand-binding site seems unlikely, because
on the side of the pivot opposite from the ligand-binding site residues
are already closely packed. Thus,
3 residue Glu-297
would clash with
V residue Phe-337, and
3
residues Leu-324 and Pro-326 would clash with
V residues
Lys-308 and Leu-309; all of these
V residues are highly
conserved in
IIb. Our results therefore rule out a
significant tilting motion between the
-propeller and I-like domains
that would open up the ligand binding interface, as well as complete separation of these domains. This conclusion is in accord with high
resolution EM projection averages that show no gross change in
orientation between the
-propeller and I-like domains upon ligand
binding (5). Indeed, the only inter-domain movement observed within the
headpiece upon ligand binding is between the I-like and hybrid domains
(5). Partial head separation is also inconsistent with the crystal
structure of RGD peptide bound to
V
3,
because the Arg and Asp side chains of RGD are already extended in
opposite directions (Fig. 1) (6), and separation at this interface
would abolish one or the other of the highly specific interactions that
these side chains make with the
V- and
3-subunits, respectively.
We have suggested that swinging of the hybrid domain pulls down the
C-terminal helix of the I-like domain and as a consequence activates
the metal ion-dependent adhesion site analogously to I
domain activation (3, 36). Since physiological ligands use many
residues other than the potential metal ion-dependent adhesion site-coordinating residues to interact with integrins, it is
natural to expect that conversion to the high affinity conformation involves rearrangement of residues of the
-
interface outside of
the metal ion-dependent adhesion site. In fact, there are
activation-reporting mAbs that map to this region (41-43). These
rearrangements may involve loop remodeling and some reorientation
between the
-propeller and I-like domains, but in a more subtle
degree than proposed by head separation/tilting models. Precise
determination of the subtle conformational changes responsible for
affinity regulation of integrins awaits further study using atomic
resolution analysis.