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The Relative Influence of Metal Ion Binding Sites in the
I-like Domain and the Interface with the Hybrid Domain on
Rolling and Firm Adhesion by Integrin
␣
4

7
*
Received for publication, July 9, 2004, and in revised form, September 9, 2004
Published, JBC Papers in Press, September 24, 2004, DOI 10.1074/jbc.M407773200
JianFeng Chen‡, Junichi Takagi§, Can Xie‡, Tsan Xiao‡, Bing-Hao Luo‡,
and Timothy A. Springer‡¶
From ‡The CBR Institute for Biomedical Research and Department of Pathology, Harvard Medical School,
Boston, Massachusetts 02115 and §Institute for Protein Research, Laboratory of Protein Synthesis and
Expression, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
We examined the effect of conformational change at
the

7
I-like/hybrid domain interface on regulating the
transition between rolling and firm adhesion by inte-
grin
␣
4

7
.AnN-glycosylation site was introduced into
the I-like/hybrid domain interface to act as a wedge
and to stabilize the open conformation of this interface
and hence the open conformation of the
␣
4

7
head-
piece. Wild-type
␣
4

7
mediates rolling adhesion in Ca
2ⴙ
and Ca
2ⴙ
/Mg
2ⴙ
but firm adhesion in Mg
2ⴙ
and Mn
2ⴙ
.
Stabilizing the open headpiece resulted in firm adhe-
sion in all divalent cations. The interaction between
metal binding sites in the I-like domain and the inter-
face with the hybrid domain was examined in double
mutants. Changes at these two sites can either coun-
terbalance one another or be additive, emphasizing
mutuality and the importance of multiple interfaces in
integrin regulation. A double mutant with counterbal-
ancing deactivating ligand-induced metal ion binding
site (LIMBS) and activating wedge mutations could
still be activated by Mn
2ⴙ
, confirming the importance
of the adjacent to metal ion-dependent adhesion site
(ADMIDAS) in integrin activation by Mn
2ⴙ
. Overall,
the results demonstrate the importance of headpiece
allostery in the conversion of rolling to firm adhesion.
Integrins are a family of heterodimeric adhesion molecules
with noncovalently associated
␣
and

subunits that mediate
cell-cell, cell-matrix, and cell-pathogen interactions and that
signal bidirectionally across the plasma membrane (1, 2). The
affinity of integrin extracellular domains is dynamically regu-
lated by “inside-out” signals from the cytoplasm. Furthermore,
ligand binding can induce “outside-in” signaling and activate
many intracellular signaling pathways (3–6). Integrin extra-
cellular domains exist in at least three distinct global confor-
mational states that differ in affinity for ligand (5, 7); the
equilibrium among these different states is regulated by the
binding of integrin cytoplasmic domains to cytoskeletal compo-
nents and signaling molecules (4, 6).
Integrin affinity regulation is accompanied by a series of
conformational rearrangements. Electron micrographic studies
of integrins
␣
V

3
and
␣
5

1
demonstrate that ligand binding, in
the absence of restraining crystal lattice contacts, induces a
switchblade-like extension of the extracellular domain and a
change in angle between the I-like and hybrid domains (5, 7).
Recent crystal structures of integrin
␣
IIb

3
in the open, high
affinity conformation demonstrate that the C-terminal
␣
7-helix
of the

I-like domain moves axially toward the hybrid domain,
causing the

hybrid domain to swing outward by 60°, away
from the
␣
subunit (8). This conversion from the closed to the
open conformation of the ligand-binding domains in the inte-
grin headpiece also destabilizes the bent conformation and
induces integrin extension in which the headpiece extends and
breaks free from an interface with the leg domains that connect
it to the plasma membrane. To stabilize the outward swing of
the hybrid domain and the high affinity open headpiece con-
formation, glycan wedges have been introduced into the inter-
face between the hybrid and I-like domains of

3
and

1
inte-
grins (9). The relation between hybrid domain swing-out and
high integrin affinity has also been strongly supported by the
study of an allosteric inhibitory

1
integrin antibody SG19,
which binds to the outer side of the I-like hybrid domain inter-
face and prevents hybrid domain swing-out as shown by elec-
tron micrographic image averages (10). Allosteric inhibition by
this mAb
1
was confirmed because it did not inhibit ligand
binding to the low affinity state but rather inhibited conversion
to the high affinity state. Binding of SG19 mAb to the

1 wedge
mutant was dramatically decreased compared with wild-type,
further supporting induction of hybrid domain swing-out by the
wedge mutant. Conversely, allosteric activating mAbs have
been shown to map to the face of the

hybrid domain that is
closely opposed to the
␣
subunit in the closed conformation and
therefore appear to induce the high affinity state by favoring
hybrid domain swing-out (11). Disulfide cross-links in the

6–
␣
7 loop (12) and shortening of the
␣
7-helix in the I-like
domain (13) also support the conclusion that downward dis-
placement of the
␣
7-helix induces high affinity for ligand. A
homologous
␣
7-helix displacement in integrin
␣
subunit I do-
mains similarly induces high affinity for ligand (14).
It has long been known that integrin affinity for ligand is
strongly influenced by metal ions, and recently the basis for
this regulation has been deduced for the integrin
␣
4

7
(15). The
integrin
␣
4

7
binds the cell surface ligand mucosal cell adhe-
sion molecule-1 (MAdCAM-1) and mediates rolling adhesion by
* This work was supported by National Institutes of Health Grant
HL48675. The costs of publication of this article were defrayed in part
by the payment of page charges. This 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: Harvard Medical
School, 200 Longwood Ave., Boston MA, 02115. Tel.: 617-278-3225; Fax:
617-278-3232; E-mail: springeroffice@cbr.med.harvard.edu.
1
The abbreviations used are: mAb, monoclonal antibody; MIDAS,
metal ion-dependent adhesion site; LIMBS, ligand-induced metal bind-
ing site; ADMIDAS, adjacent to MIDAS; LMA, LIMBS, MIDAS, and
ADMIDAS; MAdCAM-1, mucosal cell adhesion molecule-1.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 53, Issue of December 31, pp. 55556–55561, 2004
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org55556
at National Institutes of Health Library on March 18, 2008 www.jbc.orgDownloaded from
lymphocytes in postcapillary venules in mucosal tissues and
the subsequent firm adhesion in endothelium and trans-endo-
thelial migration. These key steps in lymphocyte trafficking in
vivo can be mimicked in vitro by introducing
␣
4

7
transfected
cells into parallel wall flow chambers with MAdCAM-1 coated
on the lower wall. In Ca
2⫹
and Ca
2⫹
/Mg
2⫹
,
␣
4

7
mediates
rolling adhesion, whereas in Mg
2⫹
,Mn
2⫹
, or when
␣
4

7
is
activated from within the cell,
␣
4

7
mediates firm adhesion
(15–17). Unliganded-closed and liganded-closed structures of
␣
V

3
have revealed a linear array of three divalent cation
binding sites in the I-like domain (18, 19). The metal coordi-
nating residues in

3
are 100% identical to those in

7
. Muta-
tion of these residues in

7
, and studies of synergy between
Ca
2⫹
and Mg
2⫹
and competition between Ca
2⫹
and Mn
2⫹
,
revealed the following (15). 1) The middle of the three linearly
arrayed sites, the metal ion-dependent adhesion site (MIDAS),
is absolutely required for rolling and firm adhesion, and it can
bind to MAdCAM-1 (and presumably coordinate) either
through Ca
2⫹
,Mg
2⫹
,orMn
2⫹
. 2) The adjacent to MIDAS
(ADMIDAS) metal ion binding site functions as a negative
regulatory site that stabilizes rolling adhesion. Its mutation
results in firm adhesion in Ca
2⫹
,Mg
2⫹
,orMn
2⫹
. Furthermore,
Ca
2⫹
exerts negative regulation at high concentrations at this
site by favoring the closed I-like domain conformation, and
Mn
2⫹
activates integrins by competing with Ca
2⫹
at this site
and favoring an alternative coordination geometry seen in the
open I-like domain conformation. 3) The ligand-induced metal
binding site (LIMBS) functions as a positive regulatory site
that favors firm adhesion. Its mutation results in rolling adhe-
sion in Ca
2⫹
,Mg
2⫹
,orMn
2⫹
. Furthermore, synergism between
low concentrations of Ca
2⫹
and Mg
2⫹
results from their binding
to the LIMBS and MIDAS, respectively.
Despite these advances in understanding the mechanism by
which metal ions stabilize alternative conformations of integrin

I-like domains, several issues remain unresolved. How do the
closed and open conformations of the
␣
4

7
headpiece affect
rolling and firm adhesion? Does metal ion occupancy at the
LIMBS and ADMIDAS or outward swing of the hybrid domain
have the strongest effect on I-like domain conformation? If
changes occur at both metal binding sites and the I-like/hybrid
domain interface, does one dominate the other, or can they be
counterbalancing or additive? Here we address these questions
and the importance of allostery at the I-like/hybrid domain
interface by introducing a glycan wedge mutation into the

7
subunit to stabilize the open conformation of this interface.
MATERIALS AND METHODS
Monoclonal Antibodies—The human integrin
␣
4

7
-specific mono-
clonal antibody Act-1 was described previously (20, 21).
cDNA Construction, Transient Transfection, and Immunoprecipita-
tion—The

7
site-directed mutations were generated by using
QuikChange (Stratagene). Wild-type human

7
cDNA (22) in vector
pcDNA3.1/Hygro(⫺) (Invitrogen) was used as the template. All muta-
tions were confirmed by DNA sequencing. Transient transfection of
293T cells using calcium phosphate precipitation was as described (23).
Transfected 293T cells were metabolically labeled with [
35
S]cysteine
and -methionine, and labeled cell lysates were immunoprecipitated
with 1
l of Act-1 mAb ascites and 20
l of protein G agarose, eluted
with 0.5% SDS, and subjected to non-reducing 7% SDS-PAGE and
fluorography (24). The selected protein bands were quantified using a
Storm PhosphorImager after3hofexposure to storage phosphor
screens (Amersham Biosciences).
Immunofluorescence Flow Cytometry—Immunofluorescence flow cy-
tometry was as described (23) using 10
g/ml purified antibody.
Flow Chamber Assay—A polystyrene Petri dish was coated with a
5-mm diameter, 20-
l spot of 5
g/ml purified h-MAdCAM-1/Fc in
coating buffer (phosphate-buffered saline, 10 mMNaHCO
3
, pH 9.0) for
1 h at 37 °C followed by 2% human serum albumin in coating buffer for
1 h at 37 °C to block nonspecific binding sites (16). The dish was
assembled as the lower wall of a parallel plate flow chamber and
mounted on the stage of an inverted phase-contrast microscope (25).
293T cell transfectants were washed twice with Ca
2⫹
- and Mg
2⫹
-free
Hanks’ balanced salt solution, 10 mMHepes, pH 7.4, 5 mMEDTA, 0.5%
bovine serum albumin and resuspended at 5 ⫻10
6
/ml in buffer A (Ca
2⫹
-
FIG.1. Design of the N-glycosylation site (wedge) mutation
and confirmation by immunoprecipitation. A, sequence alignment
of the relevant portion of the human integrin

3
and

7
I-like domains.
The N-glycosylation sites in

3
and

7
wedge mutants are underlined,
and the residues mutated to Thr are shown in red. Helices and

-strands are labeled and overlined.Band C, the

3
I-like/hybrid
domain interface. The interfaces are shown in the closed (B) (19) and
open (8) (C) conformations, with the I-like and hybrid domains shown as
cyan and yellow ribbons, respectively. The structures are shown in the
same orientation after superposition using allosterically invariant por-
tions of the I-like domain (8). The side chain of the Asn that is N-
glycosylated in

3
and

7
wedge mutants is shown. D, lysates from
35
S-labeled 293T cell transfectants were immunoprecipitated with
Act-1 mAb. Precipitated wild-type (WT) and glycan wedge mutant
(Q324T) materials were subjected to non-reducing 7.5% SDS-PAGE and
fluorography. Positions of molecular mass markers are shown on the
left, and the integrin bands are indicated on the right.
TABLE I
Expression of
␣
4

7
mutants
Integrin
␣
4

7
cell surface expression in 293T transient transfectants
was determined with Act-1 mAb and immunofluorescence flow cytom-
etry. The data are mean specific fluorescence intensity as percent of
wild type (WT) ⫾difference from the mean for two independent exper-
iments.
Name Mutation Expression
%WT
Wild type 100 ⫾12
Glycan wedge Q324T 47 ⫾3
LIMBS D237A 99 ⫾20
Wedge/LIMBS Q324T/D237A 38 ⫾10
ADMIDAS D147A 70 ⫾9
Wedge/ADMIDAS Q324T/D147A 35 ⫾8
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and Mg
2⫹
-free Hanks’ balanced salt solution, 10 mMHepes, and 0.5%
bovine serum albumin) and kept at room temperature. Cells were
diluted to 1 ⫻10
6
/ml in buffer A containing different divalent cations
immediately before infusion in the flow chamber using a syringe pump.
Cells were allowed to accumulate for 30 s at 0.3 dyne cm
⫺2
. Then,
shear stress was increased every 10 s from 1 up to 32 dynes cm
⫺2
in
2-fold increments. The number of cells remaining bound at the end of
each 10-s interval was determined. Rolling velocity at each shear stress
was calculated from the average distance traveled by rolling cells in 3 s.
To avoid confusing rolling with small amounts of movement due to
tether stretching or measurement error, a velocity of 2
m/s, which
corresponds to a movement of 1/2 cell diameter during the 3-s meas-
urement interval, was the minimum velocity required to define a cell as
rolling instead of firmly adherent (26). Microscopic images were re-
corded on Hi8 videotape for later analysis.
Surface Calculations—Accessible surfaces were calculated with
probes of the indicated radii using the buried surface routine of Crys-
tallography and NMR System software (27).
RESULTS
Activation of
␣
4

7
with a Glycan Wedge Mutation—The mu-
tation Gln-324 3Thr in

7
introduced an N-glycosylation site
at Asn-322 in the
␣
4–

5 loop of the I-like domain (Fig. 1A), the
same position as used previously for the wedge mutant in the
highly homologous

3
subunit (9). Crystal structures have been
defined for the

3
I-like/hybrid domain interface in both closed
low affinity and open high affinity conformations (5, 8, 19). In
the

3
subunit, the identical Asn residue has 5-fold more sol-
vent-accessible surface area as determined with a 1.4-Å probe
radius to simulate a water molecule in the open conformation
(Fig. 1C) than in the closed conformation (Fig. 1B)ofthe
hybrid/I-like interface. To approximate the size of the first four
carbohydrate residues of an N-linked glycan, we used a 10-Å
probe radius (see “Materials and Methods”), and we found that
the Asn side chain was accessible in the open but not the closed
conformation, as would be expected from visual inspection of
the interface (Fig. 1, Band C). Similarly to other wedge mu-
tants (9), the
␣
4

7
wedge mutant was expressed somewhat less
well than wild type in 293T transfectants (Table I). Immuno-
precipitation and SDS-PAGE of [
35
S]cysteine- and -methio-
nine-labeled
␣
4

7
showed a 3,000 M
r
increase for the Q324T
mutant

7
subunit compared with wild type, confirming N-
glycosylation of the introduced site (Fig. 1D).
During maturation and processing of
␣
4

1
and
␣
4

7
, a por-
tion of the intact
␣
4
subunit, which migrates at 150,000 and
180,000 M
r
, is cleaved to fragments of 80,000 and 70,000 M
r
(28–31). Cleavage occurs after a dibasic Lys-Arg sequence in
the
␣
4
thigh domain (29, 31). Activation of T lymphocytes
increases cleavage of the
␣
4
subunit (29, 32, 33). Interestingly,
addition of the glycan wedge markedly increased
␣
4
subunit
cleavage from 51% (wild type) to 90% (Q324T mutant) (Fig. 1C
and Table II). This finding directly demonstrates that
␣
4
inte-
grin activation (see below) enhances proteolytic processing of
the
␣
4
subunit. This could result either from greater exposure
of the cleavage site in open, extended
␣
4

7
or longer residence
in the post-endoplasmic reticulum compartments where proc-
essing occurs (34).
The adhesive behavior in shear flow of 293T
␣
4

7
cell trans-
fectants was characterized by allowing them to adhere to
MAdCAM-1 in a parallel wall flow chamber, incrementally in-
creasing the wall shear stress, and determining the velocity of
the adherent cells. In 1 mMCa
2⫹
, 293T transient transfectants
expressing wild-type
␣
4

7
rolled with increasing velocity as shear
stress was increased (Fig. 2). By contrast, wild-type transfectants
were firmly adherent in 1 mMMg
2⫹
(Fig. 2). In Mn
2⫹
, adhesive-
FIG.2.Adhesion in shear flow of wild-type and glycan wedge mutant
␣
4

7
cell transfectants on MAdCAM-1 substrates. Cells were
infused into the flow chamber in buffer containing 1 mMCa
2⫹
,1mMCa
2⫹
⫹1mMMg
2⫹
,1mMMg
2⫹
, or 0.5 mMMn
2⫹
. Cells transfected with
␣
4
cDNA alone (Mock)or
␣
4

7
transfectants treated with 5 mMEDTA did not accumulate on MAdCAM-1 substrates. Rolling velocities of individual
cells were measured at a series of increasing wall shear stresses, and cells within a given velocity range were enumerated to give the population
distribution. dyn, dynes.
TABLE II
Effect of the glycan wedge in
␣
4

7
on cleavage of
␣
4
subunit
293T cells were transiently transfected with wild-type (WT) or mu-
tant integrin
␣
4

7
using calcium phosphate precipitation and metabol-
ically labeled with [
35
S]cysteine and -methionine as in Fig. 1D. Labeled
cell lysates were immunoprecipitated with Act-1 antibody and sub-
jected to non-reducing 7% SDS-PAGE and fluorography. The selected
protein bands were quantified using a Storm PhosphorImager after 3 h
of exposure to storage phosphor screens. The percent of radioactivity in
each
␣
4
subunit band was calculated, and cleavage was calculated as
percent of (
␣
4/80
⫹
␣
4/70
)/(
␣
4/180
⫹
␣
4/150
⫹
␣
4/80
⫹
␣
4/70
).
Radioactivity in each
␣
4
subunit band Cleavage
␣
4/180
␣
4/150
␣
4/80
␣
4/70
%
WT 10 39 25 26 51
Q324T 2 8 41 49 90
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ness was more activated than in Mg
2⫹
because more cells accu-
mulated and fewer cells detached at the highest wall shear stress
of 32 dynes/cm
2
. By contrast with wild type, the
␣
4

7
Q324T
glycan wedge mutant mediated firm adhesion regardless of the
divalent cation present (Fig. 2). Furthermore, the accumulation
efficiency and shear resistance of the wedge mutant was identical
in Ca
2⫹
,Ca
2⫹
/Mg
2⫹
,Mg
2⫹
, and Mn
2⫹
and similar to that of the
wild-type
␣
4

7
293T transfectants in Mn
2⫹
. Thus, integrin
␣
4

7
was constitutively activated by the glycan wedge introduced into
the hybrid/I-like domain interface.
Mutation of the
␣
4
cleavage site residue Arg-558 abolishes
␣
4
subunit cleavage and has no effect on
␣
4

1
adhesion on fibronec-
tin or VCAM-1 (29, 35). We tested the effect of the same mutation
in
␣
4

7
transfectants, and we found it to have no effect on adhe-
sion in shear flow to MAdCAM-1 (data not shown).
As described previously (15), mutation of LIMBS residues
stabilizes integrin
␣
4

7
in the low affinity state. For example,
the LIMBS mutant D237A mediates rolling adhesion regard-
less of the divalent cations that are present (Fig. 3A). The
wedge/LIMBS double mutant (Q324T/D237A) was expressed
as well as the wedge mutant in 293T transfectants (Table I).
Compared with the LIMBS mutation, the wedge/LIMBS double
mutation reproducibly increased the number of firmly adher-
ent cells at low shear (1 and 2 dynes cm
⫺2
)inCa
2⫹
/Mg
2⫹
and
Mg
2⫹
(Fig. 3). In Mn
2⫹
, the wedge/LIMBS mutant mediated
firm adhesion, whereas the LIMBS mutant mediated rolling
adhesion (Fig. 3). These data show that the LIMBS is required
for full activation by the wedge mutation in Ca
2⫹
,Ca
2⫹
/Mg
2⫹
,
FIG.3. Interaction of glycan wedge and LIMBS mutations. A, adhesive modality and resistance to detachment in shear flow of LIMBS
(D237A) and wedge/LIMBS double mutant (Q324T/D237A)
␣
4

7
293T transfectants on MAdCAM-1 substrates in the presence of 1 mMCa
2⫹
,1mM
Ca
2⫹
⫹1mMMg
2⫹
,1mMMg
2⫹
, or 0.5 mMMn
2⫹
.B, the number of rolling and firmly adherent
␣
4

7
293T transient transfectants was measured
in the same divalent cations as in Aat a wall shear stress of 1 and 2 dynes cm
⫺2
. Data are ⫾S.D. (n⫽3). dyn, dynes.
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and Mg
2⫹
(Q324T/D237A mutant in Fig. 3Acompared with
Q324T mutant in Fig. 2). Furthermore, activation by Mn
2⫹
of
the double wedge/LIMBS Q324T/D237A mutant definitively
establishes that the LIMBS is not required for activation by
Mn
2⫹
.
Increased Firm Adhesion by Double ADMIDAS/Wedge Mu-
tant—Mutation of the negative regulatory ADMIDAS activates
firm adhesion even in Ca
2⫹
(15) (D147A mutant in Fig. 4
compared with wild type in Fig. 2). The double wedge/
ADMIDAS Q324T/D147A mutant was somewhat less well ex-
pressed than the ADMIDAS D147A mutant (Table I). Nonethe-
less, the double Q324T/D147A mutant showed more firmly
adherent cells in Ca
2⫹
and Mn
2⫹
than did the single D147A
mutant (Fig. 4) or the single Q324T mutant (Fig. 2).
DISCUSSION
Allosteric transition to the high affinity integrin headpiece
conformation is proposed to involve rearrangement of the

I-like LIMBS, MIDAS, and ADMIDAS (LMA) sites, downward
displacement of the

I-like
␣
7-helix that connects to the hybrid
domain, and outward swing of the

hybrid domain (5, 7–9, 11,
12, 36). Outward swing of the hybrid domain has been demon-
strated by electron micrographic studies of liganded
␣
V

3
and
␣
5

1
integrins and crystal studies of liganded
␣
IIb

3
but not in
an
␣
V

3
crystal structure in which crystal lattice and head-
piece-leg interactions presumably prevented swing-out when a
ligand was soaked into crystals (8, 18). Conversely, introduc-
tion of a glycan wedge into the

1
and

3
subunits has been
demonstrated to induce high affinity for ligand by
␣
5

1
and
␣
IIb

3
integrins (9). Both of these integrins recognize ligands
with RGD sequences. We have extended these results here to
the
␣
4

7
integrin, which does not recognize RGD in its ligands
and which mediates both rolling and firm adhesion. In
␣
4

7
,
the glycan wedge converted rolling adhesion in Ca
2⫹
and Ca
2⫹
/
Mg
2⫹
to firm adhesion, demonstrating that stabilizing the open
conformation at the I-like/hybrid domain interface is sufficient
to stabilize high affinity firm adhesion.
Furthermore, we examined here for the first time the inter-
play between the LMA metal binding sites at the ligand-bind-
ing interface on the “top” of the

I-like domain and the inter-
face with the hybrid domain on the opposite, “bottom” face of
the I-like domain. We asked whether one of these two inter-
faces would dominate regulation of rolling or firm adhesion, or
whether there would be mutuality in which mutations in each
of these interfaces influenced the equilibrium between rolling
and firm adhesion. The results demonstrate the latter. That is,
stabilization of rolling adhesion by LIMBS mutation was par-
tially counteracted by the wedge mutation in Ca
2⫹
/Mg
2⫹
and
Mg
2⫹
and fully counteracted in Mn
2⫹
, where firm adhesion
occurred. Conversely, stabilization of firm adhesion by the
wedge mutation was fully counteracted by the LIMBS muta-
tion in Ca
2⫹
, where rolling occurred, and largely counteracted
in Ca
2⫹
/Mg
2⫹
and Mg
2⫹
. Therefore, the equilibrium at the
LMA sites strongly influences that at the

I-like/hybrid do-
main interface and vice versa, and changes in equilibrium at
one site can counterbalance those at the other. The combined
effects of the ADMIDAS and wedge mutations also demon-
strated additive effects at the LMA sites and I-like/hybrid
interface because changes at both of these sites stabilized firm
adhesion more strongly than changes at either alone.
Another notable finding of these studies is that Mn
2⫹
can
still activate firm adhesion when the LIMBS is mutated. Pre-
viously, the LIMBS and ADMIDAS were found to be positive
and negative regulatory sites, respectively, and positive regu-
lation by low Ca
2⫹
concentrations was found to be intact when
the ADMIDAS was mutated (15). This, together with struc-
tural considerations, suggested that negative regulation by
high Ca
2⫹
concentrations was effected at the ADMIDAS. Scat-
chard plots showed competitive rather than noncompetitive
inhibition by Ca
2⫹
of stimulation by Mn
2⫹
, suggesting that the
ADMIDAS was also the stimulatory site for Mn
2⫹
. However, it
was not possible to confirm the role of the ADMIDAS in stim-
ulation of firm adhesion by Mn
2⫹
because rolling adhesion
occurred in LIMBS mutants even in Mn
2⫹
. By contrast, in the
double LIMBS/wedge mutant, the equilibrium between rolling
and firm adhesion is not far from that in wild type, and it is
regulated by divalent cations. Mn
2⫹
was found to fully activate
FIG.4.Interaction of glycan wedge and ADMIDAS mutations. Adhesive modality and resistance to detachment in shear flow of ADMIDAS
(D147A) and double wedge/ADMIDAS (Q324T/D147A) mutant
␣
4

7
transfectants on the MAdCAM-1 substrates in the presence of the indicated
divalent cations. The divalent cation concentrations are the same as in Fig. 2. dyn, dynes.
Conversion of Rolling to Firm Adhesion55560
at National Institutes of Health Library on March 18, 2008 www.jbc.orgDownloaded from
firm adhesion by the LIMBS/wedge mutant, showing that the
LIMBS is not required for regulation by Mn
2⫹
and providing
strong support for the previous conclusion that the ADMIDAS
is the site for activation by Mn
2⫹
.
Although much progress has been made recently in defining
different integrin conformational states, questions remain
about how signals are transduced from the cytoplasm to the
ligand binding site and whether intermediate conformational
states have intermediate affinity for ligand. It appears that to
mediate rolling adhesion, integrins must be in one of the ex-
tended conformations rather than in the bent conformation (15,
37). The extended conformation with the closed headpiece is an
intermediate in the conformational pathway between the bent
conformation, which contains a closed headpiece, and the ex-
tended conformation with the open headpiece (5). The current
study demonstrates that stabilization of the open headpiece by
a glycan wedge at the

I-like/hybrid interface is sufficient to
convert low affinity rolling adhesion to high affinity firm adhe-
sion. It appears that the glycan wedge converts the extended
conformation with the closed headpiece to the extended confor-
mation with the open headpiece. Therefore, this study strongly
suggests that within the extended integrin conformation, con-
version of the closed to the open headpiece is sufficient to
convert rolling adhesion to firm adhesion. In an intact integrin,
marked separation in the plane of the membrane of the trans-
membrane domains of the integrin
␣
and

subunits would also
stabilize the open headpiece and therefore may be the mecha-
nism for converting rolling adhesion to firm adhesion.
Acknowledgment—We thank Dr. Michael J. Briskin for providing the
human MAdCAM-1/Fc.
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