THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 5, pp.
3239 –3249, January 30, 2009
Disruption of the Integrin ␣L␤2 Transmembrane Domain
Interface by ␤2 Thr-686 Mutation Activates ␣L␤2 and
Promotes Micro-clustering of the ␣L Subunits*
Received for publication, April 10, 2008, and in revised form, October 17, 2008 Published, JBC Papers in Press, November 23, 2008, DOI 10.1074/jbc.M802782200
Ardcharaporn Vararattanavech1, Xin Lin, Jaume Torres, and Suet-Mien Tan2
From the School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore
Integrins are type I heterodimeric cell adhesion molecules
that mediate a wide array of biological processes. Integrin bi-
directional signaling allows communication between the cell
interior with its microenvironment. The integrin transmem-
brane domains (TMs) are the transducers of activation signal
that is relayed from the cytoplasmic domains to the distal ligand
binding site located in the ectodomain of the integrin and vice
versa. In this study, we showed that the disruption of the ␣L␤2
TMs by mutation of a key interface residue Thr-686 in the ␤2
TM promoted ␣L␤2 activation with ICAMs binding properties
that are reminiscent of an intermediate affinity receptor. The
activated ␣L␤2 TM mutants, however, showed minimal reactiv-
ity with the reporter mAb KIM127 that recognizes a highly
extended ␣L␤2. Two models of ␣L␤2 TM interaction were pro-
posed previously. One with GXXXG-type interaction, and
another that is based on TM cysteine-scanning analyses. Our
data are consistent with a GXXXG-type interaction of the ␣L␤2
TMs. Finally, we observed by FRET analyses that perturbation of
the ␣L␤2 TMs by ␤2 Thr-686 mutation facilitated ␣L micro-
cluster formation. This was diminished by linking the ␣L␤2
TMs with a disulfide bond, which served to clasp the TMs. These
data suggest that disruption of the TM interface changes ␣L␤2
ligand binding affinity, and it may contribute to ␣L micro-clus-
ter formation.
The metazoan cell adhesion molecules integrins are a large
family of type I membrane proteins form by noncovalent and
specific pairing of ␣ and ␤ subunits (1). Integrins have a large
ectodomain, two transmembrane domains (TMs),3 and short
cytoplasmic tails with the exception of the ␤4 subunit. Devoid
of intrinsic enzymatic activities, the integrins trigger intracel-
* This work was supported by Singapore Agency for Science, Technology,
and Research (A*STAR) BMRC Grant 04/1/22/19/358. The costs of publica-
tion of this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked “advertisement” in accord-
ance with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported by A*STAR BMRC Grant 03/1/22/19/238.
2
To whom correspondence should be addressed: School of Biological Sci-
ences, Nanyang Technological University, 60 Nanyang Dr., Singapore
637551, Singapore. Tel.: 65-6316-2834; Fax: 65-6791-3856; E-mail:
smtan@ntu.edu.sg.
3
The abbreviations used are: TM, transmembrane domain; BSA, bovine
serum albumin; CFP, cyan fluorescent protein; FRET, fluorescence reso-
nance energy transfer; ICAM, intercellular adhesion molecule; IEGF, inte-
grin epidermal growth factor; mAb, monoclonal antibody; PBS, phos-
phate-buffered saline; YFP, yellow fluorescent protein; DTT, dithiothreitol;
DPC, dodecylphosphocholine.
JANUARY 30, 2009 • VOLUME 284 • NUMBER 5
This is an Open Access article under the CC BY license.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
lular signaling circuits by selective recruitment of cytosolic
molecules to their cytoplasmic tails (2). The phosphorylations
of integrin cytoplasmic tails are also important events in mod-
ulating integrin activity and signaling capacity (3, 4). Together,
these cytosolic interactions and post-translational modifica-
tions are required for integrin bi-directional signaling that
allows communication between the interior of the cell with its
external microenvironment. The TMs of the integrins provide
the connection for signal transmission between their cytoplas-
mic tails and their ectodomains. Electron cryomicroscopy
study of human integrin ␣IIb␤3 suggests that the integrin TMs
interact with each other in the resting or low affinity state (5).
Conceivably, structural changes in the integrin ectodomain
elicited by ligand binding or the separation of the integrin cyto-
plasmic tails by cytosolic molecule such as talin will trigger
reorientation or/and separation of the TMs (6), events that are
required to relay signal from one end of the integrin molecule to
the other. Indeed, in the absence of activation signal impinging
on the integrin ectodomain or its cytoplasmic tails, the disrup-
tion of the TM interface alone is sufficient to promote ␣IIb␤3
activation and intermolecular homomeric interactions (7–9).
In line with these observations, the introduction of a disulfide
bond linking the ␣ and ␤ TMs constrained the movement of the
TMs, which blunted both inside-out and outside-in signaling in
␣IIb␤3 (10, 11).
To fully characterize integrin bi-directional signaling, the
structure of the TMs, and more importantly the orientation of
the ␣ and ␤ TMs with respect to each other require investiga-
tions. Based on computational studies, it was proposed that a
GXXXG-like motif in the ␣ and ␤ TMs, in particular those of
␣IIb␤3, favors interactions similar to that of the homodimer
glycophorin A (GpA) (12, 13). However, different models for
the packing of integrin ␣IIb␤3 TMs have also been suggested (5,
10). These models of TM heterodimers may represent distinct
stages of ␣ and ␤ TM rearrangements during integrin activation
or affinity change (10, 12, 14, 15). Another interesting but
debatable concept is the involvement of TM in the process of
integrin clustering. Homo-oligomerization of ␣IIb and ␤3 TM-
cytoplasmic tail fragments in dodecylphosphocholine (DPC)
micelles were detected by methods of ultracentrifugation,
NMR, and SDS-PAGE (16). The same group also reported
dimerization of the ␣IIb TM by TOXCAT assay that measures
oligomerization in the inner membrane of Escherichia coli (17).
However, using the GALLEX assay, a LexA DNA binding
domain/lacZ reporter system in specific E. coli strains, the inte-
grin ␤7 TM but not the ␤1 or ␤3 TM showed strong propensity
JOURNAL OF BIOLOGICAL CHEMISTRY 3239
Activation and Micro-clustering of ␣L␤2 with Disrupted TMs
to form homo-oligomers comparable to that of the GpA TMs;
homo-oligomerization of ␣IIb was only weakly detected (18).
Recently, NMR spectroscopy analysis of ␤3 TM peptide
embedded in bicelles and micelles also showed monomeric TM
segments (19). In CHO cell system in which full-length ␣IIb␤3
was investigated, disruption of the ␣IIb␤3 TM interface by the
point mutation G708N in ␤3 promoted receptor activation,
homo-oligomerization, and facilitated ␣IIb␤3 macro-cluster
formation (7). In a separate study, the same mutation increased
the ligand-binding affinity of ␣IIb␤3, but failed to form macro-
clusters (8). What accounts for these different observations is
not known.
Integrin ␣L␤2 (LFA-1, CD11aCD18) is being studied exten-
sively because of its primary function in immune homeostasis
involving leukocyte adhesion, migration, and proliferation
(20 –22). Many studies investigate the ectodomain confor-
mation of ␣L␤2, and the membrane proximal events imping-
ing on its cytoplasmic tails with regard to its functional reg-
ulation (23, 24). Although computational and modeling
studies provide a guide to ␣L and ␤2 TM interactions that
are possible, they remain to be tested in a full-length recep-
tor in a cell-based system (15). Unlike ␣IIb␤3, much less is
known about the ␣L␤2 TM interface. Previously, we showed
by TM exchange that replacing ␤2 TM with ␤4 TM gener-
ated an ␣L␤2 mutant that exhibited wild type activity (25).
This was in contrast to ␣L␤2 mutants with ␤2 TM replaced
by other ␤ TMs, many of these were activated. In this study,
we show that the ␤2 TM residue Thr-686 within the
GXXXG-like motif contributes toward the formation of the
␣L␤2 TM interface. Mutation of Thr-686 induced ␣L␤2 acti-
vation, and promoted ␣L micro-cluster formation.
EXPERIMENTAL PROCEDURES
Antibodies and Reagents—The mAb MHM23 (␤2 integrins
heterodimer-specific) (26, 27) hybridoma was a kind gift from
A. J. McMichael (John Radcliffe Hospital, Oxford, UK). The
anti-Golgi matrix protein GM130 mAb (28) was kindly pro-
vided by W. J. Hong (Institute of Molecular and Cell Biology,
A*STAR, Singapore). The hybridomas of mAb KIM185 (␤2-
specific and activating; its epitope is in the ␤2 integrin epider-
mal growth factor fold 4 and the ␤-tail domain (29, 30), mAb
KIM127 (␤2-specific and reporter of an extended ␤2 integrin;
its epitope is in the ␤2 integrin epidermal growth factor fold 2,
IEGF-2) (31, 32), and mAb IB4 (␤2 integrins heterodimer-spe-
cific and function-blocking; its epitope is in the ␤2 inserted-like
domain) (33, 34) were obtained from American Type Culture
Collection (ATCC, Manassas, VA). mAbs from culture super-
natant were purified using Hi-Trap protein A/G columns
(Amersham Biosciences). Recombinant human intercellular
adhesion molecule (ICAM)-1/Fc and ICAM-3/Fc were kindly
provided by S. K. A. Law (School of Biological Sciences, Nan-
yang Technological University, Singapore). All general chemi-
cals and reagents were from Sigma-Aldrich unless otherwise
indicated.
Expression Plasmids—The positions of the integrin amino
acids are numbered accordingly to Barclay et al. (35). Expres-
sion plasmids encoding full-length human integrin ␣L and ␤2
subunits were described previously (36). The integrin TM chi-
3240 JOURNAL OF BIOLOGICAL CHEMISTRY
mera ␤24, in which the ␤2 TM is replaced with the correspond-
ing TM segment from ␤4, was reported previously (25). All
point mutations were generated using the QuikChangeTM site-
directed mutagenesis (SDM) kit (Stratagene, La Jolla, CA) with
relevant primers. For fluorescence resonance energy transfer
(FRET) experiments, the expression plasmids containing ␣L
monomeric CFP (mCFP) and ␤2 monomeric YFP (mYFP) have
been described, and ␣LmYFP was generated by the same pro-
cedure (25). Briefly, mCFP was fused to the C terminus of the
␣L cytoplasmic tail with a linker that contains the amino acids
GPVAT, and the mYFP was fused to the C terminus of the ␤2
cytoplasmic tail with a linker that contains amino acids GGP-
VAT as described (6). All expression plasmids were verified by
sequencing (Research Biolabs, Singapore).
Cell Culture and Transfection—The 293T and K562 cells
were obtained from ATCC. 293T cells were cultured in Dulbec-
co’s modified Eagle’s medium containing 10% (v/v) heat-inac-
tivated fetal bovine serum (HI-FBS), 100 international units/ml
of penicillin and 100 ␮g/ml of streptomycin (Hyclone, South
Logan, UT). K562 cells were cultured in RMPI 1640 containing
10% (v/v) HI-FBS, 100 international units/ml of penicillin and
100 ␮g/ml of streptomycin. The Polyfect transfection reagent
(Qiagen) was used for 293T transient transfection. K562 cells
were transiently transfected by the method of electroporation
using the Amaxa Nucleofector device, and reagents per the
manufacturer’s instructions (Amaxa Gmbh, Germany).
Flow Cytometry—The surface expression levels of integrins
on transfectants were determined by immunostaining cells
with mAb IB4 (20 ␮g/ml) and secondary fluorescein isothiocya-
nate-conjugated sheep anti-mouse IgG (Sigma) (1:400), fol-
lowed by analysis on a FACS Calibur with the software
CellQuest (Becton Dickinson Biosciences, Mountain View,
CA) essentially as described previously (37). Expression index
(EI) was calculated by % gated positive ⫻ geo-mean fluores-
cence intensity (37).
Biotin Labeling of Cell Surface Proteins and Immuno-
precipitation—In brief, transfectants were washed twice in PBS
and incubated in PBS containing 0.5 mg/ml sulfo-NHS-biotin
(Pierce) for 15 min at room temperature. The biotin-labeling
reaction was terminated by washing cells in PBS containing 10
mM Tris-HCl, pH 8.0 and 0.1% (w/v) bovine serum albumin. For
the analyses of ␣L␤2 conformation, cells were maintained in
Dulbecco’s modified Eagle’s medium containing 5% (v/v) HI-
FBS, 10 mM HEPES (pH 7.4), and 2 ␮g each of mAb MHM23 or
KIM127 at 37 °C for 30 min. 5 mM MgCl2 and 1 mM EGTA were
included for the ␣L␤2 activating condition. Unbound mAb was
removed by washing cells twice in medium. Cells were lysed in
lysis buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% (v/v)
Nonidet-P 40) containing protease inhibitors at 4 °C for 30 min.
Immunoprecipitation was performed using rabbit anti-mouse
IgG (Sigma) coupled to protein A-Sepharose beads (Amersham
Biosciences) as described previously (37). Bound proteins were
resolved on a 7.5% SDS-PAGE gel under reducing conditions
(⫹DTT), and electroblotted onto Immobilon P membrane
(Millipore, Bedford, MA). Biotinylated protein bands were
probed with streptavidin-horseradish peroxidase and detected
by enhanced chemiluminescence (ECL) using the ECL-plus kit
(Amersham Biosciences).
VOLUME 284 • NUMBER 5 • JANUARY 30, 2009
 Activation and Micro-clustering of ␣L␤2 with Disrupted TMs
For the analyses of disulfide bond formation between the ␣L
and ␤2 TMs by the introduction of a cysteine pair, biotin label-
ing of ␣L␤2, immunoprecipitation, and protein bands detec-
tion were performed as described above with the following
changes. The mAb IB4 (2 ␮g) was used for immunoprecipita-
tion. Proteins were resolved on 7% SDS-PAGE gels under
reducing (⫹DTT) or non-reducing (⫺DTT) conditions.
Cell Adhesion Assays—Adhesion of transfectants to immobi-
lized ICAMs was performed essentially as described (37). Poly-
sorb microtiter well (Nunc, Roskilde, Denmark) was coated
with 0.5 ␮g of goat anti-human IgG Fc-specific (Sigma) in 50
mM bicarbonate buffer (pH 9.2), and nonspecific binding sites
blocked with 0.5% (w/v) bovine serum albumin in PBS. There-
after, ICAM-Fc (50 ng) in PBS was added to each well and
incubated at room temperature for 2 h. Cells were labeled
with 2⬘,7⬘-bis-(2-carboxyethyl)-5-(and-6) carboxyfluores-
cein (Molecular Probes, Eugene, OR), and ⬃2 ⫻ 104 cells in
medium containing 5% (v/v) HI-FBS and 10 mM HEPES (pH
7.4) were added to each ICAM-coated well and incubated at
37 °C for 30 min in a humidified 5% CO2 incubator. The
activating agents mAb KIM185 (10 ␮g/ml) and/or
Mg/EGTA (ME)(5 mM MgCl2, 1 mM EGTA) were included in
the experiments. Non-adherent cells were removed by wash-
ing the wells twice with medium. The fluorescence signal
that corresponds to the number of adherent cells was meas-
ured with a fluorescent plate reader (FL600) (Bio-Tek Instru-
ments, Winooski, VT). In all cases, the level of adhesion to
the ICAMs was determined and expressed as relative adhe-
sion to ligand with respect to that of wild-type ␣L␤2 in the
absence of activation.
Fluorescence Resonance Energy Transfer (FRET) Analyses—
For FRET experiments, K562 transfectants were adhered onto
poly-L-lysine coated glass slides by brief spinning. Acceptor
photobleaching, FRET (38) was performed on a Zeiss LSM510
confocal microscope (Carl Zeiss, Inc., Thornwood, NY) to
detect ␣L micro-clusters using similar procedures as described
previously (25). When ␣LmCFP oligomerizes with ␣LmYFP,
FRET will be detected. The settings for FRET used were: mCFP,
excitation wavelength 458 nm; emission filter BP 470 –500 nm;
mYFP, excitation wavelength 514 nm; emission filter LP 530
nm. Cells were visualized with an oil immersion 63⫻ objective.
The mYFP of an entire cell was photobleached by scanning the
entire cell 20 times using the 514 argon laser line that was set at
maximum intensity. The mCFP signal of the region of interest
(ROI), which is the cell membrane, was acquired before and
after the photobleach. The high density of ␣L␤2 in the center of
the cell (Golgi compartment) was excluded from the analyses
(6). FRET efficiency (EF) was calculated as a percentage using
the equation EF ⫽ (I6 ⫺ I5) ⫻ 100/I6, where In is the mCFP
intensity at the nth time point. Bleaching was performed
between the 5th and 6th time points. The mean noise computed
as NF ⫽ (I5 ⫺ I4) x 100/I5 in which the mCFP signals at the 4th
and 5th time points before the bleaching process was close to
zero in all cases.
Immunofluorescence Co-localization Analyses—K562 cells
transfected with ␣LmCFP, ␣LmYFP, and ␤2 or ␤2T686F were
adhered onto poly-L-lysine slides by brief spinning, and fixed in
3.7% (w/v) paraformaldehyde in PBS for 10 min at room tem-
JANUARY 30, 2009 • VOLUME 284 • NUMBER 5
perature. Cells were washed in HBSS, followed by incubation in
HBSS containing 0.3%(v/v) Triton X-100 for 3 min at room
temperature. Cells were stained with anti-Golgi matrix protein
GM130 mAb (1:50 dilution) for 1 h at room temperature. Cells
were washed in HBSS followed by staining with Alexa Fluor威
594-conjugated goat anti-mouse antibody (1:500 dilution)
(Molecular Probes) for 1 h at room temperature. Cells were
mounted in Vector shield and examined on a Zeiss LSM510
confocal microscope for co-localization of integrins with
GM130.
Models of ␣L␤2 TMs—The two backbone models for ␣L␤2
TMs interaction were obtained from a previous conformational
search that used the TMs of all integrin types (15), where only
these two models were found to be evolutionarily conserved,
and consistent with previous studies (10, 18).
RESULTS
Thr-686 of the ␤2 Subunit Participates in the Interaction of
the ␣L␤2 TMs—We reported previously that the specific pair-
ing of the integrin TMs is required for the proper expression of
the receptor, and the maintenance of the ␤2 integrins in a low
affinity state (25). Conceivably, non-permissive TM pairing
leads to disrupted TM packing of the ␣ and ␤ subunits. An
interesting observation was made with regard to the TM
mutant ␤24 in which the ␤2 TM was replaced with the ␤4 TM.
Unlike other ␤2 mutants with TM of ␤1, ␤3, ␤5, or ␤6 that
generated constitutively activated receptors when combined
with ␣L, the ␣L␤24 mutant showed wild-type ligand binding
activity (25). The ␤2 having its TM replaced by ␤7 or ␤8 TM
were excluded from the analyses because of the aberrant glyco-
sylation of ␤27 and defective expression of ␤28. Inspection of
the TM sequences of ␤1, ␤3, ␤5, and ␤6 shows a conserved Val
within the GXXXG-like motif of these subunits (Fig. 1A). How-
ever, in ␤2 and ␤4 the corresponding residues are Thr and Leu,
respectively. Previously, we have reported that two backbone
models of interaction of ␣ and ␤ integrin TMs are evolutionar-
ily conserved (15); a GXXXG-type (GpA-like) interaction
(model I, cyan) and a model consistent with previous TM cys-
teine-scanning analyses (model II, green) (Fig. 1B) (10). The ␤2
Thr-686 is positioned at the interface of the ␣L and ␤2 TMs in
both models.
We examined whether ␤2 Thr-686 is a key residue in the
interface of the ␣L␤2 TMs. 293T cells were transiently trans-
fected with either wild-type ␣L␤2 or ␣L␤2T686V (Fig. 2A). The
expression level of ␣L␤2T686V was comparable to wild-type
␣L␤2 as determined by flow cytometry using the mAb IB4 that
recognizes ␤2 integrin heterodimers (Fig. 2B) (33). To test the
activity of ␣L␤2T686V, cell adhesion assays on immobilized
␣L␤2 ligands ICAM-1 (39) and ICAM-3 (40) were performed.
Cells expressing wild-type ␣L␤2 showed minimal adhesion to
ICAM-1, but adhesion was markedly enhanced by the addition
of the ␤2 integrin activating mAb KIM185 (Fig. 2C) (29).
Importantly, cells expressing ␣L␤2T686V showed significant
constitutive adhesion to ICAM-1, albeit at a lower level when
compared with that of KIM185 activation. Adhesion was medi-
ated by ␣L␤2 because it was abrogated in the presence of the
mAb IB4 that is also function-blocking (25).
JOURNAL OF BIOLOGICAL CHEMISTRY 3241
Activation and Micro-clustering of ␣L␤2 with Disrupted TMs
FIGURE 1. Sequence alignment of integrin ␤ subunit TM sequences and
model of ␣L␤2 TMs. A, TM sequences of the human ␤ subunits were aligned.
The GXXXG-like motif is in the green box. Thr-686 (pink) of the ␤2 subunit and
the corresponding residue Val (yellow) in ␤1, ␤3, ␤5, and ␤6 are shown. The
amino acid number of the putative first residue in each TM sequence is shown
on the left. It should be noted that the membrane boundaries of the TM
segments remain to be fully defined for each subunit (19, 60, 61). Val-683 of
the ␤2 subunit is indicated and is discussed in the latter section. B, two models
of ␣L␤2 TM interactions described previously are shown (15). Model I (cyan) is
consistent with a GXXXG-type (GpA-like) interaction (18). Model II (green) is
consistent with the proposed TM interface of ␣IIb␤3 based on cysteine-scan-
ning analyses (10). The side chain of Thr-686 in these models is shown as a
stick.
The investigation was extended to examine the adhesion
profiles of these transfectants to ICAM-3 (Fig. 2D). It was pro-
posed that an intermediate affinity ␣L␤2 binds ICAM-1 but not
ICAM-3, and effective binding to ICAM-3 requires a high affin-
ity ␣L␤2 (41). Binding studies with isolated recombinant ␣L
inserted (I) domain, and the ICAMs showed that the binding
affinity is in the order of ICAM-1⬎ICAM-2⬎ICAM-3 (42).
Further, we showed that the conformational changes in ␣L␤2
that are required to bind ICAM-1 and ICAM-3 are different
(43). In our previous studies, the adhesion of ␣L␤2 expressing
3242 JOURNAL OF BIOLOGICAL CHEMISTRY
cells to ICAM-3 can be induced by a combination of KIM185
and Mg/EGTA (25, 41, 44, 45). In line with these observations,
transfectant expressing wild-type ␣L␤2 only adhered signifi-
cantly to ICAM-3 when both activating KIM185 and
Mg/EGTA were included (Fig. 2D). Interestingly, cells express-
ing ␣L␤2T686V showed significant adhesion to ICAM-3 in the
presence of KIM185 alone, and the level of adhesion was aug-
mented when KIM185 and Mg/EGTA were both included.
These data suggest that substitution of Thr-686 with Val in ␤2
TM promotes an activated ␣L␤2 with properties of an interme-
diate affinity receptor.
Based on these observations, it may be inferred that Thr-686
of the ␤2 subunit contributes to the packing of the ␣L and ␤2
TMs. To further verify these findings, we asked whether substi-
tution of Leu-686 to Val in the mutant ␤24, which retains a wild
type activity when paired with ␣L (25), could also induce an
intermediate affinity receptor. The expression level of ␣L␤24
was comparable to that of wild-type ␣L␤2 and ␣L␤24L686V as
determined by flow cytometry using mAb IB4 (Fig. 2E). Trans-
fectants expressing wild-type ␣L␤2 and ␣L␤24 showed similar
adhesion profiles to ICAM-1 for minimal adhesion was
detected in the absence of activating KIM185 (Fig. 2F). This was
consistent with our previous observations (25). By contrast,
cells expressing ␣L␤24L686V showed constitutive ICAM-1
adhesion, albeit at a lower level when compared with cells
treated with KIM185 (Fig. 2F). When ICAM-3 adhesion assay
was performed, cells bearing wild-type ␣L␤2 and ␣L␤24
showed similar adhesion profiles. Both KIM185 and Mg/EGTA
were required to induce significant cell adhesion to ICAM-3.
However, cells expressing ␣L␤24L686V showed significant
adhesion in the presence of KIM185 alone, which could be fur-
ther enhanced by the addition of Mg/EGTA (Fig. 2G). Taken
together, these data suggest that the ligand-binding properties
of ␣L␤2T686V and ␣L␤24L686V are similar, and that Thr-686
in the GXXXG-like motif of ␤2 has a role in maintaining the
␣L␤2 TM interface.
It was also apparent that the precocious activity of
␣L␤2T686V was not specific to the substituted Val because
substitution of Thr-686 with a conserved residue Ser or a
hydrophobic and aromatic residue Phe generated receptors
that were expressed at comparable levels (Fig. 3A) and had sim-
ilar ICAM binding properties as the ␣L␤2T686V mutant (Fig. 3,
B and C), although the activity of ␣L␤2T686S was compara-
tively less than ␣L␤2T686V and ␣L␤2T686F. We went further
to assess the conformation of these receptors by performing
immunoprecipitation analyses of transfectants that were sur-
face labeled with biotin (Fig. 4). The mAb KIM127 is a confor-
mational-sensitive mAb that recognizes the IEGF-2 fold, and
reports a highly extended ␣L␤2 (31, 32). A half-bent ␣L␤2 with
an engineered disulfide lock in the ␤2 flexion failed to react with
KIM127 (46). In the absence of activation by Mg/EGTA, wild-
type ␣L␤2 and mutants reacted minimally with KIM127 with
traces of unassociated ␤2 detectable that were attributed to free
␤2 subunits as reported previously (44). Addition of Mg/EGTA
markedly increased the level of receptors immunoprecipitated
with KIM127. The mAb MHM23 that is ␤2 heterodimer-spe-
cific reacted with wild-type ␣L␤2 and mutants even in the
absence of receptor activation, and was included as a control
VOLUME 284 • NUMBER 5 • JANUARY 30, 2009
 Activation and Micro-clustering of ␣L␤2 with Disrupted TMs
consideration. Val-683 does not
reside in the GXXXG-like motif of
the ␤2 TM (Fig. 1A). In the model of
a GXXXG-type interaction (cyan),
Val-683 is positioned away from the
interface, while in the other model
(green), Val-683 is located closer to
the ␣L TM (Fig. 5B). Thus we have
also generated ␣L␤2V683F and
examined its ligand-binding prop-
erties compared with ␣L␤2T686F
and wild-type ␣L␤2. All transfec-
tants expressed comparable levels of
integrins as determined by flow
cytometry analyses using mAb IB4
(Fig. 5C). When cell adhesion assays
to the ICAMs were performed, cells
bearing ␣L␤2V683F did not show
constitutive adhesion to ICAM-1,
and did not adhere significantly to
ICAM-3 in the presence of a single
activating agent KIM185 (Fig. 5, D
and E). It was apparent that the
adhesion profiles of cells express-
ing ␣L␤2V683F were similar to
that of cells expressing wild-type
␣L␤2. By contrast, cells expressing
␣L␤2T686F showed intermediate
affinity ICAMs binding properties
as discussed in the preceding sec-
tions. These data are in line with
the model of a GXXXG-type inter-
action of the ␣L␤2 TMs.
Micro-clustering of ␣L␤2 with
Disrupted TM-TM Interaction—
Previous study showed that the
mutation G708N of the ␤3 TM gen-
erated an activated ␣IIb␤3, and it
induced receptor macro-cluster for-
mation (7). A separate study showed
FIGURE 2. The contribution of ␤2 Thr-686 to the ␣L␤2 TM interface. A, illustration of the ␣L and ␤2 subunits affinity modulation of ␣IIb␤3 when
without (white) or with (gray) TM substituted with ␤4. The point mutations T686V and L686V are indicated. the interface of its TMs was dis-
B, expression levels of ␣L␤2 and ␣L␤2T686V on 293T transfectants were assessed by flow cytometry using the ␤2
integrin heterodimer-specific mAb IB4 (shaded histogram). Irrelevant mAb (open histogram). C and D, adhesion
rupted, but ␣IIb␤3 macro-cluster
of transfectants to immobilized ICAM-1 or ICAM-3 was performed with or without activating agents. The formation was not detectable (8).
activating agents are mAb KIM185 and Mg/EGTA (ME). Adhesion specificity was verified using mAb IB4, which Our present data are in line with the
is also a function-blocking mAb. The level of adhesion to the ICAMs was determined and expressed as relative
adhesion to ligand with respect to that of wild-type ␣L␤2 in the absence of activation. Data points represent concept that a disrupted TM-TM
means ⫾ S.D. of triplicates. E, expression levels of ␣L␤2, ␣L␤24, ␣L␤24L686V on 293T transfectants were interaction changes the ligand bind-
determined by flow cytometry using mAb IB4 (shaded histogram). Irrelevant mAb (open histogram). F and G, ing affinity of the integrin. Next, we
adhesion of transfectants expressing wild-type ␣L␤2 and mutants to the ICAMs is as described before. Data
points represent means ⫾ S.D. of triplicates. EI, expression index. sought to determine whether the
disruption of ␣L␤2 TMs interaction
(26, 27). These data suggest that the mutation of ␤2 Thr-686 could induce subunit oligomerization. To detect micro-cluster
promotes activation of the ␣L␤2, but does not induce an formation in which the individual molecules are ⬃10 nm in the
extended conformation detectable by KIM127. proximity of one another (47) instead of macro-cluster with a
A GXXXG-type Packing of the ␣L␤2 TMs—We note that the length scale of ⬎200 nm, we made use of photobleach FRET
orientation of the ␣L␤2 TMs in both models are different. In analysis. The C terminus of the ␣L cytoplasmic tail was fused
these models, Thr-686 is located at the interface of the TMs but with either monomeric cyan fluorescent protein (mCFP) or
with different degrees of rotation relative to the ␣L TM (Fig. 5, monomeric yellow fluorescent protein (mYFP) generating
A and B). This is more pronounced when Val-683 is taken into ␣LmCFP or ␣LmYFP, respectively. These will be referred to as

JANUARY 30, 2009 • VOLUME 284 • NUMBER 5 JOURNAL OF BIOLOGICAL CHEMISTRY 3243
Activation and Micro-clustering of ␣L␤2 with Disrupted TMs
FIGURE 3. Substitution of ␤2 Thr-686 with Phe or Ser generated an acti-
vated ␣L␤2. A, flow cytometry analyses of 293T transfectants expressing
wild-type ␣L␤2 and three mutants using the mAb IB4 (shaded histogram).
Irrelevant mAb (open histogram). B and C, cell adhesion assays of transfectants
bearing wild-type ␣L␤2 and mutants to the ICAMs. IB4 was included as the
function-blocking mAb to demonstrate adhesion specificity. Data points rep-
resent means ⫾ S.D. of triplicates. EI, expression index.
␣LCFP and ␣LYFP henceforth. K562 cells were used as the
surrogate cells for the transfection of ␣LCFP, ␣LYFP, and ␤2 as
described previously (25), and wild-type ␣L␤2 in K562 trans-
fectants has minimal propensity to oligomerize (6). When ␣L
micro-cluster forms, the proximity of ␣LCFP with ␣LYFP
(⬍10 nm apart) should allow FRET to be detected although there
will also be populations of ␣LCFP with ␣LCFP, and ␣LYFP with
␣LYFP that will not lead to FRET (Fig. 6A). In the absence of
YFP photobleach (referred to as unbleach), no significant
changes in the fluorescence intensity of CFP was detected in
transfectants bearing ␣LCFP, ␣LYFP, and ␤2 (Fig. 6, B and C).
When photobleach was performed, a marked decreased in YFP
signal was detected, but there was minimal increase in CFP
signal in these cells. By contrast, cells expressing ␣LCFP,
␣LYFP, and ␤2T686F showed an increase in CFP signal after
3244 JOURNAL OF BIOLOGICAL CHEMISTRY
FIGURE 4. The ectodomain conformation of the ␣L␤2 TM mutants as
determined by immunoprecipitation. Transfectants expressing the indi-
cated integrins were surface-labeled with biotin. Cells were incubated in
media containing the indicated mAbs, including the mAb KIM127 that
reports a highly extended ␣L␤2 (32). The mAb MHM23, which reacts with ␤2
integrin heterodimers and is not activation-dependent, was included as a
control (26, 27). Mg/EGTA (ME) was included to induce ␣L␤2 activation. Cells
were lysed and immunocomplexes precipitated as described under “Experi-
mental Procedures.” Proteins were resolved on a 7.5% SDS-PAGE under
reducing conditions. The ␣L and ␤2 bands are indicated.
YFP photobleach. Consistent with the lack of constitutive activ-
ity in ␣L␤2V683F shown in the previous section, cells bearing
␣LCFP, ␣LYFP, and ␤2V683F, showed minimal change in CFP
signal after YFP photobleach. The % FRET efficiency for each
condition was also calculated as described under “Experimental
Procedures” and plotted (Fig. 6D). We also examined FRET in
cells transfected with ␣LCFP and ␣LYFP (Fig. 6E). FRET signal
detected in these cells was low and comparable to cells express-
ing ␣LCFP, ␣LYFP, and ␤2. Cells expressing ␣LCFP, ␣LYFP,
and ␤2T686F consistently showed an increase in FRET signal. It
should be noted that the ␣L subunit may not fold properly in its
␤-propeller domain when expressed in the absence of the ␤2
subunit (48). Thus, we made use of cells transfected with
␣LCFP, ␣LYFP, and ␤2 as the control in the FRET analyses. As
reported previously (6), the fluorescence detected in the central
region of the cells was attributed to integrins in the Golgi
because immunofluorescence staining of the Golgi matrix pro-
tein GM130 (28) showed co-localization of the integrins with
GM130 (Fig. 6F). In this study, only the rim of each cell was
taken as the region of interest (ROI) in all the FRET analyses.
These observations suggest that mutation of Thr-686 in the ␤2
TM not only disrupts the packing of the ␣L␤2 TMs resulting in
receptor activation, micro-clustering of the ␣L subunits was
detected.
A Disulfide Bond Linking the ␣L␤2 TMs Attenuates ␣L
Micro-clustering Induced by the Disruption of the ␣L␤2 TM
Interface—To further verify that the disruption of the TMs in
␣L␤2 facilitates ␣L micro-cluster formation, a disulfide
clasp in the ␣L␤2 TMs was generated to prevent TM sepa-
ration, and its effect on ␣L micro-clustering examined. The
␣LLeu1067 and ␤2Ile679 were selected for mutation to Cys
based on model I (GXXXG-type interaction) (Fig. 7A). First,
we examined formation of the intersubunit disulfide bond
within ␣L␤2. Transfectants expressing wild-type ␣L␤2,
␣L␤2T686F, and ␣LL1067C␤2T686F/I679C were surface
labeled with biotin, and integrins precipitated with mAb IB4
VOLUME 284 • NUMBER 5 • JANUARY 30, 2009
 Activation and Micro-clustering of ␣L␤2 with Disrupted TMs
␣LL1067CCFP, ␣LL1067CYFP,
and ␤2T686F/I679C), a reduction
in FRET signal was detected (Fig.
8A). The reduction in FRET signal
was not due to an aberrant effect of
the introduced Cys because the
introduction of Cys into the ␣L
TM or the ␤2T686F TM alone
(cells expressing ␣LL1067CCFP,
␣LL1067CYFP, ␤2T686F or cells
expressing ␣LCFP, ␣LYFP,
␤2T686F/I679C, respectively) did
not reduced significantly the
FRET signal (Fig. 8B). FRET in
cells expressing ␣LL1067CCFP,
␣LL1067CYFP, ␤2I679C was also
comparable to cells expressing
␣LCFP, ␣LYFP, and ␤2. These
data suggest that the disulfide
clasp could effectively reduce the
disrupting effect of ␤2T686F
mutation on ␣L␤2 that facilitates
␣L micro-cluster formation.

DISCUSSION
A functional integrin is formed by
noncovalent and specific associa-
tion of an ␣ and a ␤ subunit (1). The
ligand binding properties and the
cytoplasmic signaling capacities of
integrins require conformational
changes that are transmitted from
their headpieces to their cytoplas-
mic tails and vice versa (23, 24). The
integrin TMs serve as the signal
transducers for the relay of activa-
FIGURE 5. Testing the GXXXG-type model of interaction of ␣L␤2 TMs. A, drawing illustrating the positions of tion signals between the extracellu-
part of the TM residues in ␣L and ␤2. Val-683 and Thr-686 are shaded gray. B, superimposition of the two models lar and intracellular regions of the
of ␣L␤2 TM interaction. Two views of the GXXXG-type model (cyan) and the model based on cysteine-scanning
analyses (green) are shown. The side chains of Val-683 and Thr-686 are shown as sticks. Note the difference in molecule. Unlike the well known
the orientation of Val-683 with respect to the ␣L TM in both models. C, flow cytometry analyses of 293T mechanisms of receptor tyrosine
transfectants expressing wild-type ␣L␤2 and mutants using IB4 as the primary mAb (shaded histogram). Irrel- kinases activation that involves
evant mAb (open histogram). D and E, transfectants were allowed to adhere to ICAMs under different condi-
tions aforementioned. The ␣L␤2-activating agents used were mAb KIM185 and Mg/EGTA (ME). Adhesion receptor dimerization (49), the sep-
specificity was demonstrated using the mAb IB4. Data points represent means ⫾ S.D. of triplicates. EI, expres- aration of the integrin TMs is
sion index.
required for integrin bi-directional
signaling (6, 7, 11). It appears that
(Fig. 7B). Proteins were resolved on reducing or non-reduc- the specific pairing and packing of integrin TMs via parallel
ing SDS-PAGE gels. Under reducing conditions, the ␣L and evolution is required to maintain integrins in a low affinity con-
the ␤2 protein bands were detected. Under non-reducing formation (25). Indeed, mutations that disrupt the interface
conditions, the ␣L and ␤2 subunits were detected for ␣L␤2 between integrin ␣IIb␤3 TMs have a propensity to generate
and ␣L␤2T686F. However, a single high MW protein band activated receptors (7, 8). Further, an engineered disulfide bond
that is equivalent to the MW of ␣L␤2 combined was detected that clasps the integrin TMs and prevents their separation
in ␣LL1067C␤2T686F/I679C. Thus, a disulfide bond linking attenuated ␣IIb␤3 outside-in signaling (11).
the ␣L␤2T686F TMs was generated. Next, FRET analyses Here, we showed that the TM residue Thr-686 of the integrin
were performed. As observed previously, effective FRET ␤2 subunit participates in the interaction of the ␣L␤2 TMs
was detected in cells expressing ␣LCFP, ␣LYFP, and because mutations of this residue induced ␣L␤2 activation with
␤2T686F after YFP photobleach as compared with cells ligand binding properties of an intermediate affinity receptor.
expressing ␣LCFP, ␣LYFP, and ␤2 (Fig. 8, A and B). By intro- This is based on the capacity of TM disrupted ␣L␤2 to bind
ducing a disulfide clasp in ␣L␤2T686F (cells expressing constitutively to ICAM-1 and not ICAM-3, which requires an

JANUARY 30, 2009 • VOLUME 284 • NUMBER 5 JOURNAL OF BIOLOGICAL CHEMISTRY 3245
Activation and Micro-clustering of ␣L␤2 with Disrupted TMs

FIGURE 6. The mutation T686F in the ␤2 TM promoted ␣L micro-cluster formation. A, an illustration of FRET in ␣LCFP␤2 and ␣LYFP␤2 micro-cluster. When
␣LCFP␤2 and ␣LYFP␤2 do not form micro-clusters, and the distance between the CFP and YFP is ⬎10 nm, none or minimal FRET will be detected. The
disruption of the TM interface by T686F leads to ␣LCFP␤2 and ␣LYFP␤2 micro-cluster formation (case I) in which ␣LCFP and ␣LYFP are ⬍10 nm apart, and FRET
will be detected. There will also be cases II and III in which no FRET will be detected. B, photobleach FRET analyses of K562 cells transfected with the indicated
integrin subunits. A representative cell is shown for each sample. The unbleached sample was included to illustrate the effectiveness of the YFP photobleach
procedure. C, fluorescence intensity plots of these transfectants with YFP photobleach occurring between the 5th and 6th time points. D, % FRET efficiency plot
of 30 cells analyzed for each sample. Two independent experiments were performed. Data from a representative experiment are shown. Data points represent
means ⫾ S.E. of 30 cells analyzed. *, p ⬍ 0.05 versus cells expressing ␣LCFP, ␣LYFP, ␤2, and cells expressing ␣LCFP, ␣LYFP, ␤2V683F. E, % FRET efficiency plot
of 20 cells analyzed for each sample. *, p ⬍ 0.05 versus cells expressing ␣LCFP, ␣LYFP, ␤2, and cells expressing ␣LCFP, ␣LYFP. F, co-localization of integrins with
the Golgi matrix protein GM130. GM130 was stained with anti-GM130 mAb and Alexa Fluor姞 594 goat anti-mouse conjugate. Bar, 10 ␮m.

3246 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 5 • JANUARY 30, 2009
 Activation and Micro-clustering of ␣L␤2 with Disrupted TMs
FIGURE 7. An engineered disulfide bond linking the ␣L␤2 TMs. A, posi-
tions of ␣L Leu-1067 and ␤2 Ile-679 are shown in the GXXXG-type model of
␣L␤2 TM interaction. The side chains of these residues are shown as sticks.
B, analyses of disulfide bond formation in the ␣L␤2 mutant. Transfectants
expressing the indicated integrins were surface-labeled with biotin, followed
by immunoprecipitation with mAb IB4. Proteins were resolved on 7% SDS-
PAGE under either non-reducing (⫺DTT) or reducing (⫹DTT) conditions. The
positions of the ␣L, ␤2, and disulfide-bonded ␣L␤2 (c-c clasp) protein bands
are indicated.
additional activation signal as reported previously (41, 43).
However, these ␣L␤2 mutants did not adopt an extended con-
formation that was detectable by reporter mAb KIM127 (32).
Based on negative stain electron microscopy of ␣L␤2 and
␣X␤2, it is evident that an extended conformation is one of the
hallmarks of integrin activation (50). Nonetheless, conforma-
tional breathing of a bent ␣L␤2 may also provide certain degree
of unbending that will not be poised to react with KIM127, but
could allow ligand-engagement. Interestingly, the ligand bind-
ing capacity of bent integrin ␣V␤3 has also been described and
discussed (24, 51). Using the method of FRET-based detection,
it was shown that integrin ␣4␤1 on U937 cells did not undergo
full extension when activated by Mn2⫹ or by formyl peptide
receptor activation (52). Recently, it was also shown that an
engineered disulfide bond linking the plexin-semaphorin-inte-
grin domain and IEGF-2 of the ␤2 subunit generated a mutant
␣L␤2 that was KIM127 negative, but showed a propensity to
adhere to ICAM-1, suggesting that full extension need not be an
absolute requirement for ␣L␤2 ligand-binding (46). Taken
together, it may be inferred that the disrupted packing of the
JANUARY 30, 2009 • VOLUME 284 • NUMBER 5
FIGURE 8. The TM disulfide clasp attenuated the propensity of ␣L micro-
cluster formation induced by ␤2 T686F mutation in ␣L␤2. A and B, % FRET
efficiency plots of K562 cells transfected with different ␣L and ␤2 constructs.
20 cells were analyzed for each sample. Two independent experiments were
performed. Data from a representative experiment are shown. Data points
represent means ⫾ S.E. of 20 cells analyzed. *, p ⬍ 0.05 versus cells express-
ing ␣LCFP, ␣LYFP, ␤2, or cells expressing ␣LL1067CCFP, ␣LL1067CYFP,
␤2T686F/I679C.
␣L␤2 TMs by mutations employed in this study induces con-
formational changes that do not culminate to full extension of
the receptor, but these changes are sufficient to activate ␣L␤2.
We have previously generated two models for integrin ␣/␤
TM interface independently from any experimental data (15)
that are consistent with both a GXXXG-type (cyan) interaction,
suggested from experiments using the GALLEX assay (18), and
that based on cysteine-scanning analyses (green) (10) (Fig. 1B).
The orientation of the ␤2 TM residues Val-683 and Thr-686 are
different in these models. Whereas both residues are located in
the interface of the TMs based on the cysteine-scanning ␣IIb␤3
model (green), Val-683 is projected away from the interface in
the GXXXG-type model (cyan) (Fig. 5B). Indeed, mutation
T686F but not V683F induced ␣L␤2 activation, which corrob-
orates well with a GXXXG-type interaction. This was further
exemplified in the FRET-based analyses of ␣L oligomerization.
Other than the heterodimeric TM-TM interaction, homomeric
TM-TM oligomerization of integrin subunits have been
reported using protein fragments containing integrin TMs
(16 –18). In a CHO cell-based system, the mutation G708N in
the ␤3 TM increased the ligand binding affinity of full-length
␣IIb␤3 and induced formation of macro-clusters (7). Further,
in the same study, oligomerization of purified full-length
␣IIb␤3 under activating conditions was detected by transmis-
sion electron microscopy (7). However, macro-cluster forma-
tion was not detected using the same system by another group
JOURNAL OF BIOLOGICAL CHEMISTRY 3247
Activation and Micro-clustering of ␣L␤2 with Disrupted TMs
(8). What accounts for the difference in observations is not
known. Nevertheless, in both studies, the ability of ␣IIb␤3 to
form micro-clusters rather than macro-clusters in a cell-based
system was not addressed. Integrin macro-cluster contains an
area with a diameter of ⬎200 nm that is detectable by micros-
copy, and it can only provide information on integrin localiza-
tion but not homo-oligomerization (8). However, micro-cluster
of integrins is defined as integrins that are within ⬃10 nm of
each other, which is a more accurate assessment of integrin
homo-oligomerization (47, 53).
FRET-based studies are suitable to detect integrin micro-
cluster because effective FRET requires the CFP and YFP that
are fused to the integrin subunits to be ⬍10-nm apart. Using
FRET by the method of photobleach, we detected ␣L micro-
cluster formation as a result of direct disruption of the ␣L␤2
TM interface. In line with the up-regulated ligand binding
activity of ␣L␤2T686F and not ␣L␤2V683F, ␣L micro-cluster
was detected only in ␣L␤2T686F. Thus, we conjectured that
direct disruption of the ␣L␤2 TM interface not only induces
receptor affinity change but also facilitates ␣L micro-cluster
formation. Because the study was conducted with a protocol of
using ␣LCFP, ␣LYFP, and ␤2 in the same cell, the detection of
FRET could only suggest oligomerization of the ␣L subunits.
Whether the ␤2 subunit homo-oligomerizes as a result of the
disrupted ␣L␤2 TMs is not known at present, but would be
interesting future studies to pursue.
The micro-cluster formation of ␣L in cells expressing
␣L␤2T686F may be attributed to the separation of ␣L␤2 TMs,
but it is also possible that ␣L␤2T686F has greater lateral mobil-
ity in the membrane as compared with wild-type ␣L␤2, hence a
higher propensity for ␣L to micro-cluster in ␣L␤2T686F cells.
This requires further investigations. Indeed, an interesting
study reports that different conformational states of ␣L␤2 dif-
fer in their lateral mobility in T cells (54). Recently, we have
reported a high propensity of ␣M and ␤2 synthetic TM homo-
meric interactions (55). Also homomeric associations of these
subunits could be detected in CHO cells by FRET (56). Addi-
tional studies addressing TM homomeric interactions of the ␤2
integrins, and the contribution of TM-TM interactions to
receptor lateral mobility will provide useful insights into
immune cell homeostasis, as in antigen-presentation and toler-
ance, in which cytoplasmic signaling emanating from clusters
of ␤2 integrins modulates the immune response (57–59).
Acknowledgments—We thank M. Cooray and K. G. Lim for technical
assistance. The authors have no financial conflict of interest.
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