THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 49, pp. 42115–42122, December 9, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. The C-Terminal ␣I Domain Linker as a Critical Structural
Element in the Conformational Activation of ␣I Integrins*
Received for publication, July 15, 2011, and in revised form, September 22, 2011 Published, JBC Papers in Press, September 30, 2011, DOI 10.1074/jbc.M111.282830 Gabriele Weitz-Schmidt‡§1, Thomas Schu¨rpf‡, and Timothy A. Springer‡2
From the ‡Immune Disease Institute, Children’s Hospital Boston and Department of Pathology, Harvard Medical School, Boston
Massachusetts 02115 and the §University Basel, PharmaCenter, Klingelbergstr. 50-70, 4056 Basel, Switzerland Integrins are a large family of ␣/␤ heterodimeric cell surface
receptors that mediate interactions with other cells or the
extracellular matrix. They are important therapeutic targets in
a wide range of diseases, including cardiovascular and immune
disorders (1). Integrin activation is dynamically regulated by
signals from within the cell in a process termed inside-out signaling. In addition, outside-in signaling induced by ligand binding directs signals from the extracellular domains to the cytoplasm. This bidirectional signaling is associated with highly
coordinated domain rearrangements in both the ␣ and the ␤
subunits (2).
Several studies indicate that inside-out signaling converts
integrins from a bent conformation with a closed headpiece
into an extended conformation with an open headpiece and * This work was supported, in whole or in part, by National Institutes of Health
Grant CA31798.
To whom correspondence may be addressed: University Basel, PharmaCenter, Klingelbergstr. 50-70, 4056 Basel, Switzerland. E-mail: gabriele.
weitz-schmidt@unibas.ch.
2
To whom correspondence may be addressed: Immune Disease Institute, 3
Blackfan Circle, Center for Life Sciences Boston, Third Floor, Boston, MA
02115. Fax: 617-713-8232; E-mail: springer@idi.harvard.edu. thereby activates ligand binding (2) (Fig. 1, A–C). Central to
ligand recognition are von Willebrand factor type A domains
that are present in all integrin ␤ subunits (termed ␤ inserted
(␤I) domains) and in some ␣ subunits (termed ␣ inserted (␣I)
domains). In integrins that lack ␣I domains, the activated
␤I domain directly interacts with the ligand through a metal
ion-dependent adhesion site (MIDAS).3 In ␣I integrins, the ␣I
domain serves as the ligand-binding domain instead of the
nearby ␤I domain. Interestingly, in these integrins, the ␤I
domain appears to regulate the activation of the ␣I domain and,
thus, ligand binding. In the current model, an invariant Glu
residue (Glu-310 in ␣L) located at the C-terminal end of the ␣I
domain is thought to bind to the MIDAS of the active ␤I
domain (Fig. 1C). This interaction is hypothesized to lead to an
axial displacement of the ␣7 helix of the ␣I domain in the C-terminal direction, as seen in crystal structures of isolated ␣I
domains (3). As a consequence, the ␣I MIDAS is turned into a
high-affinity, ligand-binding state (2).
Additional support for a structural link between the ␣I and ␤I
domains comes from studies that characterize small molecule
inhibitors of the integrin ␣L␤2 at a molecular level (4). One class
of ␣L␤2 inhibitors, termed ␣I allosteric inhibitors, has been
shown to bind underneath the C-terminal ␣7 helix of the isolated ␣I domain and stabilize it in the closed, low-affinity state
(4). On the basis of this finding it is hypothesized that these
inhibitors lock the integrin in its inactive form by preventing
the downward axial shift of the ␣7-helix required for ␣I
domain/␤I domain interactions (4) (Fig. 1A). Another class of
␣L␤2 allosteric inhibitors, termed ␣/␤ I allosteric inhibitors,
appear to bind to the MIDAS of the ␤I domain (4, 5). Thereby
they are hypothesized to competitively antagonize the binding
of ␣L Glu-310 to the ␤I domain. As a result, the ␣I domain
remains in an inactive state, whereas the ␤I domain together
with the “leg” region of the integrin is stabilized in a pseudoliganded, active state, as shown by the induction of activationdependent epitopes and induction of the extended conformation with the open headpiece (as seen with electron
microscopy) (6) (Fig. 1D).
Crystal structures of the ␣I integrin ␣X␤2 revealed unanticipated flexibility of the ␣I domain (7). One possible function of
this flexibility would be to enable two ␤I domain conformational states to couple with three ␣I domain states (7). A key 1 DECEMBER 9, 2011 • VOLUME 286 • NUMBER 49 3 The abbreviations used are: MIDAS, metal ion-dependent adhesion site;
ICAM-1, intercellular adhesion molecule-1; DMSO, dimethyl sulfoxide; MFI,
mean fluorescence intensity; LFA-1, lymphocyte function-associated
antigen-1. JOURNAL OF BIOLOGICAL CHEMISTRY 42115 Downloaded from http://www.jbc.org/ by guest on February 16, 2017 The activation of ␣/␤ heterodimeric integrins is the result of
highly coordinated rearrangements within both subunits. The
molecular interactions between the two subunits, however,
remain to be characterized. In this study, we use the integrin
␣L␤2 to investigate the functional role of the C-linker polypeptide that connects the C-terminal end of the inserted (I) domain
with the ␤-propeller domain on the ␣ subunit and is located at
the interface with the ␤I domain of the ␤ chain. We demonstrate
that shortening of the C-linker by eight or more amino acids
results in constitutively active ␣L␤2 in which the ␣I domain is no
longer responsive to the regulation by the ␤I domain. Despite
this intersubunit uncoupling, both I domains remain individually sensitive to intrasubunit conformational changes induced
by allosteric modulators. Interestingly, the length and not the
sequence of the C-linker appears to be critical for its functionality
in ␣/␤ intersubunit communication. Using two monoclonal antibodies (R7.1 and CBR LFA-1/1) we further demonstrate that shortening of the C-linker results in the gradual loss of combinational
epitopes that require both the ␣I and ␤-propeller domains for full
reactivity. Taken together, our findings highlight the role of the
C-linker as a spring-like element that allows relaxation of the ␣I
domain in the resting state and controlled tension of the ␣I domain
during activation, exerted by the ␤ chain. C-Terminal ␣I Domain Linker and Activation of ␣I Integrins EXPERIMENTAL PROCEDURES
Antibodies, Small Molecules, and Recombinant ICAM-1—
The sources of the mouse anti-human ␣L mAbs TS2/4, TS1/22,
CBR LFA-1/1, and the mouse anti-human ␤2 mAb CBR LFA1/2 have been described previously (9, 10). The mouse antihuman ␤2 mAb KIM127 was a kind gift from Martyn Robinson
at Celltech (11). The mouse anti-human ␣L mAb R7.1 was provided by Robert Rothlein (Boehringer-Ingelheim Pharmaceuticals, Ridgefield, CT) (12). LFA878 was obtained from Novartis
Pharma, Basel, Switzerland. XVA143 was synthesized according to example 345 of the patent (13) and was obtained from
Paul Gillespie (Hoffmann-La Roche, Inc., Nutley, NJ). LFA878
and XVA143 were dissolved in DMSO at 10 mM or 1 mM,
respectively, and stored at ⫺20 °C. Recombinant ICAM-1
D1-D5 was produced as described (14) using a C-terminal His
tag and purification by nickel-nitrilotriacetic acid-agarose.
Cell Culture—Human embryonic kidney 293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented
with 2 mM L-glutamine, 10% fetal bovine serum, nonessential
amino acids, and penicillin-streptomycin at 37 °C with 5% CO2
(all reagents from Invitrogen). The day before transfection lowpassage 293T cells were transferred into 24-well plates.
cDNA Constructs and Transient Transfections—cDNA of
WT ␣L was inserted into pcDNA3.1/Hygro(-) (Invitrogen) and
used as template for mutagenesis. ␣L C-linker deletion and
swap mutants were generated by overlap extension PCR (15).
For the ␣L C-linker deletion mutants ⌬ 8 and ⌬ 10, the mutant
⌬ 6 was used as a template. Human-mouse ␣L chimeras in
expression vector AprM8 and I-less ␣L␤2 (lacking residues
129 –308) in the same expression vector were described previously (9, 16). The chimeras were named according to the species origin of their segments. For example, h217m248h indicates that residues 1–217 are from human (h) ␣L, residues from
218 to 248 are from mouse (m) ␣L, and residues from 249 to the
C terminus are from human ␣L. ␣L I domain expressed on the
cell surface with N-terminal or C-terminal transmembrane 42116 JOURNAL OF BIOLOGICAL CHEMISTRY domains have been described previously (17, 18). Resequencing
of these vectors demonstrated that they contain ␣L residues Val130 to Val-339 and Gly-128 to Tyr-307, respectively. All constructs were confirmed by sequencing. 293T cells (80% confluent)
were transfected with empty vector (mock) or cotransfected with
mutant ␣L and wild-type (WT) ␤2 plasmids (either inserted into
pcDNA3.1/Hygro(-), AprM8, or pcDNA3.1(⫹)) using Lipofectamine 2000 according to the manufacturer’s instructions. Two
days after transfection the cells were harvested for flow cytometric
analysis, adhesion, and binding assays.
Immunofluorescence Flow Cytometry—Immunofluorescence
flow cytometry was performed as described previously (19).
Briefly, transfected 293T cells were detached and washed once
in 20 mM HEPES (pH 7.3) containing 150 mM NaCl, 1 mM
CaCl2, 1 mM MgCl2, and 1.5% BSA (assay buffer A). Cells were
then resuspended in assay buffer A containing 10 ␮g/ml primary antibody and incubated on ice for 30 min. mAb KIM127
was used at a concentration of 7 ␮g/ml and incubated at 37 °C
for 20 min. After a washing step, the cells were exposed to
FITC-conjugated goat anti-mouse IgG (Invitrogen) diluted
1:500 in assay buffer A for 20 –30 min on ice. After two washing
steps, cells were resuspended in cold assay buffer A and analyzed on a FACScan (BD Biosciences). Mean fluorescence
intensities were calculated using the CellQuest software.
Cell Adhesion to ICAM-1—The cell adhesion assay was performed in V-bottom 96-well plates (Corning) as described previously (20). Briefly, the plates were coated with 10 ␮g/ml
recombinant human ICAM-1 or 10 ␮g/ml BSA as a control in
20 mM Tris (pH 8), 150 mM NaCl, and 2 mM MgCl2 at 4 °C
overnight (or 37 °C for 2 h) and then blocked with 20 mM Tris
(pH 7.5) containing 150 mM NaCl, 1.5% BSA, and 5 mM glucose
(assay buffer B) at 37 °C for 2 h. The transfected 293T cells were
detached, resuspended in assay buffer B, and labeled with 1–2
␮g/ml 2⬘,7⬘-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein
acetoxymethyl ester (BCECF AM) (Invitrogen) at 37 °C for 30
min in the dark. After this labeling step, the cells were washed
once and resuspended in assay buffer B containing 1 mM CaCl2
and 1 mM MgCl2 (resting condition) or 1 mM CaCl2, 1 mM
MgCl2, and activating mAbs (10 ␮g/ml KIM127 and 10 ␮g/ml
CBR LFA-1/2) or 1 mM MnCl2 alone (activating conditions).
After incubation at 37 °C for 25 min in the dark, cells were
vigorously pipetted up and down and added to the ICAM-1and BSA-coated plates (the cell number varied from 3000 to
10,000 cells/well from experiment to experiment). The plates
were immediately centrifuged at 100 ⫻ g for 10 min (Beckman
CS centrifuge, brake off). After centrifugation, nonadherent
cells that accumulated in the center of the V bottom were quantified using the Fluoroskan Ascent microplate fluorometer
(Thermo Scientific) with the “small beam” setting and filter sets
allowing excitation at 485 nm and quantification of emission at
535 nm. The percentage of cell adhesion was calculated according to the following formula: 冋 1⫺ 册 FIICAM-I
⫻100 ⫽ % of adhesive cells
FIBSA (Eq. 1) where Fl ICAM-1 is the fluorescent signal (arbitrary units)
when cells bind to ICAM-1 (low signal) and Fl BSA is the
VOLUME 286 • NUMBER 49 • DECEMBER 9, 2011 Downloaded from http://www.jbc.org/ by guest on February 16, 2017 question remaining to be answered is which structural features
enable the conformational flexibility of the ␣I domain needed
for the activation of I domain integrins. Linkers connect the N
termini (N-linker) and C termini (C-linker) of the ␣I domain to
the ␤-propeller domain in which the ␣I domain is inserted (7).
The role of the N-linker appears to be limited by its short length
of three residues. In contrast, the C-linker, which follows the
␣7-helix of the ␣I domain and contains the invariant Glu-310, is
ten residues long and is flexible, as shown by weak electron
density in the crystal structure (7).
Here we use the leukocyte integrin ␣L␤2 to test the hypothesis that the C-linker acts as a spring-like element that, when
mutationally shortened, activates the ␣I domain. ␣L␤2 is selectively expressed on all leukocytes and is among the best characterized of ␣I integrins (8). The ligands of ␣L␤2 are members of
the Ig superfamily, including intercellular adhesion molecule-1
(ICAM-1). The ␣L␤2/ICAM-1 interaction plays a major role in
inflammatory and immune responses by regulating cell adhesion, leukocyte trafficking, and T cell costimulation (8). The
present study provides important insights into how the
C-linker regulates ␣I integrin adhesiveness. C-Terminal ␣I Domain Linker and Activation of ␣I Integrins fluorescent signal in absence of ICAM-1 and presence of
BSA (high signal).
Binding of Multimeric Soluble ICAM-1—The binding of soluble ICAM-1 was assessed as described previously (21). Transfected 293T cells were detached using 20 mM HEPES (pH 7.3)
supplemented with 150 mM NaCl and 5 mM glucose (assay
buffer C) and transferred into V-bottom 96-well plates (Corning). The cells were washed in assay buffer C and resuspended
in assay buffer C containing 2 mM CaCl2 and 2 mM MgCl2 (50
␮l/well). Multimeric ICAM-1 complexes were prepared by
mixing human ICAM-1/Fc (R&D Systems) with affinity-purified goat anti-human IgG (H⫹L)-FITC antibodies (Invitrogen)
(1:10 w/w) and incubated at room temperature for 30 min. The
ICAM-1 complexes were diluted 1:6 in assay buffer C and
added to the plates (50 ␮l/well), yielding a final concentration of
DECEMBER 9, 2011 • VOLUME 286 • NUMBER 49 1 mM for each cation. The cells were incubated at room temperature for 30 min, washed in assay buffer C containing 1 mM
CaCl2/1 mM MgCl2 and subjected to immunofluorescence flow
cytometry. As a control, soluble multimeric human myeloma
IgG1␬ complexes with anti-human IgG were prepared as
described above and exposed to the transfected cells. RESULTS
Design and Cell Surface Expression of ␣L␤2 C-linker Mutants—
We designed five ␣L␤2 mutants in which the C-linker of the ␣I
domain (residues 309 –318) (Fig. 1F) was shortened by 2, 4, 6, 8,
or 10 amino acids (Fig. 1G). In ⌬ 10, the C-linker is completely
removed, and thus, this mutant is the only mutant that lacks the
invariant Glu-310 residue thought to be important for ␣I/␤I
domain communication (Fig. 1F). The C-linker of ␣L␤2 was also
JOURNAL OF BIOLOGICAL CHEMISTRY 42117 Downloaded from http://www.jbc.org/ by guest on February 16, 2017 FIGURE 1. Model of conformational activation of ␣L␤2 with the C-linker acting as a spring-like element (modified from (2)). A, bent conformation of WT
␣L␤2 with closed headpiece, stabilized by the ␣I allosteric antagonist LFA878 (low affinity). B, extended conformation of WT ␣L␤2 with closed headpiece and
epitopes of mAbs KIM127 and CBR LFA-1/2 exposed (low affinity (23)). TM, transmembrane; PSI, plexin-semaphorin-integrin. C, extended conformation of WT
␣L␤2 with open headpiece and ligand bound (high affinity). D, extended conformation of WT ␣L␤2 with open headpiece but closed ␣ I domain induced by ␣/␤
⌱ allosteric inhibitor XVA143. E, ␣L␤2 C-linker mutant ⌬ 10 shown in its extended conformation with a constitutively active ␣ I domain that is no longer
responsive to regulation by the ␤ I domain. F, close-up of the ␣L␤2 C-linker region. The C-linker is shown in black. Upon activation, residue Glu-310 (shown in
stick) is hypothesized to bind to the MIDAS Mg2⫹ ion shown as a green sphere. The homology model is based on the ␣X␤2 crystal structure (7). G, C-linker
deletions ⌬ 2 to ⌬ 10 and replacement of the ␣L␤2 C-linker by ␣X␤2 C-linker residues. C-Terminal ␣I Domain Linker and Activation of ␣I Integrins
replaced with the C-linker of ␣X␤2 (CX mutant) to differentiate
the importance of its sequence as compared with its length (Fig.
1G). Wild-type (WT) and mutated ␣ subunits were coexpressed with WT ␤2 in 293T cells. Immunofluorescence flow
cytometry using mAb TS2/4, which binds the ␣L ␤-propeller
domain and requires ␤2 association for reactivity, demonstrated that all ␣L␤2 C-linker deletion mutants were correctly
assembled and expressed at WT levels (Fig. 2A). In contrast,
swapping the ␣L␤2 C-linker for the ␣X␤2 C-linker reduced cell- FIGURE 3. Ligand-binding activity of ␣L␤2 C-linker mutants under activating or resting conditions. A, adhesion of fluorescently labeled 293T cell transfectants to immobilized ICAM-1 induced by Mn2⫹ or by activating mAbs (KIM127 & CBR LFA-1/2) was quantified using the V-bottom adhesion assay (activating
conditions). Percentage of adhesive cells was calculated as described under “Experimental Procedures.” Each bar represents the mean value ⫾ S.D. of four
independent experiments run in triplicates (Mn2⫹) or three independent experiments run in duplicates or triplicates (mAbs). The adhesion of mock-transfected
cells varied from 12% to 36% and was subtracted before calculating the mean values. **, p ⬍ 0.01; ***, p ⬍ 0.001, paired two-tailed Student’s t test comparisons
to WT. n.d., not determined. B, the adhesion of transfectants to ICAM-1 was quantified in presence of 1 mM CaCl2/1 mM MgCl2 using the V-bottom assay (resting
conditions). Each bar represents the mean value ⫾ S.D. of three to five independent experiments run in triplicates. The binding of mock-transfected cells varied
from 2% to 23% and was subtracted before calculating the mean values. C and D, the binding of soluble multimeric ICAM-1 complexes to 293T cell transfectants
was measured in the presence of 1 mM CaCl2 and 1 mM MgCl2 using flow cytometry (resting conditions). Results are expressed as a histogram (C) and MFI values
(D). m, mock-transfected cells; wt ctrl, binding of soluble multimeric human myeloma IgG1␬ to WT ␣L␤2-expressing cells; WT, wild-type ␣L␤2; ⌬ 2 to 10, ␣L
C-linker deletion mutants; CX and CX-E, ␣L C-linker swap mutants. 42118 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 49 • DECEMBER 9, 2011 Downloaded from http://www.jbc.org/ by guest on February 16, 2017 FIGURE 2. Reactivity of ␣L␤2 C-linker mutants with mAb TS2/4 and
KIM127. A, the reactivity of mAb TS2/4 with WT or mutant ␣L␤2 transiently
expressed on the surface of 293T cells was determined by immunofluorescence flow cytometry. Each bar represents the mean ⫾ S.D. of four independent experiments. The binding to mock-transfected 293T cells was subtracted before calculating the mean values. B, the binding of mAb KIM127 to
transiently expressed WT and mutant ␣L␤2 was measured in the presence of
0.1% DMSO (-XVA143) or 1 ␮M XVA143 (⫹XVA143). Results are expressed as
MFI. The binding of KIM127 to mock-transfected 293T cells has been subtracted from the MFI values. A representative experiment of two independent
experiments is shown. surface expression by 2-fold (Fig. 2A). However, expression at
WT level was achieved when ␣X-Glu-313 of the CX mutant was
replaced by the corresponding ␣L C-linker Ser residue (mutant
CX-E) (Figs. 1G and 2A).
Impact of C-linker Shortening on Global Conformation of
the Mutants—To assess the global conformation of the deletion mutants, we tested the binding of mAb KIM127. The
epitope of this activation-dependent antibody involves residues
on the ␤2 subunit that are masked in the bent (inactive) and
exposed in the extended (active) conformation (22). Further,
the KIM127 epitope is known to be induced by ␣/␤ I allosteric
inhibitors such as XVA143 (5). Under resting conditions in the
presence of Ca2⫹ and Mg2⫹, only basal binding of mAb
KIM127 to the mutants was noted (Fig. 2B). This result suggests
that the mutants are largely in the bent conformation in the
absence of activating agents. Interestingly, KIM127 epitope
exposure in the mutants was induced by XVA143 to a degree
comparable with the WT receptor (Fig. 2B). These results show
that despite C-linker shortening, all mutants are basally bent
and are able to bind XVA143 and undergo conversion to an
extended conformation.
Impact of C-linker Shortening or Swapping on ␣L␤2 Function—
The function of the mutants was studied by investigating the
adhesion of 293T cell transfectants to immobilized ICAM-1
using the V well assay format. Under activating conditions in
the presence of Mn2⫹, the mutants ⌬ 2 and ⌬ 10 adhered comparably to the WT, whereas adhesion of 293T cells expressing C-Terminal ␣I Domain Linker and Activation of ␣I Integrins FIGURE 4. Effect of ␣L␤2 inhibitors on the adhesion of C-linker deletion
mutants ⌬ 8 and ⌬ 10. The adhesion of 293T cells expressing the constitutively active mutants ⌬ 8 and ⌬ 10 to immobilized ICAM-1 was quantified in
the absence (w/o) and presence of DMSO (0.1%), XVA143 (1 ␮M), LFA878 (10
␮M), or TS1/22 (10 ␮g/ml) using the V-bottom assay. The experiment was
performed under resting conditions. The percentage of adhesive cells was
calculated as described under “Experimental Procedures.” Each bar represents the mean value ⫾ S.D. of one to three independent experiments run in
triplicates. The adhesion of 293T cells transfected with empty vector varied
from ⫺7 to 25% and was subtracted before calculating the mean values. DECEMBER 9, 2011 • VOLUME 286 • NUMBER 49 FIGURE 5. Characterization of the binding sites of mAbs R7.1 and CBR
LFA-1/1. A, mapping studies using ␣L␤2 mouse-human chimeras. 293T cells
transiently transfected with ␣L␤2 mouse-human chimeras were stained with
mAbs CBR LFA-1/1, R7.1, TS1/22 (control mAb), and TS2/4 (as a measure for
cell surface expression) and subjected to flow cytometry. The specific MFI
values were determined by subtracting the MFI of mock-transfected cells. The
binding of CBR LFA-1/1, R7.1, and TS1/22 was expressed as a percentage of
mAb TS2/4 binding. Reactivity of mAb TS2/4 to chimeras h217m248h,
h249m303h, and h300m442h was 78%, 24%, and 97% of the reactivity to WT
␣L␤2, respectively. Each bar represents the mean value ⫾ S.D. of two independent experiments. B, binding to ␣I-less ␣L␤2. 293T cells transiently transfected with ␣I-less ␣L␤2 were stained with mAbs R7.1, CBR LFA-1/1, TS1/22,
and TS2/4 and subjected to flow cytometry. The binding of the antibodies to
mock-transfected 293T cells was subtracted from the MFI values. Results are
expressed as percentage of mAb TS2/4 control. Each bar represents the mean
value ⫾ S.D. of triplicates. *, p ⬍ 0.05, paired two-tailed Student’s t test comparison to TS1/22. n.s., not significant. C, binding of mAbs to 293T cells transiently transfected with WT ␣L␤2 or with ␣I domains with type I or type II
transmembrane (TM) domains fused to the C- or N-terminus, respectively,
measured by flow cytometry. Results are expressed as a percentage of mAb
TS1/22 MFI and are mean ⫾ S.D. of three independent experiments. *, p ⬍
0.05, paired two-tailed Student’s t test comparisons to TS1/22 binding. n.s.,
not significant. D, effect of allosteric ␣L␤2 inhibitors on R7.1 and CBR LFA-1/1
epitope expression. 293T cells transiently transfected with wt ␣L␤2 were
stained with mAb R7.1 and CBR LFA-1/1 in the presence of DMSO (0.1%),
LFA878 (10 ␮M), or XVA143 (1 ␮M). Binding of the antibodies was quantified
by flow cytometry. The specific MFI values were determined by subtracting
the MFI of mock-transfected cells. Each bar represents the mean ⫾ S.D. of
triplicates. *, p ⬍ 0.05, paired two-tailed Student’s t test comparison to DMSO
control. neighboring regions, we characterized two antibodies that recognize epitopes that appear to include both the ␣I and ␤-propeller domains. mAbs R7.1 and CBR LFA-1/1 each inhibit the
function of human ␣L␤2 and are specific for the ␣L subunit (9,
25, 26). CBR LFA-1/1 reacts with a cell surface-expressed fragment containing ␣L residues 130 –338 that includes ␣L I
domain residues 130 –308 (17, 25). Similarly, mAb R7.1 binds to
a purified fragment containing the ␣I domain (26). Speciesspecific residues recognized by these mouse anti-human antibodies were mapped within intact ␣L␤2 using mouse-human
chimeras. Loss of reactivity with the h300m442h chimera
showed that human residues 301– 442 were absolutely required
for CBR LFA-1/1 (Fig. 5A) in agreement with the previously
described requirement for residues 301–359 (9). Furthermore,
human ␣I domain residues 250 –303 were also required for full
CBR LFA-1/1 reactivity (Fig. 5A). The h300m442h chimera
JOURNAL OF BIOLOGICAL CHEMISTRY 42119 Downloaded from http://www.jbc.org/ by guest on February 16, 2017 mutants ⌬ 4, 6, or 8 was significantly lower despite normal
expression (Figs. 3A and 2A). In contrast, all...

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