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Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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L and M subunits
intracellularly. However, only the ß2 variants
terminating after Q444 promote cell surface expression of LFA-1 and
Mac-1. Thus, the major cysteine-rich region and the three highly
conserved cysteine residues at positions 445, 447, and 449 of the
ß2 subunit are not required for LFA-1 and Mac-1 surface
expression. The surface-expressed LFA-1 variants are constitutively
active with respect to ICAM-1 adhesion and these variants express the
activation reporter epitope of the mAb 24. In contrast,
surface-expressed Mac-1, both the wild type and variants, require 0.5
mM MnCl2 for adhesion to denatured BSA. These results
suggest that the role of the ß2 subunit in LFA-1- and
Mac-1-mediated adhesion may be different. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunits: LFA-1
(Lß2, CD11a/CD18),
Mac-1 (Mß2,
CD11b/CD18), p150,95
(Xß2, CD11c/CD18), and
Dß2 (2, 3). In the present study, we focus on LFA-1 and Mac-1. LFA-1 is
expressed on all leukocytes and is involved in a broad range of
immunological processes including leukocyte extravasation, Ag
presentation, and T-lymphocyte alloantigen-induced proliferation
(4). The three well-characterized LFA-1 counterreceptors
are ICAM-1 (CD54), -2 (CD102), and -3 (CD50), all of which are members
of the Ig superfamily (5, 6, 7). Mac-1 is expressed mainly on
cells of myeloid lineage and plays an important role in the
phagocytosis of infectious agents (8), the
transendothelial migration of phagocytes (9, 10), and the
activation of neutrophils and monocytes (11). Mac-1
recognizes a wide spectrum of ligands, which include ICAM-1
(12), fibrinogen (13), the blood-clotting
factor X (14), the complement fragment iC3b
(8), the hookworm neutrophil inhibitory factor
(15), and denatured proteins (16).
The extracellular domain of the common ß2
subunit of LFA-1 and Mac-1 is linearly organized into a short
N-terminal cysteine-rich region
(CRR),5 a highly
conserved region (HCR) predicted to have an Inserted (I)-domain-like
fold, a mid-region, and a major CRR followed by a transmembrane segment
and a cytoplasmic tail (Fig. 1
). The N-terminal CRR is predicted to
form a plexin-semaphorin-integrin (PSI) domain (17).
Functional roles have been assigned to the HCR and the major CRR.
Mutations of selected residues in the HCR abolished the ligand-binding
capacity of LFA-1 and Mac-1 (18). In addition, of the 10
missense mutations reported to date for patients with leukocyte
adhesion deficiency type-1, nine reside in the HCR, the exception being
the R593C mutation, which is located in the CRR. These mutations either
abrogate the expression of the CD11/CD18 integrins or lead to the
expression of dysfunctional integrins (19, 20). These
observations underscore the importance of the HCR in the expression and
ligand-binding capacity of the ß2 integrins.
The major role of the CRR appears to be regulation of adhesion
functions. Replacing the major CRR of the ß2
subunit with that of ß1, which does not form
heterodimers with either the L or
M subunits, results in a chimeric
ß2/ß1(CRR) subunit that
forms a constitutively active LFA-1 with respect to ICAM-1-binding
(21). Furthermore, the epitopes of
ß2 integrin-activating mAbs KIM185, KIM127,
MEM48, and CBR LFA-1/2 reside in the major CRR of the
ß2 subunit (22, 23, 24, 25). Thus,
perturbation of the major CRR affects the activity of the
ß2 integrins. The ß2
cytoplasmic tail interacts with a number of actin-binding proteins
through which the adhesion properties of LFA-1 and Mac-1 are regulated
(26, 27, 28, 29, 30, 31, 32).
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IIb subunit and formed a functional soluble
receptor (33). The extracellular domains of LFA-1, Mac-1,
1ß1 and
3ß1 have also been
expressed as soluble molecules and shown to be able to bind ligands
(34, 35, 36, 37). In this article, we report the determination of
a minimal fragment of the integrin ß2 subunit
required for the surface expression of LFA-1 and Mac-1, and the effects
of ß2 subunit truncations on the functions of
the resultant variants. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following mAbs were generous gifts from various sources:
MHM23 (dimer specific) and MHM24 (anti-L;
Refs. 38 and 39) were obtained from A.
J. McMichael (John Radcliffe Hospital, Oxford, U.K.); LPM19c
(anti-M; Ref. 40) was
obtained from the Leukaemia Research Fund Diagnostic Unit (John
Radcliffe Hospital); KIM185 (anti-ß2,
activating mAb; Ref. 41) was obtained from Dr. M. K.
Robinson (Celltech, Slough, U.K.); mAb 24 (directed against an
activation reporter epitope on ß2 integrins;
Ref. 42) was obtained from Dr. N. Hogg (Imperial Cancer
Research Fund, London, U.K.); MEM148
(anti-ß2; Ref. 43) was
obtained from Dr. V. Horejsí (Institute of Molecular Genetics,
Prague, Czech Republic); and MRC OX33 and MRC OX34 were obtained from
M. J. Puklavec (Medical Research Council Cellular Immunology Unit,
Oxford, U.K.). The mAb H52 was previously described (44).
Purified IgGs of MHM23, MHM24, LPM19c, and H52 were prepared from
hybridoma supernatants using HiTrap protein-G columns (Pharmacia
Biotech, Uppsala, Sweden). ICAM-1/Fc was prepared as described
previously (45).
cDNA expression constructs
The L cDNA in expression vector pcDNA3
(Invitrogen, Groningen, The Netherlands) was described previously
(46). Full-length M cDNA was
obtained by tailoring partial cDNA clones screened from a HPB-ALL
library (45) using probes constructed by PCR based on a
published sequence of M (47). The
ß2 cDNA J8.1E (21) in pBluescript KS-
(Stratagene, La Jolla, CA was used as a template for the construction
of the truncated ß2 variants. They were named
according to the amino acids at which stop codons (*) were introduced
through site-directed mutagenesis using PCR or the QuikChange
Site-Directed Mutagenesis kit (Stratagene). In all cases, the modified
PCR fragments were re-introduced into pBS-J8.1E. The authenticity of
the truncated ß2 variants was confirmed by DNA
sequencing before subsequent subcloning into expression vector pcDNA3.
The initiation methionine is assigned number "1" in the protein
sequence.
Cell culture and transfection
COS-7 cells were cultured in complete media containing the
following: RPMI 1640 with L-glutamine (Life Technologies,
Paisley, U.K.), 10% (v/v) heat-inactivated FCS (Sigma, St. Louis, MO),
and 100 µg/ml kanamycin. Transfections were performed using the
DEAE-dextran method (45). COS-7 cells grown to 
90%
confluency in 80 cm2 tissue culture flasks were
washed twice with RPMI 1640 followed by incubation in 10 ml of RPMI
1640 containing 0.4 mg/ml DEAE-dextran, 0.1 mM chloroquine, and 5 µg
each of the respective expression vectors for the
L, M, and ß
subunits for 4 h at 37°C in a 5% CO2
incubator. Thereafter, cells were washed once in PBS, shocked in 5 ml
of 10% (v/v) DMSO in PBS for 3 min, and washed twice in PBS. The cells
were then returned to fresh complete media. The following day, cells
were detached with trypsin and transferred to new tissue culture flasks
for another 24 h. Cells were harvested by detachment with 0.5 mM
EDTA in PBS for subsequent analyses.
Cell surface and metabolic labeling
Twenty-four hours after transfection, COS-7 transfectants were
detached with trypsin and 1 x 106 cells
were transferred into wells of six-well tissue culture plates, and
cultured for 24 h at 37°C in a 5% CO2
incubator. Cell surface labeling was conducted by washing adherent
cells twice in PBS followed by incubation in sulfo-NHS-biotin (Pierce,
Rockford, IL) at 0.5 mg/ml in PBS for 20 min on ice. The reaction was
quenched by washing the surface-labeled cells once in PBS containing 10
mM Tris-HCl (pH 8.0) and 0.1% (w/v) BSA, and cell lysates were
prepared.
For metabolic labeling, adherent cells were washed twice with PBS and incubated for 30 min in Met/Cys-free RPMI 1640 (ICN Biomedicals, Irvine, CA) at 37°C. Thereafter, cells were incubated in 1.5 ml of Met/Cys-free RPMI 1640 containing 300 mg/L L-glutamine, 5% (v/v) dialyzed heat-inactivated FCS, and 120 µCi/ml of Translabel 35S-labeled Met/Cys (ICN Biomedicals). After 2 h, 1.5 ml of complete media was added to each well. After 4 h at 37°C, the cells were washed twice in PBS and cell lysates were prepared.
For detecting soluble ß2 variants, COS-7 transfectants were incubated for 30 min in Met/Cys-free RPMI 1640 (ICN Biomedicals) at 37°C, washed with PBS, and incubated for 6 h in 3 ml of Met/Cys-free RPMI 1640 containing 300 mg/L L-glutamine, 5% (v/v) dialyzed heat-inactivated FCS, and 120 µCi/ml of Translabel 35S-labeled Met/Cys (ICN Biomedicals) at 37°C in a 5% CO2 incubator. The spent medium was collected and centrifuged at 12,000 x g for 5 min to remove cell debris. A total of 1 ml spent medium was used for immunoprecipitation.
Immunoprecipitation
Labeled cells were lysed by incubating on ice for 30 min in lysis buffer (10 mM Tris-HCl (pH 8), 150 mM NaCl, 1% (v/v) Nonidet P-40, 0.5 mM MgCl2, 0.15 mM CaCl2, 1 mg/ml BSA, 0.5 mM PMSF, 75 µg/ml aprotinin, 2.5 mM iodoacetamide, and 3 mM NaN3). Cell nuclei were removed by centrifugation for 15 min, 12,000 x g at 4°C. Cell lysate (200 µl) was precleared with an irrelevant Ab, either MRC OX33 (IgG1) or OX34 (IgG2a; 50 µl of hybridoma culture supernatant), and rabbit anti-mouse IgG (Sigma) was coupled to protein A-Sepharose beads (Sigma) before immunoprecipitation with the desired mAb (3 µg) for 1 h at 4°C. Beads were washed once with lysis buffer containing 500 mM NaCl, followed by another wash with lysis buffer, and then were loaded onto a 30% (w/v) sucrose cushion. After centrifugation for 3 min at 12,000 x g at 4°C, beads were washed once with 10 mM Tris-HCl (pH 8.0) containing 0.05% (w/v) SDS. Bound proteins were eluted with SDS-sample buffer containing 30 mM DTT at 85°C for 5 min. Protein samples were subjected to SDS-PAGE. For surface labeling studies, proteins were transferred to immobilon P membranes (Millipore, Bedford, MA) by electrophoresis and biotinylated protein bands were detected with streptavidin-HRP followed by ECL-plus (Amersham Life Sciences, Little Chalfont, U.K.). For metabolic labeling analysis, the protein gel was fixed in 10% (v/v) acetic acid and 20% (v/v) isopropanol for 20 min, incubated in Amplify (Amersham Life Sciences) for 15 min, vacuum-dried at 80°C onto a 3 MM Whatman chromatography paper, and exposed to Fuji (London, U.K.) x-ray film for 48 h at -70°C.
Cell adhesion assay
For analysis of LFA-1-mediated adhesion to ICAM-1, the wells of Polysorb microtiter plates (Nunc, Roskilde, Denmark) were coated with 100 µl/well of goat anti-human IgG (Fc specific) at 5 µg/ml in 50 mM sodium bicarbonate buffer (pH 9.2) for 16–20 h at 4°C. Nonspecific binding sites were blocked with 0.5% (w/v) BSA (Sigma) in PBS for 30 min at 37°C. Thereafter, 50 µl of 1 µg/ml ICAM-1/Fc in PBS containing 0.1% (w/v) BSA was added to each well of the coated plates and incubated for 2 h at room temperature. This coating was shown to be saturating for detection of LFA-1-mediated adhesion (21). For analysis of Mac-1-mediated adhesion to denatured proteins, plates were coated with 100 µl/well of 50 µg/ml BSA in sodium bicarbonate buffer for 24 h at 4°C. Nonspecific binding sites were blocked with 150 µl/well of 0.2% (w/v) polyvinyl-pyrrolidone (m.w. 10,000; Sigma) in PBS for 30 min at 37°C. In all cases, coated plates were washed twice with wash buffer (RPMI 1640 containing 5% (v/v) heat-inactivated FCS, 10 mM HEPES (pH 7.4)) before being used. Transfected cells were harvested and labeled with 3.0 mM 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester fluorescent dye (Molecular Probes, Eugene, OR) by incubation for 20 min at 37°C. Labeled cells were washed once in wash buffer, resuspended, and dispensed into each well of the ligand-coated plates at 1–3 x 104 cells/well followed by incubation for 30 min at 37°C in a 5% CO2 incubator. For different adhesion assays, cells were incubated with various combinations of activating and blocking reagents. The mAbs MHM24, LPM19c, and/or KIM185 were used at 10 µg/ml. Mg2+/EGTA (5 mM MgCl2 and 1 mM EGTA) and 0.5 mM MnCl2 were used to activate LFA-1- and Mac-1-mediated adhesion, respectively. Total cell fluorescence was determined using a fluorescence plate reader (CytoFluor 4000; PerSeptive Diagnostics, Framingham, MA). Plates were washed three times with wash buffer and the fluorescence of bound cells was determined.
Flow cytometry
Cells were incubated with 20 µg/ml primary mAb in wash buffer for 1 h at 4°C unless otherwise stated. Thereafter, cells were washed twice and incubated with FITC-conjugated sheep anti-mouse F(ab')2 secondary Ab (1:400 dilution; Sigma) for 45 min at 4°C. Stained cells were washed once and fixed in 1% (v/v) formaldehyde in PBS. Cells were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Data were analyzed using CellQuest Software (Becton Dickinson). Expression index was calculated by (% cells gated positive x geo-mean fluorescence intensity).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The initial set of truncated ß2 variants
are shown in Fig. 1
(upper
part of the diagram). ß2(I701*) contains
the entire extracellular domain of the ß2
subunit with both the transmembrane and cytoplasmic domains removed.
ß2(C481*) corresponds to a truncated
ß3 subunit which contained the first
469 expressed residues and was reported to be capable of
forming a functionally active platelet integrin
IIbß3 heterodimer
complex (33). ß2(C459*) was
designed to terminate at the putative N-terminal boundary of the CRR.
However, as will be shown later, this boundary assignment may have to
be revised. ß2(A430*): During the
characterization of a ß integrin subunit from sea sponge
(phylum Porifera), a splice variant was found to code for a
truncated ß subunit (48).
ß2(A430*) was constructed accordingly.
ß2(K414*) and
ß2(Y301*): The HCR of the
ß2 subunit was postulated to have an
I-domain-like fold. However, two different boundaries of the HCR were
predicted to have the same N terminus but different C termini
(49, 50). ß2(Y301*) was
constructed such that it contains the shorter version of the HCR, and
ß2(K414*) was generated encompassing the longer
version. ß2(K414*) also included a portion of
the mid-region to which the epitope of mAb H52 was mapped
(44). Construction of the
ß2(R450*) and ß2(C445*)
variants will be described in later sections.
Cell surface expression and heterodimer assembly of LFA-1 and Mac-1 variants
COS-7 cells transfected with L or
M in combination with the
ß2 variants were analyzed by flow cytometry
using the dimer-specific mAb MHM23 (Fig. 2). Single L or
M transfectants showed minimal staining with
MHM23. Similarly, the MHM23 epitope is not expressed on single
ß2 transfectants (data not shown), although the
ß2 subunit is expressed at high levels in COS-7
transfectants (21). Positive staining was detected for
transfectants expressing L or
M in combination with wild-type
ß2 or the variants
ß2(I701*), ß2(C481*),
and ß2(C459*), although the expression levels
of the integrins with the truncated ß2 variants
were lower than that with wild-type ß2. Minimal
MHM23 staining was detected on cells transfected with
L or M, or with
ß2(A430*), ß2(K414*),
and ß2(Y301*), suggesting that these variants
fail to support heterodimer expression on the cell surface.
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L specific)
or mAb LPM19c (M specific). Both
L and ß2 wild-type or
variant subunits were precipitated by MHM24 (Fig. 3A, left panel)
from the cell lysates of
Lß2wt,
Lß2(I701*),
Lß2(C481*), and
Lß2(C459*)
transfectants. In contrast, only low levels of
L were precipitated by MHM24 from lysates of
L,
Lß2(A430*),
Lß2(K414*), and
Lß2(Y301*)
transfectants. An identical profile was obtained using the
heterodimer-specific mAb 6.5e (not shown), whose epitope on the
ß2 subunit has been mapped to a region within
the shortest of the ß2-truncated subunits
(i.e., ß2(Y301*); Ref. 24). A
similar profile was observed for Mac-1 (Fig. 3A, right
panel) using LPM19c with the exception that the
M band was prominent in all lanes. This is
concordant with the expression properties of the
L and M subunits as
monomers on COS-7 cell transfectants; whereas the
L subunit is expressed at a suboptimal level
(21), the expression level of the
M subunit is high (51) as
compared with Lß2 and
Mß2 transfectants,
respectively.
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L and M might be due
to defects in protein biosynthesis or heterodimer assembly. To examine
these possibilities, the same set of transfectants was metabolically
labeled with [35S]-labeled Met/Cys and cell
lysates were immunoprecipitated with MHM24 or LPM19c. In all cases, the
ß2 variants coprecipitated with the
L or M subunits (Fig. 3B). Hence, ß2(A430*),
ß2(K414*), and
ß2(Y301*) are expressed and can combine with
the L or M subunit,
but they do not promote cell surface expression of the resultant
heterodimers.
The size of each of the following ß2 variants
precipitated was in agreement with their predicted molecular mass:
ß2wt (95 kDa),
ß2(I701*) (83 kDa),
ß2(C481*) (50 kDa),
ß2(C459*) (48 kDa),
ß2(A430*) (46 kDa),
ß2(K414*) (43 kDa), and
ß2(Y301*) (38 kDa).
Functional analyses of the LFA-1 and Mac-1 variants
LFA-1-mediated adhesion was assayed on immobilized ligand ICAM-1
(Fig. 4A). The wild-type
Lß2 transfectants
showed minimal adhesion to ICAM-1 but adhesion was augmented in the
presence of Mg2+/EGTA (42) or the
activating mAb KIM185 (41). In contrast,
Lß2(I701*),
Lß2(C481*), and
Lß2(C459*)
transfectants adhered strongly to ICAM-1 irrespective of the presence
or absence of Mg2+/EGTA. KIM185 was only included
for the analysis of
Lß2wt and
Lß2(I701*) because its
epitope was mapped to the ß2 major CRR and,
therefore, was absent from the remaining ß2
variants (41). In all cases, adhesion was specific because
it was abrogated in the presence of the function-blocking mAb MHM24.
Consistent with the lack of cell surface expression,
Lß2(A430*),
Lß2(K414*), and
Lß2(Y301*)
transfectants showed minimal adhesion to ICAM-1 under all conditions
(data not shown).
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Lß2(I701*),
Lß2(C481*), and
Lß2(C459*)
transfectants. Lß2wt
transfectant only showed positive staining with mAb 24 in the presence
of Mg2+/EGTA at 37°C (Fig. 4B). In
contrast, Lß2(I701*),
Lß2(C481*), and
Lß2(C459*)
transfectants expressed the mAb 24 epitope in the absence of
Mg2+/EGTA at 4°C. Taken together, these
observations indicate that
Lß2(I701*),
Lß2(C481*), and
Lß2(C459*) are
expressed in an active conformation and, therefore, display
constitutive ICAM-1 adhesion.
We next examined the adhesion properties of Mac-1 variants on denatured
BSA (Fig. 5).
Mß2wt transfectant
showed a low level of adhesion, which was augmented in the presence of
0.5 mM Mn2+. Adhesion was specific because it was
abolished by the function-blocking mAb LPM19c. A similar adhesion
profile was observed for the
Mß2(I701*),
Mß2(C481*), and
Mß2(C459*)
transfectants. In addition,
Mß2wt can also be
activated to adhere to denatured BSA with the mAb KIM185. It should be
noted that Mß2(I701*)
cannot be activated with the mAb. Thus, unlike LFA-1 variants, the
Mac-1 variants did not show any constitutive ligand-binding properties.
Similar results were obtained for adhesion of the Mac-1 variants on
iC3b (data not shown).
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The above results showed that there is a clear distinction between
the two sets of ß2 truncation variants; the
variants longer than ß2(C459*) support LFA-1
and Mac-1 surface expression and those shorter than
ß2(A430*) do not. Therefore, there may be
critical residues within the A430-L458 region of
ß2 that are important for LFA-1 and Mac-1 cell
surface expression. The prime candidates are the three highly conserved
cysteine residues found in all integrin ß subunits sequentially
arranged in the pattern of CXCXC. In the case of
ß2 subunit, they are C445, C447, and C449 (Fig. 1, lower part of the diagram). This led us to construct the
ß2(C445*) and ß2(R450*)
variants which flank the boundaries of the CECRC sequence.
Both ß2(C445*) and
ß2(R450*) support LFA-1 and Mac-1 cell surface
expression (Fig. 6A). In
addition, the LFA-1 variants are constitutively active with respect to
ICAM-1 adhesion, and the Mac-1 variants require
Mn2+ for adhesion to denatured BSA (Fig. 6B). Thus, the conserved cysteines C445, C447, and C449 of
the ß2 subunit are not required for LFA-1 and
Mac-1 cell surface expression. Furthermore, deletion of these cysteines
did not alter the functional properties of the resultant variant
ß2(C445*) as compared with
ß2(R450*) or to longer versions of the
ß2 variants.
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We next examined whether the ß2 variants
that support LFA-1 and Mac-1 cell surface expression could be secreted
into the tissue culture media (Fig. 7).
Single ß2 variant transfectants were
metabolically labeled with [35S]-labeled
Met/Cys and the spent media collected.
ß2(I701*), ß2(C481*),
ß2(C459*), ß2(R450*),
and ß2(C445*) were immunoprecipitated from the
spent media of the respective transfectants with the mAbs H52 and
MEM148. However, only ß2(I701*) was
precipitated with the mAb KIM185, which was consistent with the KIM185
epitope mapping to the major CRR of the ß2
subunit (41).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5ß1 and
IIbß3 (52, 53). The N-terminal regions of both subunits interact with each
other to form an ovoid head from which their respective C-terminal
regions extend. The biochemical data presented in this paper suggest
that the structures of LFA-1 and Mac-1 are in general agreement with
this model. The truncated constructs can be segregated into two groups.
The shorter constructs, ß2(Y301*),
ß2(K414*), and
ß2(A430*) can only interact with the
L and M subunit
intracellularly but the longer constructs (i.e.,
ß2(I701*), ß2(C481*),
ß2(C459*), ß2(R450*),
and ß2(C445*)) also support cell surface
expression of LFA-1 and Mac-1. The expression indices of the LFA-1 and
Mac-1 variants, as determined by the expression of the epitope of the
dimer-specific mAb MHM23 (Figs. 2 and 6), are generally lower than that
of their wild-type counterparts. Exactly how different truncations
affect the level of LFA-1 and Mac-1 expression remains to be
investigated. As a general trend, the truncated
ß2 variants support LFA-1 expression better
than Mac-1. In both cases, the most ineffective variant is
ß2(I701*), which is composed of the entire
extracellular domain of the ß2 subunit. There
is no gross structural change in the ß2(I701*)
variant, because the conformational epitopes of the four mAbs, KIM185
(22), KIM127 (22), MEM48 (23),
and MEM148 (43) are expressed (data not shown).
It is intriguing that the shorter ß2 subunits
can interact with the L and
M subunits intracellularly but cannot support
cell surface expression of LFA-1 and Mac-1. The evidence for
intracellular interaction came from the immunoprecipitation of the
biosynthetically labeled cell lysates using mAbs specific for the
L and M subunits. The
shorter ß2 subunits were found in the
immunoprecipitate in addition to the subunits (Fig. 3B).
The evidence that the shorter subunits cannot support surface
expression of LFA-1 and Mac-1 came from two sets of experiments. First,
the epitope of heterodimer-specific mAb MHM23 was not detected by flow
cytometry (Fig. 2). Second, the shorter ß2
subunits were not coprecipitated with the subunits from
surface-labeled cell lysates using the subunit-specific mAbs (Fig. 3A). Similar flow cytometry and immunoprecipitation profiles
were obtained using the heterodimer-specific mAbs IB4 (54)
and 6.5e (Ref. 24 ; data not shown). It is unlikely that
the shorter ß2 subunits are not detected on the
cell surface because they are not biotinylated. There is no difference
in the number of lysine residues of the shortest
ß2 subunit detected by surface labeling (i.e.,
ß2(C459*)) and the longest
ß2 subunit not detected (i.e.,
ß2(A430*)). Furthermore, when we permeabilized
the Lß2(A430*)
transfectant before labeling, we were able to detect the intracellular
biotinylated ß2(A430*) subunit by
coprecipitation with the L subunit using the
L-specific mAb MHM24 (data not shown). It is
interesting to note that most of the mAbs mapped to the N-terminal
region of the human ß2 subunit only recognize
the ß heterodimer but not the free ß2
subunit (24). For this reason we are at present unable to
determine the fate of the shorter truncated ß2
subunits in particular, whether they are secreted as monomers into the
tissue culture medium. In contrast, the longer variants can be found in
the tissue culture medium of their respective transfectants by
immunoprecipitation with the mAbs H52 and MEM148 (Fig. 7). These
soluble ß2 variants will be most useful for
future studies on the structure and function of the
ß2 integrin subunit.
A characteristic feature of the integrin ß subunits is the very
conserved pattern of cysteine residues in their extracellular domains.
A disulfide-bond model has been proposed for the integrin
ß3 subunit (55). Of relevance to
this investigation are the three cysteines in positions 445, 447, and
449 in the ß2 subunit. On the assumption that
the disulfide bonding patterns are identical in the
ß2 and ß3 integrin
subunits, C445 and C449 engage in a disulfide bond, and C447 bonds to
C25 near the N terminus of the subunit. This arrangement suggests that
these cysteines are likely to have an important role in the overall
structure and function of the integrin heterodimers. Intriguingly, the
ß2(C445*) variant in which the three cysteine
residues were deleted retains the ability to assemble with
L and M. Furthermore,
the resultant LFA-1 and Mac-1 molecules did not exhibit any significant
alteration in cell surface expression and ligand-binding properties as
compared with other longer ß2 variants (R450*,
C459*, C481*, and I701*). Therefore, the importance of these three
cysteine residues and their possible disulfide bond engagements needs
to be re-evaluated.
We have stated that the extracellular domain of the integrin ß subunit is composed of four regions. However, the boundaries between these regions are not well defined. Based on secondary structure analyses, it was suggested that the HCR has an I-domain-like fold. Two C-terminal boundaries have been proposed (49, 50). The experiments presented in this paper did not provide any distinction between the two possibilities because both ß2(Y301*) (containing the N-terminal PSI domain and the short version of the HCR) and ß2(K414*) (extending beyond the long version of the HCR) behaved identically in all experiments, and in particular, both failed to support LFA-1 or Mac-1 cell surface expression. Correspondingly the mid-region has two alternative N termini. The CRR is generally described as having four repeating elements of about 45 residues, each containing 8 cysteines (56, 57). Again, the boundaries of these repeating elements are uncertain. According to the disulfide bonding pattern proposed for the ß3 integrin (54), the repeating element of the CRR starts at C459 (ß2 sequence numbering), thus we constructed the variant ß2(C459*) which contains the N-terminal PSI, the HCR (either version), and the mid-region. The ß2(C459*) variant supports LFA-1 and Mac-1 surface expression, but so do the shorter constructs of ß2(C445*) and ß2(R450*). If we argue that the breakpoint, the point at which the constructs differ in their ability to support integrin heterodimer expression, must lie between discrete subdomains of the ß subunit, then the boundary between the mid-region and the CRR must lie between the residues A430 and Q444. Secondary structure prediction indicated that there is one ß strand within this region (50). Experiments are under way to determine whether there is a single residue break in this region to pinpoint the boundary between the mid-region and the CRR.
The cell surface-expressed LFA-1 variants (i.e., LFA-1 with the
ß2(C445*), ß2(R450*),
ß2(C459*), ß2(C481*),
and ß2(I701*) subunits) bind ICAM-1 more
effectively than the wild type. In particular, whereas the
transfectants expressing wild-type LFA-1 require either the activating
mAb KIM185 or Mg2+/EGTA for adhesion to
immobilized ICAM-1, the transfectants expressing the LFA-1 variants are
constitutively active with respect to ICAM-1 adhesion, which cannot be
promoted with either KIM185 or Mg2+/EGTA. This
cannot be due to the difference in the level of LFA-1 expression on the
cell surface because all variants are expressed at lower levels as
compared with the wild type. These variants also expressed the
active-LFA-1 reporter epitope of mAb 24 in the absence of
Mg2+/EGTA and elevated temperature, suggesting
that they are expressed in a conformational active state. It has been
reported from different laboratories that removal of the cytoplasmic
tail of the ß2 subunit led to the expression of
constitutively active LFA-1 (27, 28). This is possibly due
to a constraint imposed on the wild-type LFA-1 by interactions between
the and ß subunits. In the platelet integrin
IIbß3, the opposing
charges of an arginine residue (R995) in the
IIb subunit and an aspartic residue (D723) in
the ß3 subunit are important in maintaining the
integrin in a resting state (58). It was proposed that
disruption of this salt bridge may be a critical feature of integrin
activation via inside-out signaling. There are no transmembrane and
cytoplasmic domains in the truncated ß2
subunits described in this paper, thus the absence of a restraining
salt bridge may have been the cause of the constitutive active
properties of the LFA-1 variants. However, Mac-1 with the same set of
ß2 variants is not constitutively active with
respect to adhesion to denatured BSA but, like the wild-type Mac-1,
adhesion could be promoted with 0.5 mM Mn2+.
Because L and M only
share 35% sequence homology, it is reasonable to postulate that the
ß2 subunit interacts differently with the
L and M subunits.
Recently, we have found that two mutations, A341P in the HCR and R593C
in the major CRR, of the integrin ß2 subunit of
patients with leukocyte adhesion deficiency type-1, which can support
LFA-1 but not Mac-1 expression (S. K. A. Law, manuscript in
preparation). Together with the results presented in this article,
these observations suggest that the ß2 subunit
may have different roles in the function of LFA-1 and Mac-1.
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Acknowledgments |
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Footnotes |
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2 Current address: Tenovus Research Laboratory, Southampton General Hospital, Southampton, U.K.
3 Current address: Roche Products, Welwyn Garden City, U.K.
4 Address correspondence and reprint requests to Dr. Alex Law, Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
5 Abbreviations used in this paper: CRR, cysteine-rich region; HCR, highly conserved region; I, inserted; PSI, plexin-semaphorin-integrin.
Received for publication November 29, 2000. Accepted for publication June 20, 2000.
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subunits and a common ß subunit: the lymphocyte function-associated antigen (LFA-1), the C3bi complement receptor (OKM1, Mac-1), and the p150,95 molecule. J. Exp. Med. 158:1785.Dß2, binds preferentially to ICAM-3. Immunity 3:683.[Medline]
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