Association of the Membrane Proximal Regions of the alpha  and beta  Subunit Cytoplasmic Domains Constrains an Integrin in the Inactive State*

Chafen LuDagger, Junichi Takagi, and Timothy A. Springer§

From the Center for Blood Research and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, January 22, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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The adhesiveness of integrins is regulated through a process termed "inside-out" signaling. To understand the molecular mechanism of integrin inside-out signaling, we generated K562 stable cell lines that expressed LFA-1 (alpha Lbeta 2) or Mac-1 (alpha Mbeta 2) with mutations in the cytoplasmic domain. Complete truncation of the beta 2 cytoplasmic domain, but not a truncation that retained the membrane proximal eight residues, resulted in constitutive activation of alpha Lbeta 2 and alpha Mbeta 2, demonstrating the importance of this membrane proximal region in the regulation of integrin adhesive function. Furthermore, replacement of the alpha L and beta 2 cytoplasmic domains with acidic and basic peptides that form an alpha -helical coiled coil caused inactivation of alpha Lbeta 2. Association of these artificial cytoplasmic domains was directly demonstrated. By contrast, replacement of the alpha L and beta 2 cytoplasmic domains with two basic peptides that do not form an alpha -helical coiled coil activated alpha Lbeta 2. Induction of ligand binding by the activating cytoplasmic domain mutations correlated with the induction of activation epitopes in the extracellular domain. Our data demonstrate that cytoplasmic, membrane proximal association between integrin alpha  and beta  subunits, constrains an integrin in the inactive conformation.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Integrins are heterodimeric adhesion molecules that mediate important cell-cell and cell-extracellular matrix interactions. To date, 25 different integrin alpha beta heterodimers have been reported (1). The leukocyte integrin subfamily consists of four members that share the common beta 2 subunit (CD18) but have distinct alpha  subunits, alpha L (CD11a), alpha M (CD11b), alpha X (CD11c), and alpha D for LFA-1, Mac-1, p150,95 and alpha D/beta 2, respectively (2-4). LFA-1 is expressed on all leukocytes and is the receptor for three Ig superfamily members, intercellular adhesion molecule-1, -2, and -3 (ICAM1-1, -2 and -3). Mac-1 and p150,95 are primarily expressed on myeloid lineage cells and bind ligands including ICAM-1, the complement component iC3b, and fibrinogen. The leukocyte integrins mediate a wide range of adhesive interactions that are essential for normal immune and inflammatory responses (5). Patients with lymphocyte adhesion deficiency have defective expression of leukocyte integrins on the cell surface because of mutations in the common beta 2 subunit. This disease causes an inability of phagocytic cells to bind to and migrate across the endothelium at sites of inflammation, resulting in severe bacterial and fungal infections (3).

The adhesiveness of leukocyte integrins is dynamically regulated in cells by cytoplasmic signals, a process termed inside-out signaling. For example, T-cell receptor cross-linking or activation of leukocytes with phorbol esters rapidly increases adhesiveness through LFA-1 (6-8). The enhanced adhesion is transient, and by 30 min after stimulation, cells lose their ability to bind to ICAM-1. This may provide a mechanism for regulating T cell adhesion and de-adhesion with antigen-presenting and target cells. In addition to activation by intracellular signals, divalent cations can directly modulate the ligand-binding function of leukocyte integrins (9-11). Activation can also be mimicked by certain antibodies that bind to the extracellular domain of the leukocyte integrins (12-15).

A key question on integrins is how signals are transduced from the cytoplasm to the ligand-binding site in the extracellular domain. The integrin alpha  and beta  subunits are both type I transmembrane glycoproteins. Electron microscopy of integrins reveals an overall structure with a globular headpiece connected to the plasma membrane by two long stalks each about 16 nm long (16). The two stalks correspond to the C-terminal portions of the alpha  and beta  subunits. The headpiece binds ligand and contains the more N-terminal domains, including a predicted beta -propeller domain and I domain in the alpha  subunit and an I-like domain in the beta  subunit. Both conformational change (affinity regulation) and receptor clustering in the membrane (avidity regulation) have been proposed as mechanisms for the enhancement of integrin adhesiveness through inside-out signaling (17-20). Conformational change in integrin I domains has been demonstrated in structural studies (21-23) and found to regulate ligand binding affinity (23-25).

The importance of integrin alpha  and beta  subunit cytoplasmic domains in inside-out signaling has been demonstrated by mutagenesis studies. Whereas partial deletions of the alpha L cytoplasmic domain have no effect on binding to ICAM-1, complete truncation of the cytoplasmic domain or internal deletion of the conserved membrane proximal GFFKR sequence constitutively activates LFA-1 (26, 27). Truncation of the alpha IIb cytoplasmic domain before, but not after the conserved GFFKR sequence renders the alpha IIbbeta 3 integrin constitutively active (28, 29). These findings demonstrate the importance of the membrane proximal alpha  subunit GFFKR sequence in the regulation of integrin adhesiveness. Partial truncations of the beta 3 cytoplasmic domain maintain alpha IIbbeta 3 in a low affinity state, but complete truncation or deletion of the membrane proximal seven residues causes constitutive ligand binding by alpha IIbbeta 3, indicating that this membrane proximal region of the beta 3 subunit is required to maintain alpha IIbbeta 3 in a low affinity state (30). It has been suggested that interactions between the alpha  and beta  subunit cytoplasmic/transmembrane domains that include complementary negatively and positively charged residues restrain integrins in an inactive state (29, 31). Several proteins that associate with integrin cytoplasmic domains have been identified (32-36); however, how these integrin-associated proteins function in physiological activation of integrins remains unclear.

Here we test the hypothesis that association between the membrane proximal regions of the integrin alpha  and beta  cytoplasmic domains constrains an integrin in the inactive state. We demonstrate that the membrane proximal region of the beta 2 cytoplasmic domain plays an important role in the formation of cell surface alpha beta heterodimers and maintenance of alpha Lbeta 2 and alpha Mbeta 2 in an inactive state. Replacement of the alpha L and beta 2 cytoplasmic domains with acidic and basic peptides that form an alpha -helical coiled coil renders the integrin inactive in cell types in which wild type alpha Lbeta 2 is either basally active or inactive, whereas replacement of the cytoplasmic domain with two basic peptides that do not form a heterodimer renders the integrin active in cell types in which wild type alpha Lbeta 2 is either basally active or inactive. Our findings directly demonstrate that association between the membrane proximal segments of the alpha  and beta  cytoplasmic domains regulates ligand binding by integrin extracellular domains.

    MATERIALS AND METHODS
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Monoclonal Antibodies-- The murine mAbs TS1/22, TS2/4 to alpha L (CD11a), CBR LFA-1/7 and CBR LFA-1/2 to beta 2 (CD18), CBRM1/33, CBRM1/20 and CBRM1/5 to alpha M (CD11b), and the nonbinding IgG X63 were described previously (15, 37-39). KIM127 (13) was kindly provided by M. Robinson (Celltech Limited, England). mAb m24 (9) was a kind gift from N. Hogg (Imperial Cancer Fund, England). mAb 2H11 (40) was generously provided by H-C Chang (Dana-Farber Institute, Boston).

Construction of Mutant alpha L and beta 2 Subunits-- beta 2 truncation mutants beta 2710* and beta 2702* were generated by introducing a stop codon at 710 and 702 in the beta 2 subunit, respectively (the 22 amino acid signal sequence was not included in beta 2 numbering; Fig. 1A). A NotI restriction site was designed in the downstream PCR primer after the stop codon. The PCR upstream primer corresponded to beta 2 cDNA sequences from 1651-1672. The wild-type beta 2 in plasmid AprM8 (41) was used as template for PCR reaction. The PCR product was cut with BstBI at nucleotide 1977 and NotI, and swapped with the corresponding BstB1-NotI fragment from wild-type beta 2 in AprM8.

The alpha Lacid and alpha Lbase constructs were generated by fusing a 30-amino acidic peptide or a 30-amino basic peptide (42) to the extracellular and transmembrane domains of the alpha L subunit following residue Tyr-1087 (Fig. 1A). The constructs were made by overlap extension PCR (43, 44) using the nucleotide sequences of the acidic and basic peptides (40). A stop codon was designed at the end of the peptide, and following the stop codon was an SphI site. The outer left PCR primer was 5' to the BstXI site at nucleotide 2963 of the alpha L cDNA. Two rounds of overlap PCR were performed. The final PCR product was cut with BstXI and SphI, and the BstXI-SphI fragment was swapped into the same sites of wild-type alpha L cDNA in plasmid AprM8. The unique NheI site in the acidic and basic peptide nucleotide sequences was used for mutant identification.

The beta 2base construct was made by fusing the basic peptide (42) to residue Trp-701 at the end of the putative beta 2 transmembrane domain with overlap extension PCR (Fig. 1A). The PCR strategy was similar to that for making the alpha Lacid and alpha Lbase constructs. The final PCR product was digested with BstBI and NotI, and the BstBI-NotI fragment was swapped into the same sites in wild-type beta 2 cDNA contained in plasmid AprM8. All mutations were verified by DNA sequencing.

Transient and Stable Transfection-- Transient transfection of 293T cells was described previously (45). Stable transfection of K562 cells and maintenance of stable cell lines were as described previously (27, 45).

Immunofluoresence Flow Cytometry-- Flow cytometry of cells was described previously (27). Briefly, cells (105) were incubated with primary antibody in 100 µl of L15/fetal bovine serum on ice for 30 min, except for KIM127 and m24. Incubation with mAbs KIM127 and m24 was carried out at 37 °C for 30 min (9, 13). mAbs, except for X63, were used as purified IgG at 10 µg/ml. The nonbinding IgG X63 was used at 1:20 dilution of hybridoma supernatant. Cells were then washed twice with L15/fetal bovine serum and incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (heavy and light chain, Zymed Laboratories Inc. Laboratories, San Francisco, CA) for 30 min on ice. After washing, cells were resuspended in cold phosphate-buffered saline and analyzed on a FACScan (Becton Dickinson, San Jose, CA).

Metabolic Labeling and Immunoprecipitation-- Metabolic labeling and immunoprecipitation was described previously (27). Briefly, 2 × 107 cells in 4 ml of labeling medium (methionine and cysteine-free RPMI 1640 containing 15% dialyzed fetal bovine serum) were labeled with 0.4 mCi of [35S]methionine and cysteine (ICN Biochemicals) overnight in a 37 °C incubator. Labeled cells were lysed, and the lysate was incubated with antibody-coupled-Sepharose beads overnight at 4 °C. The immunoprecipitates were subjected to 7.5% SDS-polyacrylamide gel electrophoresis and fluorography.

Cell Adhesion-- ICAM-1 was purified from human tonsil, and coated on 96-well plates as described previously (27). Human complement component iC3b was purchased from CalBiochem and coated at 10 µg/ml. Cell adhesion to immobilized ligand was described previously (27, 45). Bound cells were expressed as a percentage of total input cells.

    RESULTS
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The Membrane Proximal Region of the beta 2 Cytoplasmic Domain Regulates Integrin alpha beta Heterodimer Formation on the Cell Surface-- To examine the role of the membrane proximal region of the beta 2 cytoplasmic domain in the regulation of alpha beta heterodimer formation and ligand binding activity, we generated beta 2 cytoplasmic domain truncation mutants beta 2710* and beta 2702*. beta 2710* retained the membrane proximal sequence KALIHLSD, whereas beta 2702* contained a complete truncation of the beta 2 cytoplasmic domain (Fig. 1A). The beta 2 truncation mutants were transiently coexpressed with wild-type alpha L in 293T cells. Cell surface expression of heterodimeric alpha Lbeta 2 was determined by indirect immunofluorescence staining with mAbs TS1/22 to alpha L, CBR LFA-1/7 to beta 2, and TS2/4 to alpha L in the alpha Lbeta 2 complex (Table I). The level of cell surface heterodimeric alpha Lbeta 2710* was comparable with that of wild-type alpha Lbeta 2. However, the level of alpha Lbeta 2702* was greatly reduced (Table I). Complete truncation of the beta 2 cytoplasmic domain also greatly reduced cell surface expression of the alpha Mbeta 2702* heterodimer (data not shown). Thus, the membrane proximal sequence KALIHLSD plays a role in the formation of cell surface alpha beta heterodimer. Complete truncation of the alpha L cytoplasmic doman in mutant alpha L1090* reduced surface expression as previously described (27). The reduction in expression of alpha L1090* beta 2 was similar to that seen with alpha Lbeta 2702* (Table I).


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Fig. 1.   Schematic diagram of alpha L and beta 2 subunit mutants. A, the sequences of the mutants are shown in the membrane proximal regions. Numbers to the right (+16 and +28) indicate the number of C-terminal residues not shown. B, scheme for regulation of integrin adhesiveness by cytoplasmic domain association/dissociation with the acid and base peptide fusions. The acid and base peptides are shown as (---) and (+ + +), respectively.

                              
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Table I
Flow cytometric measurement of alpha Lbeta 2 expression on the surface of 293T transfectants
Wild-type or truncated beta 2 was transiently coexpressed with wild-type alpha L in 293T cells. Included for comparison is the alpha L cytoplasmic domain truncation mutant alpha L1090* (27) coexpressed with wild-type beta 2. Cell surface expression of heterodimeric alpha Lbeta 2 was determined by flow cytometry using mAb TS1/22 to alpha L, mAb TS2/4 to alpha L in the alpha Lbeta 2 complex, and mAb CBR LFA-1/7 to beta 2. Mean fluorescence intensity shown was subtracted by that of the nonbinding IgG X63. Data are mean ± difference from the mean of two independent experiments.

Complete Truncation of the beta 2 Cytoplasmic Domain Results in Constitutive Ligand Binding by alpha Lbeta 2 and alpha Mbeta 2-- To examine the effect of beta 2 cytoplasmic domain truncations on ligand binding by alpha Lbeta 2, the beta 2 truncation mutants beta 2710* and beta 2702* were stably coexpressed with wild-type alpha L in K562 cells. Clones of wild-type alpha Lbeta 2, alpha Lbeta 2710*, and alpha Lbeta 2702* transfectants that expressed similar levels of surface alpha Lbeta 2, as determined by flow cytometry (Fig. 2A), were selected and tested for their ability to bind to purified ICAM-1. Transfectants that expressed wild-type alpha Lbeta 2 and alpha Lbeta 2710* showed low basal binding to ICAM-1; however, binding was greatly increased by the activating mAb CBR LFA-1/2 to the beta 2 subunit (Fig. 3A). By contrast, alpha Lbeta 2702* showed strong constitutive binding to ICAM-1, and the level of binding was comparable with that of the constitutively active alpha L truncation mutation alpha L1090*. CBR LFA-1/2 did not further enhance ligand binding by alpha Lbeta 2702* and alpha L1090*beta 2, suggesting that these mutants are fully active without activation. All binding was specific, as shown with inhibition by mAb TS1/22 to the alpha L I domain and by comparison to mock transfectants.


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Fig. 2.   Expression of truncation mutants on the surface of K562 cell transfectants. Wild-type alpha L (A) or alpha M (B) was coexpressed with wild-type (WT) beta 2, truncation mutant beta 2710* or beta 2702* in K562 cells, or cells were transfected with vector alone (mock). Cell surface expression of the alpha Lbeta 2 (A) and alpha Mbeta 2 (B) complexes was determined by immunofluorescence flow cytometry. Numbers in parentheses after the transfectant names are clone numbers. mAbs and their specificity are indicated on the left. X63 is a nonbinding myeloma IgG control.


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Fig. 3.   Ligand binding activity of alpha Lbeta 2 and alpha Mbeta 2 mutants. A, binding to ICAM-1 of K562 stable transfectants expressing wild-type alpha Lbeta 2 or alpha Lbeta 2 with truncated beta 2 (beta 2710* and beta 2702*) or truncated alpha L (alpha L1090*). ICAM-1 was immobilized in wells of a 96-well plate, and binding of K562 transfectants was determined in the absence (control) or presence of the blocking mAb TS1/22 or the activating mAb CBR LFA-1/2 (10 µg/ml). B, binding to immobilized iC3b of K562 stable transfectants that express wild-type alpha Mbeta 2 or alpha Mbeta 2 with truncated beta 2. iC3b was immobilized, and the binding of K562 transfectants was determined in the absence (control) or presence of the blocking mAb CBRM1/33 or the activating mAb CBR LFA-1/2 (10 µg/ml). Results are mean ± S.D. of triplicate samples and are representative of three independent experiments. The expression level of cell surface alpha Lbeta 2 and alpha Mbeta 2 is shown in Fig. 2.

The beta 2 cytoplasmic domain truncation mutants beta 2710* and beta 2702* were also stably coexpressed with the wild-type alpha M subunit in K562 cells. Clones of K562 transfectants that expressed similar levels of cell surface alpha Mbeta 2 heterodimer, as determined by flow cytometry (Fig. 2B), were tested for binding to immobilized iC3b. K562 transfectants that expressed wild-type alpha Mbeta 2 or alpha Mbeta 2710* did not bind to iC3b in the absence of the activating mAb CBR LFA-1/2. By contrast, alpha Mbeta 2702* bound strongly to iC3b without activation. Binding was specific, because it was inhibited by mAb CBRM1/33 to the alpha M I domain.

Thus, complete truncation of the beta 2 cytoplasmic domain constitutively activates ligand binding by alpha Lbeta 2 and alpha Mbeta 2, whereas partial truncation that retains the membrane proximal eight residues does not. These results suggest that the membrane proximal region in the beta 2 cytoplasmic domain constrains beta 2 integrins in the inactive state.

Activating beta 2 Truncation Mutations Expose Activation-dependent Epitopes in alpha Lbeta 2 and alpha Mbeta 2-- mAb m24 has been used as a reporter for alpha Lbeta 2 activation (10, 27, 46). Recently, mAb m24 has been mapped to the I-like domain of the beta 2 subunit (47). mAb KIM127 recognizes an epitope in the beta 2 stalk region that becomes exposed upon receptor activation (48). We therefore tested expression of the m24 and KIM127 epitopes by alpha Lbeta 2 containing beta 2 truncation mutations. There was little expression of the m24 epitope by wild-type alpha Lbeta 2 or alpha Lbeta 2710* (Table II). However, the truncation mutation beta 2702* greatly induced the m24 epitope. Basal expression of the KIM127 epitope on wild-type alpha Lbeta 2 was higher than that of the m24 epitope, and there appeared to be a moderate increase in the beta 2710* mutant. However, expression of the KIM127 epitope was greatly increased by the beta 2702* mutation (Table II). Expression of the KIM127 epitope on alpha Lbeta 2702* was nearly maximal; i.e. comparable with constitutively expressed epitopes such as TS2/4.

                              
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Table II
Expression of activation epitopes by alpha Lbeta 2 mutants
Wild-type and mutant alpha Lbeta 2 were stably expressed in K562 cells, and reactivity of transfectants with mAb m24 and KIM127 was determined by immunofluorescence flow cytometry. Mean fluorescence staining of each antibody after subtraction of the mean fluorescence of the control IgG X63 is expressed as the % mean fluorescence with mAb TS2/4. mAb TS2/4 recognizes the alpha L subunit in the alpha Lbeta 2 complex and reacts with wild-type and mutant alpha Lbeta 2 equally well as shown by comparison to many other mAb specific for the alpha L and beta 2 subunits. Data are mean ± difference from the mean of two independent experiments.

mAb CBRM1/5 recognizes an activation epitope in the alpha M I-domain near the metal ion-dependent adhesion site (MIDAS) motif (39, 45). Expression of the CBRM1/5 epitope was greatly enhanced by the activating beta 2 truncation mutation beta 2702*, whereas the beta 2710* mutation did not significantly increase CBRM1/5 binding compared with wild type (Table III). The beta 2702* mutation also greatly increased mAb KIM127 binding to alpha Mbeta 2 (Table III). Thus, constitutively strong ligand binding by alpha Lbeta 2 and alpha Mbeta 2 containing the truncation mutation beta 2702* correlates with exposure of activation epitopes in the extracellular domain.

                              
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Table III
Expression of activation epitopes by alpha Mbeta 2 mutants
Reactivity of mAb CBRM1/5 and KIM127 with K562 stable transfectants that express alpha Mbeta 2 with wild-type or truncated beta 2 was determined by immunofluorescence flow cytometry. Mean fluorescence staining of each antibody was subtracted by the mean fluorescence of the control IgG X63, and is expressed as % mean fluorescence staining of mAb CBRM1/20, which binds to alpha M in the alpha Mbeta 2 complex. mAb CBRM1/20 reacts with wild-type and mutant alpha Mbeta 2 equally well. Data are mean ± difference from the mean of two independent experiments.

Replacing alpha L and beta 2 Cytoplasmic Domains with an alpha -Helical Coiled Coil Constrains alpha Lbeta 2 in the Inactive State-- The above results suggest that the membrane proximal region of the beta 2 subunit cytoplasmic domains play an important role in the regulation of beta 2 integrin function and formation of the alpha beta heterodimer. The membrane proximal GFFKR sequence in the alpha L cytoplasmic domain regulates alpha  and beta  heterodimerization and ligand binding by alpha Lbeta 2 (27). We hypothesized, therefore, that the membrane proximal regions of the alpha  and beta  cytoplasmic domains associate, and such association constrains the integrin in an inactive conformation. To test this hypothesis, we replaced the cytoplasmic domains of alpha L and beta 2 with a heterodimeric coiled coil. Peptides termed "acid" and "base" were fused to alpha L and beta 2. These peptides preferentially form heterodimeric as opposed to homodimeric alpha -helical coiled coils (42). These fusions were termed alpha Lacid and beta 2base, respectively (Fig. 1). As a control, both alpha L and beta 2 cytoplasmic domains were replaced by the basic peptide (alpha Lbase and beta 2base). Dimerization of the two basic peptides is disfavored because of interhelical electrostatic repulsion (42). K562 cell clones were selected that stably expressed alpha Lacidbeta 2base and alpha Lbasebeta 2base at similar levels on the cell surface (Fig. 4).


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Fig. 4.   Expression of alpha Lbeta 2 coiled coil fusion mutants on the surface of K562 stable transfectants. K562 cells were stably co-transfected with wild-type alpha L and beta 2, alpha Lacid and beta 2base, or alpha Lbase and beta 2base. Clones of the stable K562 transfectants were stained with the indicated mAbs (shown on the left) and analyzed by flow cytometry. Clone numbers are indicated in parentheses. X63, nonbinding IgG.

To test whether the acidic and basic peptide cytoplasmic domains indeed formed an alpha -helical coiled coil, we examined reactivity with mAb 2H11, which specifically recognizes the acidic and basic peptide heterodimer, and not monomer or homodimer (40). mAb 2H11 immunoprecipitated the alpha Lacidbeta 2base complex, but not wild-type alpha Lbeta 2 or alpha Lbasebeta 2base (Fig. 5). By contrast, mAb TS2/4 to the alpha L subunit immunoprecipitated all three types of alpha Lbeta 2 heterodimers (Fig. 5). These results demonstrate that in alpha Lacidbeta 2base, the cytoplasmic peptides noncovalently associate to form an alpha -helical coiled coil.


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Fig. 5.   The acid and base peptides of alpha Lacidbeta 2base associate in an alpha -helical coiled coil. Mock-transfected K562 cells or K562 transfectants that stably express wild-type alpha Lbeta 2, alpha Lacidbeta 2base or alpha Lbasebeta 2base were metabolically labeled with [35S]methionine and cysteine. Lysates of the labeled cells were immunoprecipitated with mAb TS2/4 specific for alpha L in the alpha Lbeta 2 complex or mAb 2H11 specific for the alpha -helical coiled coil formed by the acidic and basic peptides. Lysates from equal numbers of labeled cells were subjected to immunoprecipitation, SDS 7.5% polyacrylamide gel electrophoresis, and fluorography. The alpha L subunit with the acidic peptide or basic peptide and the beta  subunit with the basic peptide migrated slightly faster than wild-type alpha L and beta 2, respectively.

To test the hypothesis that cytoplasmic association between alpha L and beta 2 regulates ligand binding, ligand binding activity was compared of alpha Lacidbeta 2base, alpha Lbasebeta 2base, and wild-type alpha Lbeta 2. Clones of K562 stable transfectants that expressed similar levels of surface alpha Lbeta 2 (Fig. 4) were tested for binding to immobilized ICAM-1. Cells that expressed wild-type alpha Lbeta 2 or alpha Lacidbeta 2base did not bind to ICAM-1 without activation (Fig. 6A). The activating mAb CBR LFA-1/2 or Mn2+ greatly increased binding of both wild-type alpha Lbeta 2 and alpha Lacidbeta 2base to ICAM-1. By contrast, cells expressing alpha Lbasebeta 2base strongly bound to ICAM-1 in the absence of activation, and mAb CBR LFA-1/2 or Mn2+ did not further increase binding by alpha Lbasebeta 2base (Fig. 6A).


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Fig. 6.   Binding of alpha Lbeta 2 coiled coil fusion mutants to purified ICAM-1. A, binding of stable K562 transfectants to ICAM-1. Binding of cells to ICAM-1 was performed in the absence (control) or presence of 10 µg/ml of the inhibitory mAb TS1/22 or the activating mAb CBR LFA-1/2 in L15 medium that contains Mg2+ and Ca2+. For the Mn2+ experiment, the binding assay was conducted in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl supplemented with 1 mM MnCl2. Cell surface expression of alpha Lbeta 2 is shown in Fig. 4. The results are mean ± S.D. of triplicate samples and are representative of three independent experiments. B, binding of 293T transient transfectants to ICAM-1. 293T cells were transiently transfected, and binding of the transfectants was determined in the absence (control) or presence of the inhibitory mAb TS1/22 or the activating mAb CBR LFA-1/2 at 10 µg/ml. The percent of cells bound to ICAM-1 was divided by the fraction of transiently transfected cells expressing alpha Lbeta 2. The percent of cells expressing alpha Lbeta 2 was 90, 44, and 18% for wild-type alpha Lbeta 2, alpha Lacidbeta 2base, and alpha Lbasebeta 2base transfectants, respectively (determined by flow cytometry with mAb TS2/4 to alpha L in the alpha Lbeta 2 complex). The data are mean ± S.D. of triplicate samples and are representative of two independent experiments.

The adhesive function of alpha Lacidbeta 2base and alpha Lbasebeta 2base was further examined in 293T transfectants, in which wild-type alpha Lbeta 2 is basally active (Fig. 6B). Wild-type alpha Lbeta 2 and alpha Lbasebeta 2base in 293T transfectants constitutively bound to ICAM-1, and binding was specific as shown by inhibition with mAb TS1/22 to the alpha L I domain. By contrast, alpha Lacidbeta 2base showed little binding. However, mAb CBR LFA-1/2 increased ligand binding by alpha Lacidbeta 2base to a level comparable with wild-type alpha Lbeta 2 and alpha Lbasebeta 2base, indicating that lack of ligand binding by alpha Lacidbeta 2base was not because of loss of function (Fig. 6B).

Constitutive ligand binding by alpha Lbasebeta 2base correlated with the expression of activation epitopes in the extracellular domain (Table II). Binding of activation-dependent mAbs m24 and KIM127 to alpha Lbasebeta 2base in K562 cells was greatly increased compared with wild-type alpha Lbeta 2, whereas the level of m24 and KIM127 binding to alpha Lacidbeta 2base was similar to wild-type alpha Lbeta 2 (Table II).

    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

We have demonstrated that the membrane proximal region of eight residues in the beta 2 cytoplasmic domain is required for efficient expression of integrin alpha Lbeta 2 and alpha Mbeta 2 heterodimers on the cell surface and for maintenance of these integrins in the inactive state. Furthermore, we have provided evidence that membrane proximal cytoplasmic association between the alpha  and beta  subunits constrains an integrin in the inactive state, whereas a lack of association between membrane proximal regions activates an integrin.

It has been shown previously that partial truncations of the beta 2 subunit cytoplasmic domain, or mutation of the three contiguous threonines at amino acid residues 736-738 or the phenylalanine at residue 744, abolishes LFA-1 activation by phorbol ester, suggesting that the membrane distal region is involved in the inducible activation of LFA-1 (26, 49). A more recent study showed that complete truncation of the beta 2 cytoplasmic domain activated binding through alpha Lbeta 2 of K562 transfectants to ICAM-1 (50). However, the previous studies did not localize the region in the beta 2 cytoplasmic domain that maintains LFA-1 in the default inactive state. We have shown that the function of restraining LFA-1 in an inactive state can be localized to a membrane proximal segment with the sequence KALIHLSD. Complete truncation of the beta 2 cytoplasmic domain, but not truncation after the KALIHLSD sequence, constitutively activated ligand binding by alpha Lbeta 2. Furthermore, we generalized this observation to another beta 2 integrin, alpha Mbeta 2. Moreover, we demonstrated that the KALIHLSD sequence is required for efficient formation of cell surface alpha Lbeta 2 and alpha Mbeta 2 heterodimers. The membrane proximal sequence in the cytoplasmic domain is conserved among integrin beta  subunits (50). Deletion of the membrane proximal seven residues in the beta 3 cytoplasmic domain results in a constitutively active alpha IIbbeta 3 integrin (30). Similarly, in integrin alpha  subunits, the conserved membrane proximal GFFKR sequence has been shown to be important for regulating integrin alpha beta heterodimer assembly and adhesive function (27, 29, 31). Thus, the conserved membrane proximal regions of both integrin alpha  and beta  cytoplasmic domains control integrin subunit association and ligand binding activity.

The mechanism by which membrane proximal cytoplasmic domain segments regulate integrin activity and efficient association of the alpha  and beta  subunits has been unclear. Our data strongly support the hypothesis that association between these segments regulates ligand binding activity. We replaced the cytoplasmic domains in alpha Lbeta 2 with peptides that either favor or disfavor association to form an alpha -helical coiled coil (42). Association of the membrane proximal segments was confirmed by reactivity with mAb 2H11 that recognizes the acid and base peptides only when they are associated with one another in a coiled coil heterodimer (40). We tested the integrin coiled coil fusions in two different cellular contexts, 293T cells and K562 cells, in which wild-type alpha Lbeta 2 is active and inactive, respectively. 293T transfectants that expressed alpha Lacidbeta 2base showed little binding to ICAM-1, whereas cells expressing wild-type alpha Lbeta 2 strongly bound to ICAM-1. The activating mAb CBR LFA-1/2 to the beta 2 subunit or Mn2+ could activate ligand binding by alpha Lacidbeta 2base, showing that its extracellular domain is competent for activation. Furthermore, alpha Lbasebeta 2base, in which association of the cytoplasmic basic peptides is disfavored, was constitutively active when expressed in K562 cells in which wild-type alpha Lbeta 2 has little basal activity. These findings provide strong evidence that association of membrane proximal cytoplasmic domains renders an integrin inactive, and that lack of association renders an integrin active (Fig. 1B).

Direct association between the membrane proximal GFFKR sequence of the alpha IIb subunit and the KLLITIHD sequence of the beta 3 subunit has been proposed previously based on mutational studies (31). Mutation of Arg-995 in the alpha IIb GFFKR sequence or Asp-723 in the beta 3 KLLITIHD sequence resulted in 39-70% activation of alpha IIbbeta 3, whereas the complementary mutations alpha IIb Arg-995right-arrowAsp and beta 3 Asp-723right-arrowArg resulted in a significantly lower activation of 12% (31). However, no direct evidence for association between the alpha IIb and beta 3 cytoplasmic domains was presented. Moreover, we cannot generalize this result to beta 2 integrins, because mutation of the corresponding Asp-709 in the beta 2 cytoplasmic domain did not activate ligand binding by alpha Lbeta 2 and alpha Mbeta 2 (3 and 2% of maximal binding, respectively; data not shown). Thus, mechanisms other than a proposed salt bridge between integrin alpha  and beta  cytoplasmic domains may regulate integrin adhesiveness. Indeed, despite extensive evidence that mutations of the membrane proximal segments of the integrin cytoplasmic domains are activating, there has to date been no direct evidence that association between these segments, either direct or mediated by integrin-associated proteins, regulates adhesiveness. We have demonstrated for the first time that close spatial proximity between membrane proximal alpha  and beta  subunit segments maintains integrins in an inactive state and that a lack of association results in activation.

The activation state of an integrin is dependent on the cell type in which it is expressed. For example, beta 2 integrins in K562 cells require activation to bind to ligands, whereas beta 2 integrins are constitutively active in 293T cells, as shown here and previously (25). Presumably, this is due to differential expression of proteins or other factors that modulate integrin function in different cell types. We examined the effect of the alpha Lbeta 2 coiled coil fusions in both types of cellular environments. Interestingly, the effect of the coiled coil mutations was independent of cellular environment. Thus, alpha Lacidbeta 2base was inactive in both K562 and 293T cells, whereas alpha Lbasebeta 2base was active in both cell types. The dominance of the peptides over the cellular environments suggests that the factors that modulate differential integrin activity exert their effect by binding to the cytoplasmic domains of the integrins. Activation of these factors by cytoplasmic signals may regulate binding to integrin cytoplasmic domains and hence the transition between inactive and active wild-type integrins (Fig. 1B).

We have demonstrated that activating cytoplasmic domain mutations induce or enhance expression of activation epitopes in alpha Lbeta 2 and alpha Mbeta 2. For alpha Lbeta 2, complete truncation of the beta 2 cytoplasmic domain or replacement of the alpha L and beta 2 cytoplasmic domains with the basic peptides exposed the m24 epitope and enhanced KIM127 epitope expression. Both epitopes localize to the beta 2 subunit. The m24 epitope localizes to loops in the I-like domain that are predicted to be near its metal ion-dependent adhesion-like site (47). The KIM127 epitope localizes within cysteine-rich repeat 2, to residues 504, 506, and 508 in the C-terminal region (48). For alpha Mbeta 2, complete truncation of the beta 2 cytoplasmic domain greatly increased expression of the CBRM1/5 epitope in the alpha M I domain, as well as the KIM127 epitope in beta 2. CBRM1/5 binds to the alpha M I domain very close to the ligand binding site (39, 45). Thus, the activating cytoplasmic domain mutations cause conformational changes in diverse extracellular domains of alpha Lbeta 2 and alpha Mbeta 2. It has previously been shown that complete truncation of the beta 2 cytoplasmic domain alters the localization of LFA-1 into clusters, and hence increases cell adhesion (50). Our results suggest that conformational change (affinity regulation), as well as receptor clustering (avidity regulation) play a role in the enhanced adhesiveness of alpha Lbeta 2 and alpha Mbeta 2 induced by activating cytoplasmic domain mutations.

We propose the following model for integrin activation (Fig. 1B). The membrane proximal regions of the alpha  and beta  subunit cytoplasmic domains can associate either directly or indirectly. Under physiological conditions, there is an equilibrium between the association and dissociation of the membrane proximal segments of the cytoplasmic domains that is dynamically regulated. Association constrains the integrin in the inactive state, and dissociation results in activation. Inside-out signaling and integrin binding proteins regulate this equilibrium. Separation of the two membrane proximal regions results in activation. Truncation of the alpha  or beta  subunit cytoplasmic domain or deletion of the membrane proximal region disrupts this constraint, and activates the integrin. The inactive and active states of an integrin can be mimicked by replacing the integrin cytoplasmic domains with peptides that favor or disfavor, respectively, noncovalent association into a coiled coil. Thus, association of membrane proximal cytoplasmic segments is sufficient to regulate integrin activation and conformational change.

    ACKNOWLEDGEMENTS

We thank Dr. Hsiu-Ching Chang, Nancy Hogg, and Martyn Robinson for providing mAbs.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants CA31798 and CA31799.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a fellowship from the Cancer Research Institute. Present address: Millennium Pharmaceuticals, 75 Sidney St., Cambridge, MA 02139.

§ To whom correspondence should be addressed: Tel.: 617-278-3200; Fax: 617-278-3232.

Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M100600200

    ABBREVIATIONS

The abbreviations used are: ICAM, intercellular adhesion molecule; mAb, monoclonal antibody; PCR, polymerase chain reaction; FACS, fluorescent-activated cell sorter.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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