Amino Acid Residues in the PSI Domain and Cysteine-rich Repeats of the Integrin beta 2 Subunit That Restrain Activation of the Integrin alpha xbeta 2*

Qun Zang and Timothy A. SpringerDagger

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

Received for publication, July 5, 2000, and in revised form, September 13, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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The leukocyte integrin alpha Xbeta 2 (p150,95) recognizes the iC3b complement fragment and functions as the complement receptor type 4. alpha Xbeta 2 is more resistant to activation than other beta 2 integrins and is inactive in transfected cells. However, when human alpha X is paired with chicken or mouse beta 2, alpha Xbeta 2 is activated for binding to iC3b. Activating substitutions were mapped to individual residues or groups of residues in the N-terminal plexin/semaphorin/integrin (PSI) domain and C-terminal cysteine-rich repeats 2 and 3. These regions are linked by a long range disulfide bond. Substitutions in the PSI domain synergized with substitutions in the cysteine-rich repeats. Substitutions T4P, T22A, Q525S, and V526L gave full activation. Activation of binding to iC3b correlated with exposure of the CBR LFA-1/2 epitope in cysteine-rich repeat 3. The data suggest that the activating substitutions are present in an interface that restrains the human alpha X/human beta 2 integrin in the inactive state. The opening of this interface is linked to structural rearrangements in other domains that activate ligand binding.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The integrin alpha Xbeta 2 (p150,95, CD11c/CD18) is one of four integrins that are restricted in expression to leukocytes and have different alpha  subunits associated with a common integrin beta 2 subunit (1, 2). alpha Xbeta 2 is also known as the complement receptor type 4. Integrin alpha Xbeta 2 is expressed on the surface of macrophages, monocytes, granulocytes, and certain activated and B lymphocyte subpopulations (3-6). Upon activation, alpha Xbeta 2 binds to its ligands, complement component iC3b (7-9) and fibrinogen (5, 10), and mediates leukocyte adherence to endothelium and other cells, possibly by binding to intercellular adhesion molecule 1 (ICAM-1) (4, 11-13).

Comparisons among leukocyte integrins suggest that alpha Xbeta 2 has the highest barrier to activation of ligand binding. On cells that coexpress the integrins alpha Xbeta 2 and Mac-1 (alpha Mbeta 2), stronger cellular activation is required to activate alpha Xbeta 2 than alpha Mbeta 2 for binding to the ligand iC3b (8). When transfected into COS or 293T cells, the integrins LFA-1 (alpha Lbeta 2) and alpha Mbeta 2 are active in binding ligands; however, alpha Xbeta 2 is not (14). Construction of chimeric alpha M and alpha X alpha  subunits showed that many reciprocal exchanges activated ligand binding, suggesting that structural perturbations released restraints that otherwise held alpha Xbeta 2 in an inactive state (14). Interestingly, although alpha Xbeta 2 expressed on COS-7 cells could not bind to the ligand iC3b, an interspecies hybrid, alpha Xbeta 2, comprised of chicken beta 2 and human alpha X subunits was constitutively active in binding iC3b (7). By contrast, human alpha M/chicken beta 2 and human alpha M/human beta 2 integrin heterodimers bound iC3b equally well. Studies with mAb1 map ligand binding to the I domain of the alpha Xbeta 2 alpha X subunit (7). These findings suggest that an intersubunit restraint on alpha X conformation is loosened with the chicken beta 2 subunit so that alpha X can more readily adopt the conformation that binds iC3b. It may be significant in light of this finding and the finding that alpha Xbeta 2 is more difficult to activate than alpha Lbeta 2 or alpha Mbeta 2 that the association between alpha X and beta 2 is more difficult to disrupt with denaturing conditions than the association between alpha L and beta 2 or between alpha M and beta 2 (15).

A key question of current integrin research is the nature of the structural alterations in "inside-out signaling" that enables ligand binding by the extracellular domain in response to signals impinging on the cytoplasmic/transmembrane domains. Electron microscopy reveals an overall integrin structure of a globular head region connected to the cell membrane by two stalk regions (16). The head region binds ligand and contains domains from the N-terminal portions of both the alpha  and beta  subunits. Seven 60-amino acid repeats in the N-terminal half of the alpha  subunit have been predicted to fold into a beta -propeller domain (17). A subset of integrin alpha  subunits, including the alpha X subunit, contains an I domain inserted between beta -sheets 2 and 3 of the beta -propeller domain. The I domain has a structure like small G proteins with a metal ion-dependent adhesion site at the top of the domain where ligand is bound (18, 19). A conformational change at the MIDAS that regulates ligand binding is linked structurally to a large movement of the C-terminal alpha -helix that connects the bottom of the I domain to the beta -propeller domain (19-23). A domain in the beta  subunit has a predicted fold that is like the I domain and a MIDAS-like site (18, 24-26). This beta  subunit I-like domain associates with the side of the alpha  subunit beta -propeller domain at beta -sheets 2 and 3 (27, 28) and is thus near to the alpha  subunit I domain, which links to beta -sheets 2 and 3 at the top of the beta -propeller domain.

The stalk regions provide the crucial link between the signals impinging on the alpha  and beta  subunit transmembrane and cytoplasmic domains and the conformational changes that occur in the ligand-binding head region. In the alpha  subunit, the stalk region appears to consist of the region C-terminal to the predicted beta -propeller domain. The stalk region is predicted to consist of domains with a two-layer beta -sandwich structure (29). Four subregions of the alpha M stalk have been defined with mAb epitopes, three of which react with mAbs whether or not the beta  subunit is coexpressed. In the beta  subunit, the stalk region appears to consist of the cysteine-rich regions that precede and follow the I-like domain, i.e. residues 1-103 and 342-678 in beta 2. These cysteine-rich regions are linked by a long range disulfide bond defined in beta 3 that is predicted to link Cys-3 and Cys-425 in beta 2 (30). The N-terminal cysteine-rich region of residues ~1-50 shares sequence homology with membrane proteins including plexins, semaphorins, and the c-met receptor (31). This region has two predicted alpha -helices and has been termed the "PSI domain" for plexins, semaphorins, and integrins. The segment from residues 425 to 590 has a cysteine content of 20% and is composed of four cysteine-rich repeats. The first repeat is less similar to the others and at its N-terminal end contains the cysteine that disulfide bonds to the PSI domain. Several monoclonal antibodies that activate integrins or report conformational changes have been mapped to the C-terminal region of the beta  subunit that includes the cysteine-rich repeats (28, 32-37) and to the N-terminal cysteine-rich region (33). Many of these mAbs recognize epitopes that become exposed after integrin activation. One of these, mAb KIM127 to the beta 2 subunit, is not dependent on association with the alpha  subunit for reactivity and indeed reacts better with the free beta 2 subunit than with the integrin alpha beta heterodimer (38). Thus, structural changes in the stalk region that include exposing antibody epitopes on the integrin beta  subunit are associated with integrin activation.

Here, we have defined regions of the integrin beta 2 subunit involved in regulating ligand binding by alpha Xbeta 2. Ligand binding is activated when the human alpha X subunit is complexed with the chicken beta 2 subunit (7). We hypothesized that this reflects a release of structural contacts between the human alpha X and human beta 2 subunits that normally restrain alpha Xbeta 2 in a nonligand binding conformation. To map these contacts within the beta 2 subunit, we have utilized chicken/human beta 2 chimeras. We map the key differences and provide direct evidence that residues that restrain ligand binding by beta 2 are present in both the N-terminal cysteine-rich PSI domain and the C-terminal cysteine-rich repeats.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Cell Lines and Monoclonal Antibodies-- 293T cells (human renal epithelial transformed cells) were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS), nonessential amino acids (Life Technologies, Inc.), 2 mM glutamine, and 50 µg/ml gentamicin. The mouse anti-human alpha X mAb CBRp150/2E1 (7) and the anti-human beta 2 mAbs KIM185 (39) and CBR LFA-1/2 (40) have been previously described.

Human/Chicken or Human/Mouse Chimeric beta 2 Constructs-- Human or chicken beta 2 cDNA were inserted in vector AprM8 (41). Chimeras and substitution mutants were generated by polymerase chain reaction overlap extension (42). Briefly, 5' and 3' end primers were designed to include unique restriction sites. Mutations were introduced by a pair of inner complementary primers. After a second round of polymerase chain reaction, the products were digested and ligated with the corresponding predigested plasmids. All constructs were verified by DNA sequencing.

Transfection-- Plasmids for transfection were purified by QIAprep Spin Kit or Maxi Kit (Qiagen, Chatsworth, CA). 293T cells were transiently transfected with human alpha X and wild-type or mutant beta 2 constructs using calcium phosphate (43, 44). Medium was changed after 7-11 h. Cells were harvested for analysis 48 h after transfection.

Flow Cytometry-- Cells were washed twice with L15 medium supplemented with 2.5% fetal bovine serum (L15/FBS). Cells (106) were incubated with 50 µl of primary antibody (20 µg/ml purified mAb, or 1:100 dilution of ascites) on ice for 30 min. Cells were then washed three times with L15/FBS, followed by incubation with 50 µl of a 1:20 dilution of fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Zymed Laboratories Inc., San Francisco, CA) for 30 min on ice. After washing three times with L15/FBS, cells were resuspended in 200 µl of cold phosphate-buffered saline and analyzed on a FACScan (Becton Dickinson, San Jose, CA). Antigen expression is presented as mean fluorescence intensity of cells.

iC3b-coated Erythrocyte Binding Assay-- As described previously (7, 14)), sheep erythrocytes (Colorado Serum Co., Denver, CO) were washed, resuspended to 6 × 108 cells/10 ml in buffer 1 (Hanks' balanced salt solution, 15 mM HEPES, pH 7.3, and 1 mM MgCl2), and sensitized with 80 µl of IgM anti-Forssman mAb M1/87 culture supernatant for 1 h at room temperature (E-IgM). The cells were then washed and resuspended in 1.8 ml of buffer 2 (Hanks' balanced salt solution, 15 mM HEPES, pH 7.3, 1 mM MgCl2, and 1 mM CaCl2), supplemented with 200 µl of C5-deficient human serum (Sigma). After incubation at 37 °C for 1 h, the resulting E-IgM-iC3b were washed twice and resuspended in 6 ml of buffer 2.

To assay the binding of alpha Xbeta 2 to iC3b, 293T cells transfected with recombinant alpha Xbeta 2 were plated on 12-well polylysine-coated plates for at least 4 h prior to the experiment. After washing with buffer 2, the cells were incubated together with 200 µl of E-IgM-iC3b for 30 min at 37 °C. Unbound erythrocytes were removed by washing three times, and rosettes (>10 erythrocytes/293T cell, >100 cells examined) were scored with microscopy.


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Cysteine-rich Regions of the beta 2 Subunit Regulate Integrin alpha xbeta 2 Binding to iC3b-- To locate regions in the integrin beta 2 subunit that restrain activation of alpha Xbeta 2, interspecies human/chicken chimeric beta 2 subunits were made. Chimeras were named according to the species origin of their segments. For example, h103c indicates that residues 1 to 103 are human and residues 104 to the C-terminal end are chicken. Each construct was cotransfected with the human alpha X subunit into 293T cells. Proper expression was confirmed by immunostaining with antibody CBRp150/2E1 to the alpha X subunit. All human/chicken beta 2 chimeras studied here were expressed as well as human beta 2 in alpha Xbeta 2 complexes. The percentage of 293T transfectants expressing alpha Xbeta 2 ranged from 68 to 85% for chimeras and wild type in all experiments described below. Transfectants were assayed for activation of ligand binding by rosetting with erythrocytes sensitized with iC3b (E-IgM-iC3b). The percentage of rosetting cells was normalized to the percentage of alpha Xbeta 2+ cells for each construct. Transfectants expressing hybrid alpha Xbeta 2 (human alpha X/chicken beta 2) but not transfectants expressing human alpha Xbeta 2 formed rosettes with E-IgM-iC3b, confirming previous observations with COS-7 cell transfectants (7). The chimeras mapped activation of ligand binding by chicken beta 2 to two regions, residues 1-71 and residues 421-610 (Fig. 1). The importance of residues 421-610 was shown by activation of iC3b rosetting by chimeras h103c and h421c but not by chimeras h610c, h103c421h, and c71h610c. Residues 1-71 were not sufficient by themselves to activate iC3b binding as shown with chimera c71h; however, they augmented rosetting when present in combination with residues 421-610 (Fig. 1). Thus, with residues 421-610 of chicken origin in chimeras h103c and h421c, about 40% of transfectants rosetted with E-IgM-iC3b. With both residues 1-71 and 421-610 of chicken origin in chimeras c678h and c71h421c, about 80% of transfectants rosetted. This was the same level as with the wild-type chicken beta 2 subunit. Thus, activation was a synergistic effect of the N- and C-terminal cysteine-rich regions of the chicken beta 2 subunit.



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Fig. 1.   Regions 1-71 and 421-610 of the chicken beta 2 subunit activate alpha Xbeta 2 binding to iC3b. The indicated human/chicken beta 2 chimeras were cotransfected with the human alpha X subunit into 293T cells. The percentage of transfected cells rosetting with >10 iC3b-sensitized erythrocytes was determined with microscopy. Rosetting was calculated as the percentage of rosetting cells divided by the percentage of 293T cells expressing alpha Xbeta 2 as determined by immunofluorescence flow cytometry. Results are averages of three experiments. Bars indicate standard deviation. Hu, human; Ch, chicken

Activation of alpha xbeta 2 by Regions in the Mouse beta 2 Subunit-- We found that alpha Xbeta 2 heterodimers containing human alpha X and mouse beta 2 subunits were activated for binding to iC3b almost as well as those containing human alpha X and chicken beta 2 subunits (Fig. 2A). Human/mouse beta 2 chimeras showed that the region containing residues 344-612 was activating (Fig. 2A). The h98m chimera was less activating than mouse beta 2, suggesting that the N-terminal cysteine-rich region contributed to activation. Furthermore, the m122h, m163h, m254h, m302h, and m344h chimeras showed that the N-terminal cysteine-rich region was not sufficient for activation, similar to the results with the chicken beta 2 subunit.



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Fig. 2.   Regions 1-98 and 470-538 of the mouse beta 2 subunit activate complement receptor type 4 function of alpha xbeta 2. A, overall mapping. B, fine mapping in the C-terminal region. Chimeras of the human and mouse beta 2 subunits were cotransfected with human alpha X and tested for rosetting with iC3b-sensitized erythrocytes as described in Fig. 1. Hu, human; Mo, mouse.

The activating region in the C-terminal cysteine-rich region in the mouse beta 2 subunit was defined with a further series of chimeras (Fig. 2B). These narrowed activation by the C-terminal cysteine-rich region to residues 470-538 since chimera m122h470m was activating, whereas m122h538m was not (Fig. 2B). Furthermore, residues in two different segments, 470-502 and 502-538, were activating because chimera m122h502m was partially activating, whereas m122h470m was fully activating, and m122h538m was not activating.

Chicken Residues in Cysteine-rich Repeats 2 and 3 Activate alpha xbeta 2-- Mapping of the C-terminal cysteine-rich repeat region of chicken beta 2 was refined with five further chicken/human chimeric beta 2 constructs. Each construct contained N-terminal residues 1-71 and various lengths of the C-terminal cysteine-rich repeats from chicken beta 2 (Fig. 3A). Rosetting of chimeras c71h446c, c71h470c, and c71h498c with E-IgM-iC3b was similar to that of wild-type chicken beta 2. However, chimeras c71h527c and c71h562c did not bind to iC3b. Therefore, residues within region 498-527 can activate binding to the ligand iC3b.



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Fig. 3.   Specific chicken residues in cysteine-rich repeats 2 and 3 that activate binding of alpha xbeta 2 to iC3b. A, mapping within the C-terminal region. B, mapping of individual or groups of C-terminal residues that activate alpha Xbeta 2 alone or in combination with the N-terminal region. Chimeric beta 2 subunits were cotransfected with human alpha X and tested for rosetting with iC3b-sensitized erythrocytes as described in Fig. 1. Hu, human; Ch, chicken.

Within the activating region defined in chicken beta 2 of 498-527, 11 residues differ between human and chicken. Groups of one to three chicken amino acid residues in this region were introduced into the human beta 2 subunit and their effect on binding to iC3b was examined (Fig. 3B). In combination with chicken residues 1-71, four groups of amino acid substitutions were activating: Q510T/Y511F/E513D in repeat 2 and T516N/I517M, R521F/Y522H, and Q525S/V526L in repeat 3. Chimera c71h/Q525S/V526L was as active as chicken beta 2. The four activating groups of residues were also tested in the absence of any other chicken residues. In this situation, only the mutation Q525S/V526L was activating, and its activity was reduced compared with c71h/Q525S/V526L (Fig. 3B).

Residues 4 and 22 in the N-terminal Cysteine-rich Region of Chicken beta 2 Activate alpha xbeta 2 in Synergy with the C-terminal Cysteine-rich Region-- Mapping of the N-terminal cysteine-rich region was refined with three chicken/human chimeras that included different portions of the N-terminal region in combination with the synergistic C-terminal segment (Fig. 4A). All three beta 2 chimeras, c71h421c, c50h421c, and c29h421c, activated binding to E-IgM-iC3b to the same extent as chicken beta 2. Thus, residues within the first 29 amino acids of the beta 2 subunit are sufficient to synergistically activate alpha Xbeta 2.



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Fig. 4.   Chicken residues 4 and 22 of the beta 2 subunit activate iC3b binding in synergy with residues 525 and 526. A, fine mapping of the N-terminal region in synergy with the C-terminal region. B, mapping of individual N-terminal residues in synergy with residues 525 and 526. Details are as described in Fig. 1. Hu, human; Ch, chicken.

In region 1-29 of the beta 2 subunit, 11 residues differ between the human and chicken. Groups of these residues were substituted with chicken sequence in combination with the mutation Q525S/V526L in the C-terminal cysteine-rich region in each construct (Fig. 4B). Most of the mutants rosetted E-IgM-iC3b no better than the parent Q525S/V526L mutant. However, mutants Q1A/T4P/Q525S/V526L and T4P/Q525S/V526L but not Q1A/Q525S/V526L were more active than Q525S/V526L, implicating the substitution T4P in activation. Similarly, mutants T22A/Q25K/Q525S/V526L and T22A/Q525S/V526L but not Q25K/Q525S/V526L were more active than Q525S/V526L, implicating T22A. Moreover, the combination of mutations T4P and T22A was even more active, and the mutant T4P/T22A/Q525S/V526L was as active as chicken beta 2 (Fig. 4B). Therefore, four chicken residues, two each in the N-terminal and C-terminal cysteine-rich regions of beta 2, are sufficient to maximally activate iC3b rosetting by alpha Xbeta 2.

Activating Mutations Expose the CBR LFA-1/2 Epitope in the C-terminal Cysteine-rich Region of beta 2-- Several mAbs that activate beta 2 integrins map to the C-terminal cysteine-rich region of the beta 2 subunit (28, 37). The mouse/human substitutions recognized by these mAbs map very near to the substitutions Q525S/V526L that activate alpha Xbeta 2. Specifically, mAb KIM185 recognizes residues 581-604 and mAb CBR LFA-1/2 recognizes residues 534 and 536.2 Recognition by mAb CBR LFA-1/2 correlates with the activation status of beta 2 integrins; alpha Lbeta 2 and alpha Mbeta 2, which are active in 293T cell transfectants, are recognized well by CBR LFA-1/2, whereas alpha Xbeta 2, which is inactive in 293T cells, is recognized poorly2 (Fig. 5). We examined the effect of activating mutations on expression of CBR LFA-1/2 and KIM185 epitopes (Fig. 5). The KIM185 epitope was expressed equally well by wild-type and mutant alpha Xbeta 2. By contrast, activating mutations induced exposure of the CBR LFA-1/2 epitope (Fig. 5). The Q525S/V526L mutation partially exposed the CBR LFA-1/2 epitope, whereas the T4P/T22A/Q525S/V526L mutation maximally exposed the epitope, i.e. to the same level as seen with KIM185 mAb. Exposure of the CBR LFA-1/2 epitope correlated with activation of binding to iC3b (Fig. 5). Therefore, the mutations cause structural rearrangements in the stalk region that lead to exposure of the CBR LFA-1/2 epitope and are linked to activation of ligand binding. Furthermore, binding of the CBR LFA-1/2 and KIM185 mAbs demonstrates that the mutations do not disrupt the structure of the cysteine-rich repeats.



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Fig. 5.   Activation of iC3b binding by individual amino acid substitutions in the PSI domain and cysteine-rich repeat 3 correlates with exposure of the CBR LFA-1/2 epitope. Mutant or wild-type beta 2 subunits were cotransfected with human alpha X into 293T cells. The transfectants were immunostained with mAbs KIM185 or CBR LFA-1/2 followed by immunofluorescence flow cytometry. Epitope expression is normalized to the percentage of cells binding mAb CBR p150/2E1 to the alpha X subunit. Binding to iC3b was quantified as described in Fig. 1. Hu, human.



    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Among the beta 2 integrins, alpha Xbeta 2 is the most resistant to activation and to dissociation of its alpha  and beta  subunits. Here, we have identified specific amino acid residues that restrain alpha Xbeta 2 in a conformation in which it does not bind its ligand, iC3b. We extended previous observations with the chicken beta 2 subunit (7) by showing that pairing of human alpha X with beta 2 from another species, the mouse, also activates binding to iC3b. Interspecies beta 2 subunit chimeras associated with human alpha X subunits demonstrated that the C-terminal cysteine-rich repeats from mouse or chicken were sufficient for partial activation and that the N-terminal cysteine-rich PSI domain was insufficient for activation but synergized with the C-terminal cysteine-rich repeats.

Activating substitutions in the N-terminal region were localized within the PSI domain (Fig. 6). Human/chicken substitutions T4P and T22A each synergized with the C-terminal region and, when present together, gave augmented synergy. PSI domains in integrins contain six cysteines that form intradomain disulfide bonds and one cysteine that forms a long range interdomain disulfide (30, 31). Each of the activating substitutions neighbors a cysteine residue (Fig. 6). The substitution T4P neighbors Cys-3, which forms the long range disulfide bond to Cys-425, which is at the beginning of the C-terminal cysteine-rich repeats (Fig. 6). Thus, the two regions in which activating substitutions are found, the PSI domain and cysteine-rich repeats, are linked by a disulfide bond and must be neighboring domains in the three-dimensional structure of the integrin beta  subunit.



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Fig. 6.   Sequence differences of the human beta 2 PSI domain and cysteine-rich repeats with the mouse and chicken and localization of activating mutations. Only residues that differ in the mouse or chicken sequences are shown. Underlined residues: tested, no effect. Black inverse residues: activating. Gray inverse residues: activating in synergy with other residues. Underlining or inverse font is extended to include the group of residues that was tested. The long range disulfide bond between Cys-3 and Cys-425 is shown. Dots above the sequence mark residue positions that are multiples of ten. hu, human; mo, mouse; ch, chicken.

Activating substitutions within the C-terminal region localized to cysteine-rich repeats 2 and 3 (Fig. 6). One segment containing activating mouse substitutions localized wholly within repeat 2, whereas another included portions of repeats 2 and 3 (Fig. 6). Fine mapping of three groups of chicken substitutions that activated alpha Xbeta 2 in synergy with chicken residues in the PSI domain showed that one group mapped to repeat 2 and two groups mapped to repeat 3 (Fig. 6). One pair of substitutions that was sufficient for activation of alpha Xbeta 2, Q525S/V526L, mapped to repeat 3. We cannot exclude the presence of activating substitutions in repeat 1 because all chimeras in which repeat 1 was mouse or chicken also contained repeats 2 and 3 from mouse or chicken, which were activating by themselves. However, the segments following the PSI domain and preceding repeat 1 were not activating. Furthermore, repeat 4 and more C-terminal segments were not activating. The species-specific differences in repeat 4 are greater than in repeats 2 and 3 (Table I); therefore, an insufficiency of species-specific differences cannot explain the lack of activation by repeat 4. 


                              
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Table I
C-terminal cysteine-rich repeats: variation between species and activation

What is the mechanism of integrin activation by species-specific substitutions in the PSI domain and cysteine-rich repeats 2 and 3? Many other observations suggest that integrins are restrained in their resting state in a conformation that does not bind ligand and that a wide variety of perturbations can activate ligand binding. Our results suggest that the PSI domain and cysteine-rich repeats 2 and 3 have an important function in restraining integrins in their resting, inactive state. These restraints are overcome when the alpha  and beta  subunits are from different species; therefore, it appears that there are direct interactions between these beta  subunit domains and the alpha  subunit that constrain integrins in the inactive configuration. The substitutions are unlikely to disrupt the overall conformation of these domains because they are naturally occurring variations between species. Furthermore, we demonstrated that mAb CBR LFA-1/2, which binds to species-specific residues in repeat 3,2 binds well when the activating mutations Q525S and V526L are present in repeat 3. Therefore, it appears that the activating mutations we have defined are within or near an interface between the beta 2 and alpha X subunits. The findings suggest that in resting integrins, there are contacts of the PSI domain and cysteine-rich repeats 2 and 3 with the alpha  subunit and that these contacts help restrain ligand binding. It appears that certain species-specific substitutions disrupt this interaction and, thereby, lower the activation energy required for activation of ligand binding. Binding of iC3b by alpha Xbeta 2 maps to the alpha X I domain (7). Conformational shifts around the MIDAS in I domains regulate ligand binding and are linked to a large movement of the C-terminal alpha -helix of the I domain that connects to other integrin subunits (19-23). Therefore, it appears that an alteration in contacts in the stalk region between the alpha  subunit and the PSI domain and the cysteine-rich repeats in the beta  subunit are linked to conformational rearrangements in the ligand-binding domains in the headpiece of integrins.

The loss of the restraints that keep alpha Xbeta 2 in an inactive state appears to reflect an opening up of the alpha beta interface in the stalk region based on exposure of the epitope for the mAb CBR LFA-1/2. This mAb can activate integrins alpha Lbeta 2 and alpha Mbeta 2 (40). It showed little reactivity with wild-type alpha Xbeta 2; however, introduction of activating amino acid substitutions Q525S/V526L in cysteine-rich repeat 3 exposed the CBR LFA-1/2 epitope, and addition of the T4P/T22A substitutions fully exposed the epitope. Exposure correlated with iC3b binding. The CBR LFA-1/2 mAb maps to residues 534 and 5362, and nearby residues 525 and 526, to which activating mutations map in repeat 3. It is unlikely that there is a significant conformational change in this repeat because its structure is constrained by four disulfide bonds. Therefore, we envision a movement apart or change in orientation of the alpha  and beta  subunits that exposes the CBR LFA-1/2 epitope in repeat 3.

Other studies also imply a structural restraint on integrin activation that is localized in the cysteine-rich regions of the beta  subunit. Activation of the integrin LFA-1 (alpha Lbeta 2) expressed on COS cells was induced if the C-terminal cysteine-rich repeat region of the beta 2 subunit was replaced by that of beta 1 (45). A point mutation that introduces a N-glycosylation site into the beginning of cysteine-rich repeat 4 of the beta 3 subunit activated integrins alpha IIbbeta 3 and alpha vbeta 3 (46). Furthermore, disruption of the long range disulfide bond between the PSI domain and the cysteine-rich repeats resulted in increased ligand binding affinity of alpha IIbbeta 3 (47). Moreover, treatment with reducing agents, such as dithiothreitol, induced the active conformation of beta 1 integrin (33) and increased platelet aggregation through the alpha IIbbeta 3 integrin (48). Recently, an anti-beta 1 antibody with an activation-dependent epitope has been mapped to the N-terminal cysteine-rich region, suggesting a role of this region as a regulatory site for integrin activation (33). In addition, several monoclonal antibodies against the C-terminal cysteine-rich regions of beta 1 (32, 34), beta 2 (37), and beta 3 (49) integrins have been described as activating mAbs with respect to their ability to promote ligand binding. A plausible explanation is that these mAbs selectively bind to the open conformation of the stalk region and thus stabilize integrins in this conformation and induce linked rearrangements in the ligand-binding domains. Indeed, activating mAbs to both the beta 1 and beta 2 cysteine-rich regions have been found to bind better to isolated beta  subunits than alpha beta complexes, implying that they favor an open conformation (26, 37, 38, 50).

In contrast to domains in the globular headpiece of integrins, the stalk regions do not appear to directly bind ligand but instead appear to regulate ligand binding and to relay activation signals impinging on the cytoplasmic and transmembrane domains of the integrin alpha  and beta  subunits. We have identified specific amino acid residues in the PSI domain and cysteine-rich repeats 2 and 3 of the beta  subunit that form part of the interface between the alpha  and beta  subunits in the stalk region that restrains conformational movements in the ligand-binding headpiece. It would be very interesting to learn which regions of the alpha  subunit participate in this interface and the molecular details of how structural alterations are communicated from one domain to another in integrins.


    ACKNOWLEDGEMENT

We thank Mark Ryan for assistance with fluorescence-activated cell sorter analysis.


    FOOTNOTES

* This project was supported by National Institutes of Health Grant CA 31799.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 To whom correspondence should be addressed: Center for Blood Research, Dept. of Pathology, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Tel.: 617-278-3200; Fax: 617-278-3030; E-mail: springer@sprsgi.med.harvard.edu.

Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M005868200

2 C. Lu, M. Ferzly, J. Takagi, and T. A. Springer, manuscript in preparation.


    ABBREVIATIONS

The abbreviations used are: mAb, monoclonal antibody; MIDAS, metal ion-dependent adhesion site; FBS, fetal bovine serum; c, chicken; h, human; m, mouse.


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ABSTRACT
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MATERIALS AND METHODS
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DISCUSSION
REFERENCES


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