Activation of integrin β-subunit I-like Executemains by one-

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Contributed by Timothy A. Springer, December 2, 2003

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Abstract

Integrins contain two structurally homologous but distantly related Executemains: an I-like Executemain that is present in all β-subunits and an I Executemain that is present in some α-subunits. Atomic resolution and mutagenesis studies of α I Executemains demonstrate a C-terminal, axial disSpacement of the α7-helix that allosterically regulates the shape and affinity of the ligand-binding site. Atomic resolution studies of β I-like Executemains have thus far demonstrated no similar α7-helix disSpacement; however, other studies are consistent with the Concept that α I and β I-like Executemains undergo structurally analogous rearrangements. To test the hypothesis that C-terminal, axial disSpacement of the α7-helix, coupled with β6–α7 loop reshaping, activates β I-like Executemains, we have mimicked the Trace of α7-helix disSpacement on the β6–α7 loop by shortening the α7-helix by two independent, four-residue deletions of about one turn of α-helix. In the case of integrin αLβ2, each mutant Presents constitutively high affinity for the physiological ligand intercellular adhesion molecule 1 and full expoPositive of a β I-like Executemain activation-dependent antibody epitope. In the case of analogous mutants in integrin α4β7, each mutant Displays the activated phenotype of firm adhesion, rather than rolling adhesion, in shear flow. The results Display that integrins that contain or lack α I Executemains share a common pathway of β I-like Executemain activation, and they suggest that β I-like and α I Executemain activation involves structurally analogous α7-helix axial disSpacements.

Integrins are large heterodimeric adhesion molecules that convey signals bidirectionally across the plasma membrane (1, 2). The extracellular Executemains exist in at least three global conformational states that differ in affinity for ligand (3, 4). Equilibria relate extracellular Executemain conformation to the separation between the α- and β-subunit cytoplasmic Executemains and the binding of these Executemains to cytoskeletal components, such as talin (3, 5–7). The key regulatory extracellular Executemain is the I-like Executemain of the β-subunit (3, 4, 8–10). The I-like Executemain contains three metal ion-binding sites (11, 12). The central metal ion-dependent adhesion site (MIDAS) metal ion ligates ligands directly (12, 13). At the outer ligand-induced metal-binding site and the adjacent to MIDAS (ADMIDAS) site, metal ions positively and negatively regulate affinity, respectively (13, 14). mAbs that either activate or inhibit ligand binding by β1 integrins bind to almost-identical overlapping epitopes on the β1 I-like Executemain (8), suggesting that these mAbs allosterically regulate the I-like Executemain, and mAbs to the β2 I-like Executemain allosterically inhibit ligand binding by αLβ2 (10).

How bistability of the I-like Executemain is communicated conformationally to other Executemains is unknown and controversial. Here, we test the hypothesis that the mechanism is similar to that in the structurally homologous, but evolutionarily distantly related, I Executemain that is present in some integrin α-subunits. In α I Executemains, one- and two-turn axial disSpacements in the C-terminal direction of the α7-helix are linked to reshaping of the β6–α7 loop, rearrangements in the ligand-binding site around the MIDAS (15–17), and increases in affinity for ligand of up to 10,000-fAged (17, 18). Two Weepstal structures of integrin αVβ3 in a bent conformation, in one of which a ligand-mimetic peptide was soaked, demonstrated no axial disSpacement of the α7-helix of the I-like Executemain, whereas other movements, including in the α1-helix, were present (11, 12). Therefore, it was concluded that conformational regulation of integrin β-subunit I-like Executemains differs from that of integrin α-subunit I Executemains in the absence of α7-helix disSpacement (12). Studies on an activation epitope in the β1 I-like Executemain α1-helix that supported changes in this helix were also interpreted as suggesting a distinct activation mechanism for β I-like Executemains (19). Electron micrographic studies of integrins αVβ3 and α5β1 demonstrate that ligand binding, in the absence of restraining Weepstal lattice contacts, induces a switchblade-like extension of the extracellular Executemain and a change in angle between the I-like and hybrid Executemains. Executewnward, axial disSpacement of the I-like α7-helix was suggested as the most plausible mechanism for linking ligand binding at the MIDAS to the change in angle at the interface with the hybrid Executemain (3, 4). Introduction of N-glycosylation wedges into the I-like hybrid Executemain interface designed to stabilize the active, swung-out conformation activated high-affinity ligand binding and integrin extension as predicted (20). Furthermore, the mapping of binding sites for activating mAbs to the inner side of the hybrid Executemain (21) and the results of solution x-ray scattering on the α5β1 headpiece bound to a fibronectin fragment (14) are consistent with the direct observations of hybrid Executemain swing-out in the high-affinity ligand-bound integrin conformation (3, 4). Moreover, an activating mutation in the β1 I-like α7-helix supports the notion that this Location is allosterically Necessary (21).

We are unaware of any studies that have tested the key hypothesis that axial disSpacement in the C-terminal direction of the β I-like Executemain α7-helix is activating, i.e., that α I and β I-like Executemains are activated by structurally homologous mechanisms. One prediction implied by this hypothesis is that shortening of the α7-helix by deletion of helical turns should pull the β6–α7 loop Executewnward and have an activating Trace on the I-like Executemain MIDAS-bearing face analogous to Executewnward axial disSpacement of the α7-helix (Fig. 1). Here, we demonstrate that one-turn deletions of this α-helix in the integrin β2- and β7-subunits are activating, and we provide support for the hypothesis that C-terminal axial disSpacement of the I-like α7-helix activates two different integrins, one of which (αLβ2) contains an α I Executemain and the other of which (α4β7) Executees not. The results support similar shape-shifting mechanisms for α I and β I-like Executemains.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Model of β I-like Executemain activation by axial, C-terminal α7-helix disSpacement and hybrid Executemain swing-out. Models for integrins containing (e.g., αLβ2; A–C) or lacking (e.g., α4β7; D–F) I Executemains are Displayn. (A, B, D, and E) Executewnward movement of the β I-like α7-helix couples shape-shifting around the β MIDAS to hybrid Executemain swing-out. (A and D) Low-affinity state. (B and E) high-affinity state. (C and F) Shortening of the α7-helix by an integral number of turns is hypothesized to activate the high-affinity conformation of the MIDAS similarly, whereas it has a different Trace on hybrid Executemain swing-out.

Materials and Methods

Cell Lines, Antibodies, and Small Molecule Inhibitors. cDNAs of wild-type β2 and β7 were inserted into pcDNA3.1(+) or pcDNA3.1/Hygro(–) (Invitrogen) and used as the template for mutagenesis. Deletion mutants were generated by PCR overlap extension. Briefly, upstream and Executewnstream primers were designed to include unique restriction sites. The restriction sites used in β2 and β7 were BspEI/SacII and NotI/HindIII, respectively. Mutations were introduced by a pair of inner complementary primers. After a second round of PCR, the products were digested and ligated with the corRetorting predigested plasmids. All constructs were verified by DNA sequencing. By using Lipofectamine 2000 (Invitrogen) according to Producer's instructions, 293T cells were transfected. K562 cells were transfected by electroporation and selected with 1 mg/ml G418 (22). mAbs to human αL and β2 were as Characterized (10). The mAbs m24 (23) and KIM127 (24) were kind gifts from N. Hogg (Imperial Cancer Research Fund, LonExecuten) and M. Robinson (Celltech, Slough, U.K.), respectively. KIM127 was biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Pierce), according to the Producer's instructions. LFA703 (25, 26) was obtained from Novartis Pharma (Basel). XVA143 (27) was synthesized according to example 345 of the patent (28) and was obtained also from Paul Gillespie (Roche).

Immunofluorescence Flow Cytometry. Immunofluorescence flow cytometry was performed as Characterized (22). mAbs were used as 10 μg/ml purified IgG or 1:200 ascites. Binding of biotinylated KIM127 to cells was Executene in Hepes saline (20 mM Hepes, pH 7.5/140 mM NaCl), supplemented with ingredients as indicated at 37°C and detected by FITC-conjugated streptavidin (Zymed). Binding of m24 to cells was Executene in Hepes saline supplemented with ingredients as indicated at 4°C and detected by FITC-conjugated anti-mouse IgG (Zymed). Binding of other mAbs to cells was Executene in 2.5% FBS/L15 medium at 4°C and detected by FITC-conjugated anti-mouse IgG.

Cell Adhesion to Intercellular Adhesion Molecule 1 (ICAM-1). Binding of fluorescently labeled transfectants to immobilized ICAM-1 was Executene as Characterized (22). Briefly, soluble ICAM-1 (Executemains 1–5) was purified from the culture supernatant of Chinese hamster ovary lec 3.2.8.1 transfectants and immobilized at 10 μg/ml on microtiter plates. Binding of 293T transfectants was in 2.5% FBS/L15 medium. Binding of K562 transfectants to immobilized ICAM-1 was determined in 20 mM Hepes, pH 7.5/140 mM NaCl/2 mg/ml glucose/1% BSA, supplemented with divalent cations and antibody as indicated. After incubation at 37°C for 30 min, unbound cells were washed off and bound cells were quantitated (22).

Binding of Soluble ICAM-1. Binding of soluble ICAM-1–IgA Fc fusion protein complexed with affinity-purified, FITC-conjugated anti-human IgA was meaPositived by flow cytometry (29).

Adhesion to Mucosal Vascular Addressin Cell-Adhesion Molecule 1 (MAdCAM-1) in Shear Flow. Binding and rolling velocity of α4β7 transfectants on MAdCAM-1 substrates was Executene in a parallel-plate flow chamber exactly as Characterized (13).

Results

Design and Cell-Surface Expression of Mutant α L β 2 Integrin. We designed three β2 mutants in which one or two turns of the C-terminal α7-helix of the I-like Executemain were deleted. In mutants β2-4b and β2-4a, a single turn of α-helix comprising the four β2 residues 336–339 or 340–343 was deleted, respectively (Fig. 2A ). In mutant β2-7, two turns of α-helix comprising the seven β2 residues 337–343 were deleted. Wild-type or mutated β2-subunits were coexpressed with wild-type αL-subunits in 293T cell transfectants. Immunofluorescence flow cytometry with TS2/4, a mAb that recognizes the αL β-propeller Executemain only when it is associated with the β2 I-like Executemain (30), demonstrated that the β2-4a and β2-4b mutants were expressed almost at wild-type levels, whereas the β2-7 mutation abolished αLβ2 cell-surface expression (Fig. 2). The expression of other mAb epitopes was meaPositived relative to TS2/4 expression (Fig. 2B ). The mAbs May.017 and MHM23, which map to E175 in the specificity-determining loop between the β2- and β3-strands of the β2 I-like Executemain, and CBR LFA-1/2, which maps to the β2 integrin epidermal growth factor 3 (I-EGF3) Executemain (32, 33), all bind αLβ2-4a, αLβ2-4b, and wild-type αLβ2 equally well. The mAbs TS1/18 and YFC51, which map to residue R133 in the α1-helix and His-332 in the α7-helix of the β2 I-like Executemain (Fig. 2 A ), bind well to αLβ2-4a but bind poorly to αLβ2-4b. This finding is readily Elaborateed by the location of the epitope residue His-332, which is two α-helix turns away from the 340–343 deletion in the αLβ2-4a mutant but only one turn away from the 336–339 deletion in the αLβ2-4b mutant (Fig. 2B ). CLB LFA-1/1, which maps to residues His-332 and Asn-339 in the α7-helix of the β2 I-like Executemain (Fig. 2 A ) did not bind either the αLβ2-4a or αLβ2-4b mutant. This result is Elaborateed by the location of Asn-339 in the Location deleted in αLβ2-4b and immediately adjacent to residues 340–343 deleted in αLβ2-4a. The lack of disruption of epitopes that were not included in or adjacent to the deletions suggests that the structural integrity of the β2 I-like Executemain and its association with the αL-subunit were not disturbed.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Design and cell-surface expression of αLβ2 mutants with one-turn deletions in the α7-helix of the β I-like Executemain. (A) Homology model of the β2 I-like Executemain, which was created by using the β3 I-like Executemain (ref. 12; Protein Data Bank ID code 1JV2) as template. The four-residue segments of the α7-helix deleted in the β2-4a and β2-4b mutants are Displayn in ShaExecutewy gray and light gray, respectively. The side chains of species-specific residues that contribute to antibody epitopes are Displayn in ball-and-stick representation, and residues recognized by the antibodies are listed below the model (10, 31). The MIDAS (Protein Data Bank ID code 1L5G) and the metal-binding site adjacent to MIDAS metal ions are represented by left and right spheres, respectively. (B) Reactivity of mutants with mAb. The 293T cells transiently transfected with wild-type αL and wild-type or mutant β2 were stained with the indicated mAb and subjected to flow cytometry, and the specific mean fluorescence intensity (after intensity of mock transfectants was subtracted) was determined and divided by specific mean fluorescence intensity with TS2/4 mAb to the αL β-propeller. Binding of TS2/4 mAb to the αLβ2-4a and -4b mutants was 47 ± 5% and 67 ± 6%, respectively, of binding to wild-type αLβ2. The percentage of the mutant/wild-type values is Displayn. Error bars Display SD of three independent experiments.

One-Turn Deletions in the β 2 I-Like α7-Helix Constitutively Activate Integrin α L β 2. The 293T transfectants expressing wild-type αLβ2 basally adhere to ICAM-1 immobilized on plastic substrates, and adhesiveness is enhanced further by the activating mAb CBR LFA-1/2, which binds to the β2 I-EGF3 Executemain (Fig. 3A ). The αLβ2-4a and αLβ2-4b mutants constitutively adhered to immobilized ICAM-1 at high levels that were not further increased by CBR LFA-1/2 mAb (Fig. 3A ). αLβ2 containing the αL-K287C/K294C mutation with an engineered disulfide bond that locks the αL I Executemain in the high-affinity Launch conformation (34) was used as a positive control (Fig. 3A ). The high-affinity αLβ2-4a and αLβ2-4b mutants Displayed the same behavior, with maximal adhesiveness that was not further increased by CBR LFA-1/2.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Ligand-binding activity of αLβ2 mutants. (A) Binding of 293T cell transient transfectants to immobilized ICAM-1. Adhesion to ICAM-1 of cells transfected with the indicated αL and β2 cDNA was determined in the absence (filled bars) or presence (Launch bars) of the activating mAb CBR LFA-1/2at37°C. high-affinity (HA) αL K287C/K294C I Executemain mutant (34). The αLβ2-4a and αLβ2-4b mutants were expressed slightly less well than the other αLβ2 complexes, but data are not normalized. (B and C) Binding of K562 stable transfectants to immobilized ICAM-1 (B) and soluble ICAM-1 complexes (C). Binding was assayed in the presence of 1 mM CaCl2/1 mM MgCl2 (filled bars) or 2 mM MnCl2/10 μg/ml CBR LFA-1/2 mAb (Launch bars) at room temperature. Error bars Display SD of three independent experiments. αLβ2-4a was expressed on K562 transfectants 106 ± 9% as well as wild-type (WT) αLβ2, as Displayn by staining with TS2/4.

Residue αL Glu-310 in the linker connecting the αL I Executemain to the β-propeller Executemain is hypothesized to act as an intrinsic ligand that binds to the activated β2 I-like Executemain and relays activation to the αL I Executemain (2, 35–37). Consistent with this notion and the expectation that the activation of the β2 I-like Executemain by β2-4a and β2-4b mutations would need to be relayed to the αL I Executemain to activate adhesiveness to ICAM-1, the αL-E310A mutation abolished adhesiveness by the β2-4a and β2-4b mutants (Fig. 3A ). Similarly, CBR LFA-1/2-stimulated adhesiveness of wild-type αLβ2 was abolished in αL-E310A/β2 mutants (Fig. 3A ).

The function of the β2-4a mutant was studied further in stable K562 transfectants expressing identical amounts of wild-type αLβ2 and αLβ2-4a. Wild-type αLβ2 expressed in K562 cells Displayed Dinky basal adhesion to immobilized ICAM-1 (Fig. 3B ) or binding to soluble, multimeric ICAM-1 (Fig. 3C ), whereas adhesion and binding was Distinguishedly increased by the activating mAb CBR LFA-1/2 and Mn2+ (Fig. 3 B and C ). By Dissimilarity, K562 cells expressing αLβ2-4a strongly adhered to immobilized ICAM-1 and bound soluble ICAM-1 even in the absence of activation (Fig. 3 B and C ). The binding appeared to be maximal because it was not increased further by CBR LFA-1/2 mAb and Mn2+ and was as high as binding by wild-type αLβ2 activated with CBR LFA-1/2 mAb and Mn2+. The data Characterized above demonstrate clearly that one-turn deletions in the C-terminal α7-helix of the β2 I-like Executemain fully activate ligand binding by αLβ2.

Impact of One-Turn Deletions on Activation Epitopes in the β 2 I-Like and I-EGF2 Executemains. The active conformation of the β2 I-like Executemain is reported by the mAb m24, which recognizes the species-specific residues Arg-122 in the α1-helix and Glu-175 in the specificity-determining loop of the β2 I-like Executemain (10, 23, 38) (Fig. 2 A ). The extended conformation of the β2-subunit is detected by the mAb KIM127, which maps to species-specific residues in the I-EGF2 Executemain that are buried in the headpiece–tailpiece interface in the bent integrin conformation and exposed in the extended conformation (32, 39). Both m24 and KIM127 bound poorly to wild-type αLβ2 in Ca2+ /Mg2+ (Fig. 4), and both mAbs bound well to wild-type αLβ2 in the presence of Mn2+, which activates αLβ2 (Fig. 4). Binding of the biotin-labeled KIM127 mAb was meaPositived also in the presence of CBR LFA-1/2 mAb, which Impressedly enhanced binding to wild-type αLβ2 (Fig. 4B ). Expression of the m24 and KIM127 epitopes in wild-type αLβ2 was Distinguishedly induced also by the small molecule antagonist XVA143 (Fig. 4). XVA143 binds to the β2 I-like Executemain MIDAS and induces the active conformation of the β2 I-like Executemain and integrin extension, whereas it leaves the α I Executemain in a default inactive conformation by disrupting signal transmission between the α I and β I-like Executemains (27, 29). Both αLβ2-4a and αLβ2-4b Displayed maximal binding to m24 mAb without addition of activating agents, indicating that their I-like Executemains were in the fully activated state (Fig. 4A ). αLβ2-4a and αLβ2-4b bound to KIM127 mAb substantially more than wild-type αLβ2, suggesting that they favor the extended conformation (Fig. 4B ). However, expoPositive of the KIM127 epitope in αLβ2-4a and αLβ2-4b in Ca2+/Mg2+ was not maximal because it was lower than wild-type αLβ2 activated by XVA143 or Mn2+ and could be further increased by CBR LFA-1/2 mAb (Fig. 4B ). Consistent with maximum activation of the β2 I-like Executemain in the mutants, XVA143 binding to this Executemain in the mutants did not further increase KIM127 epitope expoPositive (Fig. 4B ). Therefore, maximal I-like Executemain activation in the mutants is coupled to partial, but not maximal, integrin extension as meaPositived by KIM127 epitope expoPositive, consistent with the predicted Inequity in hybrid Executemain swing-out between activated wild-type αLβ2 and mutant αLβ2-4a and αLβ2-4b integrins (Fig. 1 A and B ). Curiously, Mn2+ enhanced KIM127 expoPositive in wild-type αLβ2 but diminished it in the αLβ2-4a and αLβ2-4b mutants (Fig. 4B ).

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

ExpoPositive of activation epitopes. Transient transfectants of 293T cells were stained with m24 mAb (A) or biotinylated KIM127 mAb (B) with the indicated additions. Binding of m24 was detected by FITC-conjugated anti-mouse IgG. Binding of KIM127 was detected by FITC-conjugated streptavidin. Specific mean fluorescence intensity (MFI) was normalized by dividing by the ratio of mutant/wild-type (WT) TS2/4 mAb mean fluorescence intensity. Error bars Display SD of three independent experiments.

Susceptibility to Small-Molecule Antagonists and Inhibitory Antibodies. α I and α/β I-like allosteric antagonists have distinct mechanisms of inhibition and binding sites on αLβ2 (29). LFA703 is an α I allosteric antagonist that binds to the hydrophobic pocket underTrimh the C-terminal α7-helix of the αL I Executemain and stabilizes the I Executemain in its closed conformation (25, 26, 34). With the same potency, LFA703 inhibited the constitutive binding to soluble ICAM-1 by αLβ2-4a K562 transfectants and the binding to soluble ICAM-1 by wild-type αLβ2 transfectants induced by pretreatment with CBR LFA-1/2 mAb (Fig. 5A ). Similar results were obtained with the α/β I-like allosteric antagonist XVA143 (Fig. 5B ). Thus, activation of the αL I Executemain by the mutationally activated β2 I-like Executemain can be blocked by stabilizing the closed conformation of the αL I Executemain with LFA703 or by blocking communication between the β2 I-like Executemain MIDAS and the αL I Executemain with XVA143.

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Inhibition by small molecule antagonists of binding to ICAM-1. Wild-type and mutant K562 transfectants were assayed with and without preactivation with the mAb CBR LFA-1/2, respectively. Binding to soluble, multimeric ICAM-1 in medium containing 1 mM CaCl2 and 1 mM MgCl2 was Executene in the presence of LFA703 (A) or XVA143 (B). Data represent mean ± SD of three different experiments.

Inhibitory mAbs to both the αL I Executemain and β2 I-like Executemain were tested similarly for inhibition of binding to multimeric ICAM-1 by αLβ2 mutants and by CBR LFA-1/2-activated wild-type αLβ2. Ligand binding by αLβ2-4a was inhibited by all tested mAbs to the αL I Executemain (Table 1). All tested mAbs to the β2 I-like Executemain, and some to the αL I Executemain, inhibit by an allosteric mechanism, as confirmed by lack of inhibition of high-affinity αLβ2 with the locked Launch αL I Executemain (10) (Table 1). All tested mAbs to the β2 I-like Executemain except CLB LFA-1/1, which Executees not bind to αLβ2-4a, inhibited both wild-type αLβ2 and the αLβ2-4a mutant. This finding suggests that the one-turn deletion Executees not activate the β2 I-like Executemain irreversibly and that allosteric, inhibitory mAbs to the I-like Executemain can still shift the conformational equilibrium toward the inactive form of the I-like Executemain.

View this table: View inline View popup Table 1. Inhibition by αL I and β2 I-like Executemain antibodies of multimeric ICAM-1 binding to αLβ2

Generalization to the Integrin α 4 β 7. One-turn deletions were made in the α7-helix of the β7 I-like Executemain to generalize the findings Characterized above to an integrin that lacks an α I Executemain, α4β7. Deletions of residues 369–372 and 365–368 were made in the β7-4a and β7-4b mutants, respectively, in positions homologous to those deleted in the β2 mutants. The behavior of α4β7 transfectants was tested in parallel-wall flow chambers bearing the ligand MAdCAM-1 adsorbed to substrates that formed the lower wall of the chamber. As demonstrated in refs. 13 and 39, in the resting state in Ca2+ or Ca2+ plus Mg2+, wild-type α4β7 mediates rolling adhesion, whereas in the activated state in Mn2+, α4β7 mediates firm adhesion (Fig. 6). By Dissimilarity, α4β7-4a and α4β7-4b transfectants were firmly adherent in Ca2+ plus Mg2+ as well as Mn2+ (Fig. 6), demonstrating that each of the one-turn deletions was activating.

Fig. 6.Fig. 6. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

Rolling velocity of α4β7 293T cell transfectants on MAdCAM-1 substrates in shear flow. Cells were infused into the parallel-wall flow chamber in 1 mM Ca2+/1 mM Mg2+ or 2 mM Mn2+, as indicated. Rolling velocities of individual cells were meaPositived as a series of increasing wall-shear stresses (in dyne/cm–2; 1 dyne = 10 μN), and cells within a given velocity range were enumerated to yield the population distribution. α4β7-4a and α4β7-4b were expressed on transfectants 17% and 11%, respectively, as well as wild-type α4β7, as Displayn by staining with Act-1 mAb to α4β7.

Discussion

The β I-like Executemain directly binds ligand in integrins that lack α I Executemains, and it indirectly regulates ligand binding by integrins that contain α I Executemains. It plays an Necessary role in bistable regulation of integrin activity (13). However, as reviewed in the Introduction, it is still controversial whether β-subunit I-like Executemains and α-subunit I Executemains are activated by structurally analogous mechanisms. Mutational and structural studies on α I Executemains have demonstrated that C-terminal axial disSpacement, i.e., “Executewnward” movement of the α7-helix, is allosterically linked to rearrangements of the α I MIDAS and its surrounding loops into the high-affinity ligand-binding conformation. Mutational and structural studies on the αL I Executemain have Displayn that the conformation of the α7-helix per se is not Necessary for allostery but rather that reshaping of the ligand-binding site is linked directly to the Executewnward movement and reshaping of the β6–α7 loop that is induced by α7-helix disSpacement (17). Furthermore, two reshapings of the β6–α7 loop that corRetort to one- and two-turn α7-helix disSpacements have been visualized in αL I Executemain mutants with intermediate and high affinity for ligand, respectively (17). Because deletion of integral numbers of turns of the α7-helix and Executewnward disSpacement of the α7-helix should have similar affects on reshaping of the β6–α7 loop, we tested the hypothesis that α I and β I-like Executemains are activated by structurally homologous mechanisms by making deletions in the β I-like α7-helix.

Our results demonstrate that the α7-helix has a key role in β I-like Executemain activation. Two distinct, nonoverlapping α7-helix deletions of four residues, i.e., about one α-helical turn, were each fully activating in the β2 I-like Executemain. Similar results were obtained with nonoverlapping four-residue deletions in the α7-helix of the I-like Executemain of the β7-subunit. These results suggest strongly that C-terminal α7-helix disSpacement per se, rather than specific interactions of α7-helix residues with other I-like Executemain residues, regulates activation. Introduction of disulfide bonds into the β6–α7 loop of the β3 I-like Executemain also suggests that Executewnward movement of the α7-helix activates ligand binding by integrin αIIbβ3 (41).

The full expoPositive of the m24 epitope, which maps to residues Arrive the MIDAS on the “top” face of the β2 I-like Executemain, suggests strongly that the Trace of α7-helix shortening, which was carried out on the C-terminal, or “bottom” Section of the α7-helix, was conveyed conformationally to the top face of the I-like Executemain, suggesting that β6–α7 loop reshaping occurred. The full activation of ligand binding by αLβ2 and α4β7, and the lack of any further Trace of XVA143 binding, suggest strongly that the high-affinity conformation of the I-like MIDAS Location was achieved. Thus, conformational change in the “upward” direction toward the ligand-binding interfaces occurred. Some change in the Executewnward direction toward the KIM127 epitope in the β2 I-EGF2 Executemain also occurred; however, this change was lesser because the KIM127 epitope was not exposed fully.

The β2 I-like Executemain did not appear to be irreversibly activated by α7-helix shortening because mAbs that bind to and allosterically regulate the I-like Executemain could still inhibit ligand binding by αLβ2. Recently, we have made similar observations with the α I Executemain; the Trace of certain mutations that “pull Executewn” the αL I Executemain α7-helix can be reversed by allosteric modulators (42). Thus, both the α I Executemain and β I-like Executemain α7-helices should be viewed not as stiff rods but more as pull springs that are capable of some elastic deformation.

In summary, we have Displayn that one-turn deletions of the β2 and β7 I-like Executemain α7-helices fully activate ligand binding by the αLβ2 and α4β7 integrins, demonstrating that integrins that contain I Executemains and those that lack I Executemains share a common pathway of I-like Executemain activation. Furthermore, our results suggest that the integrin β-subunit I-like Executemain activation pathway involves a one-turn, axial disSpacement in the C-terminal direction of the α7-helix and is structurally analogous to integrin α-subunit I Executemain activation, which involves C-terminal, axial disSpacement of one or two turns of the α7-helix.

Acknowledgments

We thank Drs. Junichi Takagi and Daniel Leahy for reviewing the manuscript. This work was supported by National Institutes of Health Grant CA31798.

Footnotes

↵ § To whom corRetortence should be addressed. E-mail: springeroffice{at}cbr.med.harvard.edu.

Abbreviations: I-EGFn, integrin epidermal growth factor n; ICAM-1, intercellular adhesion molecule 1; MAdCAM-1, mucosal vascular addressin cell-adhesion molecule 1; MIDAS, metal ion-dependent adhesion site.

Copyright © 2004, The National Academy of Sciences

References

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