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J. Biol. Chem., Vol. 278, Issue 51, 51622-51629, December 19, 2003

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Role of ADMIDAS Cation-binding Site in Ligand Recognition by Integrin ₅₁^*

A. Paul Mould, Stephanie J. Barton, Janet A. Askari, Susan E. Craig, and Martin J. Humphries

From the Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom

Received for publication, June 23, 2003 , and in revised form, September 23, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Integrin-ligand interactions are regulated in a complex manner by divalent cations, and multiple cation-binding sites are found in both and integrin subunits. A key cation-binding site that lies in the subunit A-domain is known as the metal-ion dependent adhesion site (MIDAS). Recent x-ray crystal structures of integrin _V3 have identified a novel cation binding site in this domain, known as the ADMIDAS (adjacent to MIDAS). The role of this novel site in ligand recognition has yet to be elucidated. Using the interaction between ₅₁ and fibronectin as a model system, we show that mutation of residues that form the ADMIDAS site inhibits ligand binding but this effect can be partially rescued by the use of activating monoclonal antibodies. The ADMIDAS mutants had decreased expression of activation epitopes recognized by 12G10, 15/7, and HUTS-4, suggesting that the ADMIDAS is important for stabilizing the active conformation of the integrin. Consistent with this suggestion, the ADMIDAS mutations markedly increased the dissociation rate of the integrin-fibronectin complex. Mutation of the ADMIDAS residues also reduced the allosteric inhibition of Mn²⁺-supported ligand binding by Ca²⁺, suggesting that the ADMIDAS is a Ca²⁺-binding site involved in the inhibition of Mn²⁺-supported ligand binding. Mutations of the ADMIDAS site also perturbed transduction of a conformational change from the MIDAS through the C-terminal helix region of the A domain to the underlying hybrid domain, implying an important role for this site in receptor signaling.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Integrins constitute a large family of / heterodimeric transmembrane receptors found in all metazoa (1). Cell-matrix and cell-cell interactions mediated by integrins are central to many fundamental biological processes such as embryonic morphogenesis, leukocyte trafficking, and platelet aggregation. Integrins can exist in either active (ligand competent) or inactive conformational states; the equilibrium between these two states is regulated intracellularly by the binding of cytoskeletal and signaling molecules (2, 3). Integrin-ligand interactions also require divalent cations and are regulated in a complex manner by changes in the concentrations of these ions. The effects of activation can be mimicked in vitro by cations such as Mn²⁺ or Mg²⁺, whereas Ca²⁺ typically favors the inactive state. The different effects of these cations are related to their differential abilities to induce the integrin to undergo the shape changes involved in activation (4–6).

The molecular basis of integrin function has been greatly elucidated by x-ray crystal structures of the extracellular domains of _V3 in the unliganded and liganded states (7, 8). The overall structure of the heterodimer is that of a "head" on two "legs." The head region (where ligand binding takes place) comprises a seven-bladed -propeller in the subunit and a von Willebrand factor A-type domain in the subunit (A¹; also referred to as "I-like domain"), an ,-fold, which is inserted by short N- and C-terminal linkers into a "hybrid" domain. The hybrid domain is a -sandwich fold made up of the 60 amino acid residues preceding and the 90 residues following A. Both tertiary and quaternary structural changes are observed upon the binding of a ligand mimetic peptide containing the RGD recognition sequence to the preformed integrin crystal (8), however, a pivotal conformational change appears to be an inwards movement of the 1 helix of A. This shift of the 1 helix appears to be necessary for activation (6). We have also shown that 1 helix motion is linked to a movement in the 7 helix region and a swing of the hybrid domain away from the subunit (9). These latter conformational changes were not observed in the liganded _V3 crystal structure (8), probably because of restraints imposed by lattice contacts (10, 11).

Six cation binding sites were found in the unliganded and eight in the liganded _V3 structures (7, 8). Four sites are present on the lower face of the subunit -propeller. Although originally thought to be EF-hand-like, these sites are now known to form -hairpin loops (7, 12). All four hairpin loops are linked in a chain-like arrangement and the -hairpin loop in blade 7 is probably important for stabilizing interactions with the underlying "thigh" domain. A fifth cation binding site is observed at the junction between the subunit "thigh" and "calf" domains. Three sites are present on the top face of A. The first, known as MIDAS (metal ion-dependent adhesion site), plays a central role in ligand recognition. One of the carboxylate oxygens of the aspartic acid side chain of RGD coordinates directly to a metal ion bound at the MIDAS, thus explaining the absolute dependence of ligand binding on divalent cations (8). Occupancy of the MIDAS also induces conformational changes associated with activation of this domain. The second site lies adjacent to the MIDAS and is therefore termed ADMIDAS. The third is termed LIMBS (ligand-associated metal-binding site). The MIDAS and LIMBS sites were occupied only in the liganded structure (8). Residues involved in the cation coordination of the MIDAS and ADMIDAS sites are shown in Table I.

View this table: [in this window] [in a new window]   TABLE I MIDAS and ADMIDAS sites in the 1 and 3 A-domains and their coordinating residues The cation-coordinating residues in 3 (as identified in the V3 crystal structures, Refs. 7 and 8) are shown alongside the corresponding residues in 1. Cation-binding site mutants made in 1 are shown in bold.

Here we have examined the role of the ADMIDAS site in ligand binding by ₅₁. Our findings provide evidence that the ADMIDAS is a member of the class of Ca²⁺-binding inhibitory sites and, while not essential for ligand binding, the ADMIDAS may have a role in stabilizing the active conformation through an effect on the 1 helix of A. The ADMIDAS is also important for the transduction of cation-induced conformational changes from A to the underlying hybrid domain.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Monoclonal Antibodies and Proteins—Rat mAbs 16 and 13 recognizing the human ₅ and ₁ subunits, respectively, were gifts from Dr. K. Yamada (NIDCR, National Institutes of Health). Mouse anti-human ₅ mAb P1D6 was a gift from Dr. E. Wayner (Fred Hutchinson Cancer Research Center, Seattle, WA). Mouse anti-human ₅ mAb SNAKA52 and mouse anti-human ₁ mAbs 12G10 and 8E3 were produced as described (14, 4). A Fab fragment of 12G10 was prepared as previously described (9). Mouse anti-human ₁ mAb TS2/16 was a gift from F. Sánchez (Hospital de la Princesa, Madrid, Spain). Mouse anti-human ₁ mAbs JB1A and N29 were gifts from J. Wilkins (University of Manitoba, Winnipeg, Canada). Mouse anti-human ₁ mAb 15/7 was a gift from T. Yednock (Elan Pharmaceuticals, South San Francisco, CA). Mouse anti-human ₁ mAbs 4B4 and HUTS-4 were purchased from Beckman Coulter (High Wycombe, United Kingdom) and Chemicon (Harrow, UK), respectively. Mouse anti-human ₁ mAb K20 was purchased from Immunotech. A11 mAbs were used as purified IgG. A recombinant fragment of fibronectin containing type III repeats 6–10 (3Fn6–10) was produced and purified as previously described (15). For solid phase assays 3Fn6–10 was biotinylated as before (6) using sulfo-LC-NHS biotin (Perbio, Chester, UK).

Expression Vector Construction and Mutagenesis—C-terminal truncated human ₅ and ₁ constructs encoding ₅ residues 1–613 and ₁ residues 1–455 fused to the hinge regions and C_H2 and C_H3 domains of human IgG1 (5-(1–613)-Fc and 1-(–455)-Fc)) were generated as previously described (16). C-terminal truncated constructs containing the full-length extracellular domains (5-(1–951)-Fc and 1-(1–708)-Fc)) were produced as before (16). To aid the formation of heterodimers, the C_H3 domain of the ₅ construct contained a "hole" mutation, whereas the C_H3 domain of the ₁ constructs carried a "knob" mutation as described (16, 17). The mutations in the ₁ subunit were carried out using oligonucleotide-directed PCR mutagenesis, as described (9). Oligonucleotides were purchased from MWG Biotech (Southampton, UK). The presence of the mutations (and the lack of any other changes to the wild-type sequence) was verified by DNA sequencing.

Transfection—Chinese hamster ovary cell L761h variants (16) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, and 1% non-essential amino acids (growth medium). Cells were detached using 0.05% (w/v) trypsin, 0.02% (w/v) EDTA in phosphate-buffered saline, and plated overnight into 6-well culture plates (Costar). Approximately 1 µg of wild-type or mutant 1-(–455)-Fc with 1 µg of wild-type 5-(–613)-Fc DNA/well, or 1 µg of wild-type or mutant 1-(1–708)-Fc with 1 µg of wild-type 5-(1–951)-Fc DNA/well was used to transfect the cells using LipofectAMINE PLUS reagent (Invitrogen, Paisley, Scotland) according to the manufacturer's instructions. After 4 days, supernatants were harvested by centrifugation at 1000 x g for 5 min.

For comparison of purified wild-type heterodimers with heterodimers containing the ADMIDAS or LIMBS mutations in ₁, 75-cm² flasks of subconfluent CHOL761h cells were transfected with 5 µg of wild-type or mutant ₁ construct, and 5 µg of wild-type ₅ construct as described above. After 4 days, culture supernatants were harvested by centrifugation at 1000 x g for 5 min. Wild-type or mutant heterodimers were purified using Protein A-Sepharose essentially as described before (16). Concentration measurements of wild-type or mutant heterodimers were performed using a purified ₅₁-Fc standard of known concentration (a gift from P. Stephens, M. Robinson, and H. Kirby, Celltech Chiroscience, UK).

Effect of ADMIDAS Mutations on 3Fn6–10 Binding—Solid phase ligand binding assays using either Fc captured or directly coated integrin were performed essentially as previously described (6, 9, 13, 16). In these assays 3Fn6–10 coupled to sulfo-NHS LC biotin was used at 0.1 µg/ml, unless stated otherwise. Measurements obtained were the mean ± S.D. of four replicate wells.

Surface Plasmon Resonance—Experiments were performed using the BIAcore 3000 (Biacore AB). Running buffer was 150 mM NaCl, 25 mM Tris-Cl, 1 mM MnCl₂, pH 7.4. 3Fn6–10 coupled to biotin maleimide (Sigma) was bound to the surface of a streptavidin-coated chip (Biacore AB). Dilutions of wild-type ₅₁-Fc or ₅₁-Fc with the D137A or D138A mutations in the running buffer were injected at 10 µl/min, 25 °C, into flow cells containing approximately 300 response units of 3Fn6–10 fragment. The same buffer containing 5 mM EDTA in place of MnCl₂ was used to regenerate the surface after each injection. No binding was observed when samples were injected in the presence of EDTA. All measurements were baseline-corrected by subtracting the sensorgram obtained with that from a control flow cell coated with streptavidin alone. Kinetic parameters were determined by fitting the data to a 1:1 Langmuir binding model using BIAevaluation software version 3.1.

Effect of Divalent Cations on 15/7 Binding—96-well plates (Costar -area EIA/RIA, Corning Science Products, High Wycombe, UK) were coated with goat anti-human 1 Fc (Jackson Immunochemicals, Stratech Scientific, Luton, UK) at a concentration of 2.6 µg/ml in Dulbecco's phosphate-buffered saline (50 µl/well) for 16 h. Wells were then blocked for 1–3 h with 200 µl of 5% (w/v) bovine serum albumin, 150 mM NaCl, 0.05% (w/v) NaN₃, 25 mM Tris-Cl, pH 7.4 (blocking buffer). Blocking buffer was removed and supernatant from cells transfected with wild-type 5-(–613) 1-(–455)-Fc diluted 1:1 with 150 mM NaCl, 25 mM Tris-Cl, 5 mM EDTA, pH 7.4 (25 µl/well), was added for 1–2 h at room temperature. Wells were then washed three times with 200 µl of 150 mM NaCl, 25 mM Tris-Cl, pH 7.4, containing 1 mg/ml bovine serum albumin (buffer A). Buffer A was treated with Chelex beads (Bio-Rad) to remove any small contaminating amounts of endogenous Ca²⁺ and Mg²⁺ ions. 15/7 (1 µg/ml) in buffer A with 2 mM EDTA, 2 mM Mn²⁺, 2 mM Mg²⁺, or 2 mM Ca²⁺ was added to the plate (50 µl/well). The plate was then incubated at 30 °C for 2 h. Unbound antibody was aspirated and the wells washed three times with buffer A. Bound antibody was quantitated by addition of 1:1000 dilution of goat anti-mouse IgG (Fc-specific) peroxidase conjugate (Jackson Immunochemicals) in buffer A for 30 min at room temperature (50 µl/well). Wells were then washed four times with buffer A, and color was developed using 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) substrate (50 µl/well). Absorption at 405 nm was measured using a plate reader (Dynex Technologies). Background binding to mAbs to wells incubated with supernatant from mock-transfected cells was subtracted from all measurements. Measurements obtained were the mean ± S.D. of four replicate wells.

Comparison of Epitope Expression by Wild-type and Mutant Heterodimers—Plates were coated with anti-human Fc and blocked as described above. The blocking solution was removed and cell culture supernatants were added (25 µl/well) for 1–2 h. All supernatants were assayed in triplicate, and supernatant from mock-transfected cells was used as a negative control. The plate was washed 3 times with buffer A with 1 mM MnCl₂ (buffer B) (200 µl/well), and anti-5 or anti-1 mAbs (5 µg/ml) were added (50 µl/well). The plate was incubated for 2 h and then washed 3 times in buffer B. Peroxidase-conjugated anti-rat or anti-mouse secondary antibodies (1:1000 dilution in buffer B; Jackson Immunochemicals) were added (50 µl/well) for 30 min, the plate was washed four times in buffer B, and color was developed as described above. All steps were performed at room temperature. Results shown are representative of three separate experiments.

In each assay involving a comparison between different heterodimers the binding of mAb 8E3 (5 µg/ml) was used to normalize for any differences between the amounts of the different heterodimers bound to the wells. For example, normalized A₄₀₅ for 15/7 binding = (AM_15/7 – Am_15/7) x ((AWT_8E3 – Am_8E3)/(AM_8E3 – Am_8E3)), where A_15/7M = mean absorbance of wells coated with mutant integrin, Am_15/7 = mean absorbance of wells coated with mock supernatant, AWT_8E3 = mean absorbance of 8E3 binding to wells coated with wild-type integrin, Am_8E3 = mean absorbance of 8E3 binding to wells coated with mock supernatant, and AM_8E3 = mean absorbance of 8E3 binding to wells coated with mutant integrin. 8E3 recognizes a non-functional epitope in the N-terminal region of the ₁ subunit (16). In experiments using heterodimers captured from cell culture supernatants similar results were obtained using Protein A-purified heterodimers (data not shown). Each experiment shown is representative of at least three separate experiments.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
ADMIDAS Mutants Have Low Constitutive Activity and Show Reduced Expression of Activation Epitopes—The ADMIDAS site contains residues Ser¹³⁴, Asp¹³⁷, Asp¹³⁸, and Ala³⁴¹ (or Asp²⁵⁹) (see Table I). The side chains of both Asp¹³⁷ and Asp¹³⁸ contribute two carboxylate oxygens to cation coordination, hence mutation of either residue would be expected to abrogate cation binding to this site. The other ADMIDAS residues contribute only a backbone carbonyl (except in the case of Asp²⁵⁹, which also participates in cation coordination at the MIDAS site). Hence to selectively test the role of the ADMIDAS site we made mutants D137A and D138A. For these studies we employed a recently described system for expression of recombinant soluble ₅₁ (16). We mainly used a truncated version of ₅₁, 5-(–613) 1-(–455), fused to the Fc region of human IgG1 (hereafter referred to as tr51-Fc). This heterodimer contains the subunit -propeller and thigh domain with the subunit A, PSI, and hybrid domains (7), and retains the ligand-binding properties of the full-length receptor (16). The advantages of this system have been described previously (6, 9).

The ligand binding activity of the wild-type or mutant integrins was tested after either capture of the integrin onto a 96-well plate coated with anti-human Fc polyclonal antibody, or by directly coating the purified integrin onto the plate (16). Wild-type tr51-Fc had low activity when Fc captured but had high activity after direct adsorption (Fig. 1, A and B); the activating mAb 12G10 (15) restored the activity of Fc-captured integrin but did not further increase the activity of directly coated receptor. In contrast, both ADMIDAS mutants had very low activity after either Fc capture or direct adsorption. 12G10 only partially rescued the activity of either Fc-captured or directly adsorbed D137A and D138A mutants (in the case of directly adsorbed integrin to 15 and 70% of wild-type levels, respectively).

View larger version (23K): [in this window] [in a new window]   FIG. 1. Effect of 1 subunit D137A and D138A ADMIDAS mutations on binding of the 3Fn6–10 fibronectin fragment to tr51-Fc. A, Fc-captured integrin; B, directly adsorbed integrin. Binding of 3Fn6–10 was measured in 1 mM Mn2+ in the absence of mAbs (open bars) or presence of the activating mAbs TS2/16 (cross-hatched bars) or 12G10 (diagonally hatched bars). The mutation E140A was used as a control. The non-function perturbing mAb N29 had no effect on 3Fn6–10 binding (data not shown).

View larger version (33K): [in this window] [in a new window]   FIG. 2. Effect of ADMIDAS mutations on binding of 3Fn6–10 fibronectin fragment to 51-Fc. Binding of 3Fn6–10 to wild-type integrin or D137A or D138A mutants was measured in the absence of mAbs (open bars) or presence of the activating mAbs TS2/16 (cross-hatched bars) or 12G10 (diagonally hatched bars). The non-function perturbing mAb K20 had no effect on 3Fn6–10 binding (data not shown). Assay was performed using Fc-captured integrin.

View larger version (21K): [in this window] [in a new window]   FIG. 3. Effect of ADMIDAS mutations on the kinetics of 51-Fc binding to the 3Fn6–10 fibronectin fragment. Biotinylated 3Fn6–10 (300 response units) was bound to a streptavidin-coated chip and wild-type 51-Fc (A), D137A mutant (B), or D138A mutant (C) in a running buffer containing 1 mM Mn2+ was injected over the surface for 500 s and the dissociation phase was followed for 600 s. Traces show increasing concentrations (in nanomolar) of 51-Fc analyte. D, D138A mutant (17 nM) was injected over the 3Fn6–10-coated chip after preincubation with the Fab fragment of mAb 12G10 (100 nM, thick trace) or with running buffer alone (thin trace). The dissociation rate constant (koff) in the presence of 12G10 is 1 x 10–4 s–1 compared with 1 x 10–3 s–1 in the absence of 12G10. No integrin binding was observed in the presence of 3 mM EDTA (+EDTA).

View this table: [in this window] [in a new window]   TABLE III Kinetic parameters of interaction of wild-type 51-Fc and ADMIDAS mutants with 3Fn6-10 Binding curves from four different concentrations of wild-type or mutant 51-Fc (shown in Fig. 3) were analyzed using the BIAevaluation 3.1 software (Biacore AB) by separate fitting of data from association and dissociation phases. Results shown are mean ± S.D. from a single experiment but essentially identical data was obtained in two additional experiments (not shown).

View larger version (12K): [in this window] [in a new window]   FIG. 4. Effect of divalent cations on the binding of 3Fn6–10 fibronectin fragment to wild-type tr51-Fc (A) and tr51-Fc with the D138A ADMIDAS mutation (B). Binding of 3Fn6–10 was measured in the presence of varying concentrations of Mn2+ (•), Mg2+ (), or Ca2+ (). Assay was performed using Fc-captured integrin in the presence of mAb 12G10.

ADMIDAS Mutants Show Reduced Allosteric Inhibition of Mn²⁺-supported Ligand Binding by Ca²⁺—The crystal structure of _V3 in the unliganded state showed that the ADMIDAS site could be occupied by Ca²⁺. We have previously shown that one of the effects of Ca²⁺ ions on ₅₁-fibronectin interactions is an allosteric inhibition of ligand binding supported by Mn²⁺ (13). We therefore tested whether the D138A mutation affected the ability of Ca²⁺ ions to inhibit binding of the 3Fn6–10 fragment in the presence of a constant concentration of Mn²⁺ (100 µM). The results (Fig. 5) showed that the D138A mutation reduced the ability of Ca²⁺ to inhibit ligand binding. Similar results were obtained with the D137A mutant, whereas the E140A mutation had no effect (data not shown). The inhibition of Mn²⁺-supported ligand binding by Ca²⁺ had a multiphasic pattern suggesting that this inhibition was mediated by Ca²⁺ binding to at least three separate sites of differing affinities (high, medium, and low). Comparison of the pattern of inhibition for the D138A mutant with that of the wild-type receptor showed that the effect of the medium affinity site was lost. Making the assumption that the concentration of Ca²⁺ for half-maximal inhibition in this range reflects the affinity of Ca²⁺ ions for the medium affinity site, mathematical modeling of the inhibition (not shown) indicated that this site has an apparent K_D of 160 ± 30 µM (n = 4). Taken together, these findings show that the ADMIDAS is a selective, medium affinity Ca²⁺-binding site that contributes to the allosteric inhibition of Mn²⁺-supported ligand binding.

View larger version (14K): [in this window] [in a new window]   FIG. 5. Effect of D138A ADMIDAS mutation on the inhibition of Mn2+-supported ligand binding by Ca2+. Binding of 3Fn6–10 to wild-type tr51-Fc (•) or tr51-Fc with the D138A mutation () was measured in the presence of a constant concentration of Mn2+ (100 µM) and varying concentrations of Ca2+. For ease of comparison, data for the D138A mutant were normalized to the same level of ligand binding as the wild-type integrin in the absence of Ca2+. The level of ligand binding supported by 4 mM Ca2+ alone is shown by the open symbols (, wild type; , D138A mutant). Assay was performed using Fc-captured integrin in the presence of mAb 12G10.

View larger version (11K): [in this window] [in a new window]   FIG. 6. Effect of D138A ADMIDAS mutation on modulation of Mg2+-supported ligand binding by Ca2+. A, wild-type tr51-Fc; B, tr51-Fc with the D138A mutation. Binding of 3Fn6–10 was measured in the presence of 100 µM Mg2+ alone () or 100 µM Mg2+ with varying concentrations of Ca2+ (•). The level of ligand binding supported by Ca2+ alone is also shown (). Assay was performed using Fc-captured integrin in the presence of mAb 12G10.

View larger version (14K): [in this window] [in a new window]   FIG. 7. Effect of ADMIDAS mutations on modulation of 15/7 binding to tr51-Fc by divalent cations. 15/7 binding was measured in the presence of 2 mM EDTA (open bars), 2mM Mn2+ (filled bars), 2 mM Mg2+ (grey bars), or 2 mM Ca2+ (diagonally shaded bars).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In allocating functions to the various cation-binding sites in integrins, a central issue is which sites need to be occupied for ligand binding to occur; i.e. the identity of the ligand-competent sites. Both the MIDAS and LIMBS appear to fall into this category (8). The ADMIDAS, however, belongs to the category of Ca²⁺-binding inhibitory sites. A surprising feature of the inhibition of ligand binding to ₅₁ by Ca²⁺ is that Ca²⁺ acts as a competitive inhibitor of ligand binding supported by Mg²⁺ but not of ligand binding supported by Mn²⁺. This previously led us to suggest that there are separate ligand-competent sites for Mg²⁺ and Mn²⁺ (13). However, it is now clear that the major ligand-competent site for both Mg²⁺ and Mn²⁺ is the MIDAS (6). Why Ca²⁺ is able to displace Mg²⁺ but not Mn²⁺ from the MIDAS will be the subject of future investigations.

Whereas the ligand-competent sites typically bind Mn²⁺ or Mg²⁺, both the stimulatory and inhibitory sites appear to bind Ca²⁺ selectively (13, 20, 21). The _V3 crystal structures showed that the ADMIDAS can be occupied by either Ca²⁺ or Mn²⁺. However, no insight into the specificity of this site should be inferred from these studies because the crystals were prepared in the presence of a high concentration of a single cation (5 mM Ca²⁺ or Mn²⁺). The studies presented here show that the ADMIDAS preferentially binds Ca²⁺; this feature may be because of its high content of coordinating groups from negatively charged residues (22). In addition to the ADMIDAS, other sites appear to contribute to the inhibition of Mn²⁺-supported ligand binding by Ca²⁺; these could include sites on subunit -propeller.² The LIMBS would appear to be a good candidate for a stimulatory Ca²⁺-binding site because of its close proximity to the MIDAS. However, it is difficult to test this suggestion experimentally because LIMBS mutations block ligand binding to ₅₁, and therefore it is not possible to check if the synergistic effects of low concentrations of Ca²⁺ and Mg²⁺ are abrogated by LIMBS mutations.³

A second major issue concerns the contribution of cation-binding sites to signal propagation mechanisms through the receptor molecule. We have previously shown that activation of the head region of ₅₁ involves a linked movement of the 1 and 7 helices in the A domain and an outward swing of the hybrid domain (9). The MIDAS is involved in this pathway of conformational changes because MIDAS mutations block both 1 and 7 movements (6, 9). We report here that the ADMIDAS mutations also affect the activation state of the integrin. These mutations could affect activation directly through loss of ADMIDAS cation, or indirectly through an effect on the 1 helix of A, which coordinates the ADMIDAS cation at its N-terminal end. In practice it may be difficult to distinguish between these alternatives. However, in support of at least a partial role for an indirect effect, we have recently found that other mutations in the 1 helix can either inactivate or activate the receptor.⁴ Also, if the effect of the D137A and D138A mutations was simply because of loss of the ADMIDAS cation then the two mutations should have an equal effect on ligand binding. Instead, our observation that the D137A mutation has a more severe effect on ligand binding than the D138A mutation suggests that the mutations do not only affect cation binding. The ability of 12G10 to override the effect of the ADMIDAS mutations could be explained by the ability of this mAb to stabilize the inward shift of the 1 helix observed in the active state of A (6, 8). Thus the ADMIDAS appears to be required for maintaining the active conformation of the integrin head. ADMIDAS mutants also showed decreased expression of 15/7 and HUTS-4 epitopes in hybrid domain. A plausible explanation for this is that 7 helix movement and the outward pivoting of the hybrid domain are linked to 1 helix movement (9), and therefore impeding the shift of the 1 helix causes a perturbation of this signaling pathway.

Mutagenesis of ADMIDAS residues in cell surface-expressed ₂ and ₃ integrins has produced conflicting results. In _IIb3 ADMIDAS mutations had no effect (23), whereas in _L2 the mutation D120A (equivalent to D138A) blocked function (24). In _L2 this mutation also strongly perturbed binding of mAb 24 (an antibody that recognizes an activation epitope on the 2 A domain). Hence, these results for _L2 are in overall agreement with our findings for ₅₁. The ADMIDAS could represent the site from which Ca²⁺ is removed by EGTA/Mg²⁺ activation of ₂ integrins (5, 24). In support of this suggestion, this site has an apparent affinity for Ca²⁺ in the same range as that described here for the ADMIDAS site (24). The ADMIDAS probably also represents the Ca²⁺-binding site on the 2 A domain reported by Xiong and Zhang (affinity 100 µM) (25). It is less clear whether the ADMIDAS is equivalent to the inhibitory Ca²⁺ site described by Hu et al. (21) for _V3, which had an affinity in the millimolar range.

The activation mechanism of integrins has recently been proposed to involve a switchblade-like straightening from a highly bent to an extended form (26, 27). Mn²⁺ but not Ca²⁺ is able to mediate this conformational rearrangement (27). However, here we have recapitulated the effects of divalent cations on the native receptor (with full-length extracellular domains) (13) using the truncated integrin (which lacks most of the leg regions). Therefore, although cation occupancy and receptor bending/straightening may be linked, the effects of the different cations on activation and ligand binding are not dependent on unbending of the receptor. Instead, these data suggest that the cation-binding sites regulate ligand binding mainly through effects on conformation of the head region. Hence, for example, the activating property of Mn²⁺ on integrin function appears to be primarily through its effect on the A domain rather than on straightening. This activating effect of Mn²⁺, and of mAbs such as TS2/16, appears to be due mainly to a decrease in the dissociation rate (28–30).

In summary, we have established previously unidentified roles for the ADMIDAS site in regulation of ligand binding to ₅₁. These findings also support our recently proposed model of activation of the head region involving movements of the 1 and 7 helices of A (9). Control of this shape-shifting pathway may explain, in part, why the ADMIDAS site is absolutely conserved throughout all integrin subunits sequenced to date (31, 32).

	FOOTNOTES

 
^* This work was supported by grants from the Wellcome Trust (to M. J. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, 2.205 Stopford Bldg., Oxford Road, Manchester M13 9PT, United Kingdom. Tel.: 44-161-275-5649; Fax: 44-161-275-5082; E-mail: paul.mould{at}man.ac.uk.

¹ The abbreviations used are: A, subunit von Willebrand factor A-domain; A, subunit von Willebrand factor A-domain; MIDAS, metal-ion dependent adhesion site; ADMIDAS, adjacent to MIDAS; LIMBS, ligand-associated metal-binding site; mAb, monoclonal antibody; tr51-Fc, recombinant soluble integrin heterodimer containing C-terminal truncated ₅ and ₁ subunits (₅ residues 1–613 and ₁ residues 1–455) fused to the Fc region of human IgG1; ₅₁-Fc, recombinant soluble integrin heterodimer containing full-length extracellular domains of ₅ and ₁ subunits (₅ residues 1–951 and ₁ residues 1–708); CHO, Chinese hamster ovary.

² A. D. Kline, A. P. Mould, and M. J. Humphries, unpublished results.

³ A. P. Mould, S. J. Barton, D. Valdramidou, J. A. Askari, E. J. H. Symonds, and M. J. Humphries, manuscript in preparation.

⁴ S. J. Barton, M. Travis, J. A. Askari, M. J. Humphries, and A. P. Mould, manuscript in preparation.

	ACKNOWLEDGMENTS

 
We thank A. Coe, P. Stephens, M. Robinson, and H. Kirby for the ₅ and ₁ constructs and purified ₅₁-Fc. We are grateful to J. Wilkins, T. Yednock, K. Yamada, E. Wayner, and F. Sánchez-Madrid for mAbs, K. Yamada and S. Aota for the 3Fn6–10 construct, and F. Stuart for advice on use of BIAcore 3000.

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