Role of ADMIDAS Cation-binding Site in Ligand Recognition by Integrin α5β1
2003; Elsevier BV; Volume: 278; Issue: 51 Linguagem: Inglês
10.1074/jbc.m306655200
ISSN1083-351X
AutoresA. Paul Mould, Stephanie Barton, Janet A. Askari, Susan E. Craig, Martin J. Humphries,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoIntegrin-ligand interactions are regulated in a complex manner by divalent cations, and multiple cation-binding sites are found in both α and β integrin subunits. A key cation-binding site that lies in the β subunit A-domain is known as the metal-ion dependent adhesion site (MIDAS). Recent x-ray crystal structures of integrin αVβ3 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 α5β1 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 βA domain to the underlying hybrid domain, implying an important role for this site in receptor signaling. Integrin-ligand interactions are regulated in a complex manner by divalent cations, and multiple cation-binding sites are found in both α and β integrin subunits. A key cation-binding site that lies in the β subunit A-domain is known as the metal-ion dependent adhesion site (MIDAS). Recent x-ray crystal structures of integrin αVβ3 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 α5β1 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 βA domain to the underlying hybrid domain, implying an important role for this site in receptor signaling. Integrins constitute a large family of α/β heterodimeric transmembrane receptors found in all metazoa (1Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6955) Google Scholar). 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 (2Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3829) Google Scholar, 3Vinogradova O. Velyvis A. Velyviene A. Hu B. Haas T.A. Plow E.F. Qin J. Cell. 2002; 110: 587-597Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). 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 (4Burrows L. Clark K. Mould A.P. Humphries M.J. Biochem. J. 1999; 344: 527-533Crossref PubMed Scopus (51) Google Scholar, 5Dransfield I. Cabanas C. Craig A. Hogg N. J. Cell Biol. 1992; 116: 219-226Crossref PubMed Scopus (401) Google Scholar, 6Mould A.P. Askari J.A. Barton S. Kline A.D. McEwan P.A. Craig S.E. Humphries M.J. J. Biol. Chem. 2002; 277: 19800-19806Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). The molecular basis of integrin function has been greatly elucidated by x-ray crystal structures of the extracellular domains of αVβ3 in the unliganded and liganded states (7Xiong J.-P. Stehle T. Diefenbach B. Zhang R. Dunker R. Scott D.L. Joachimiak A. Goodman S. Arnaout M.A. Science. 2001; 294: 339-345Crossref PubMed Scopus (1118) Google Scholar, 8Xiong J.-P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1410) Google Scholar). 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 β-propeller in the α subunit and a von Willebrand factor A-type domain in the β subunit (βA 1The abbreviations used are: βAβ subunit von Willebrand factor A-domainαAα subunit von Willebrand factor A-domainMIDASmetal-ion dependent adhesion siteADMIDASadjacent to MIDASLIMBSligand-associated metal-binding sitemAbmonoclonal antibodytrα5β1-Fcrecombinant soluble integrin heterodimer containing C-terminal truncated α5 and β1 subunits (α5 residues 1–613 and β1 residues 1–455) fused to the Fc region of human IgGγ1α5β1-Fcrecombinant soluble integrin heterodimer containing full-length extracellular domains of α5 and β1 subunits (α5 residues 1–951 and β1 residues 1–708)CHOChinese hamster ovary.; also referred to as "I-like domain"), an α,β-fold, which is inserted by short N- and C-terminal linkers into a "hybrid" domain. The hybrid domain is a β-sandwich fold made up of the ∼60 amino acid residues preceding and the ∼90 residues following βA. 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 (8Xiong J.-P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1410) Google Scholar), however, a pivotal conformational change appears to be an inwards movement of the α1 helix of βA. This shift of the α1 helix appears to be necessary for activation (6Mould A.P. Askari J.A. Barton S. Kline A.D. McEwan P.A. Craig S.E. Humphries M.J. J. Biol. Chem. 2002; 277: 19800-19806Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). We have also shown that α1 helix motion is linked to a movement in the α7 helix region and a swing of the hybrid domain away from the α subunit (9Mould A.P. Barton S.J. Askari J.A. McEwan P.A. Buckley P.A. Craig S.E. Humphries M.J. J. Biol. Chem. 2003; 278: 17028-17035Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). These latter conformational changes were not observed in the liganded αVβ3 crystal structure (8Xiong J.-P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1410) Google Scholar), probably because of restraints imposed by lattice contacts (10Takagi J. Springer T.A. Immunol. Rev. 2002; 186: 141-163Crossref PubMed Scopus (321) Google Scholar, 11Liddington R.C. Ginsberg M.H. J. Cell Biol. 2002; 158: 833-839Crossref PubMed Scopus (255) Google Scholar). β subunit von Willebrand factor A-domain α subunit von Willebrand factor A-domain metal-ion dependent adhesion site adjacent to MIDAS ligand-associated metal-binding site monoclonal antibody recombinant soluble integrin heterodimer containing C-terminal truncated α5 and β1 subunits (α5 residues 1–613 and β1 residues 1–455) fused to the Fc region of human IgGγ1 recombinant soluble integrin heterodimer containing full-length extracellular domains of α5 and β1 subunits (α5 residues 1–951 and β1 residues 1–708) Chinese hamster ovary. Six cation binding sites were found in the unliganded and eight in the liganded αVβ3 structures (7Xiong J.-P. Stehle T. Diefenbach B. Zhang R. Dunker R. Scott D.L. Joachimiak A. Goodman S. Arnaout M.A. Science. 2001; 294: 339-345Crossref PubMed Scopus (1118) Google Scholar, 8Xiong J.-P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1410) Google Scholar). Four sites are present on the lower face of the α subunit β-propeller. Although originally thought to be EF-hand-like, these sites are now known to form β-hairpin loops (7Xiong J.-P. Stehle T. Diefenbach B. Zhang R. Dunker R. Scott D.L. Joachimiak A. Goodman S. Arnaout M.A. Science. 2001; 294: 339-345Crossref PubMed Scopus (1118) Google Scholar, 12Springer T.A. Jing H. Takagi J. Cell. 2000; 102: 275-277Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). All four hairpin loops are linked in a chain-like arrangement and the β-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 α subunit "thigh" and "calf" domains. Three sites are present on the top face of βA. 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 (8Xiong J.-P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1410) Google Scholar). 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 (8Xiong J.-P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1410) Google Scholar). Residues involved in the cation coordination of the MIDAS and ADMIDAS sites are shown in Table I.Table IMIDAS and ADMIDAS sites in the β1 and β3 A-domains and their coordinating residuesCation binding siteResiduesMIDASβ3Asp119Ser121Ser123Glu220Asp251β1Asp130Ser132Ser134Glu229Asp259MutationD130AADMIDASβ3Ser123Asp126Asp127Met335aOnly in the unliganded structure (7).Asp251bOnly in the liganded structure (8).β1Ser134Asp137Asp138Ala341Asp259MutationD137AD138Aa Only in the unliganded structure (7Xiong J.-P. Stehle T. Diefenbach B. Zhang R. Dunker R. Scott D.L. Joachimiak A. Goodman S. Arnaout M.A. Science. 2001; 294: 339-345Crossref PubMed Scopus (1118) Google Scholar).b Only in the liganded structure (8Xiong J.-P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1410) Google Scholar). Open table in a new tab We have previously used the interaction between integrin α5β1 and fibronectin as a model system to identify and characterize cation-binding sites that can support or modulate ligand recognition (13Mould A.P. Akiyama S.K. Humphries M.J. J. Biol. Chem. 1995; 270: 26270-26277Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). Our results showed that several classes of cation-binding sites could be identified. Occupancy of the first class by Mg2+ or Mn2+ was required for ligand binding (ligand-competent sites). The second class was implicated in the allosteric inhibition of Mn2+-supported ligand binding by Ca2+ (inhibitory sites), whereas the third class was involved in the stimulation of Mg2+-supported ligand binding by Ca2+ (stimulatory sites). Recently we have shown that the ligand competent site for both Mg2+ and Mn2+ is the MIDAS of the β1 A domain (4Burrows L. Clark K. Mould A.P. Humphries M.J. Biochem. J. 1999; 344: 527-533Crossref PubMed Scopus (51) Google Scholar). The identity of the two classes of Ca2+-binding regulatory sites is currently unclear. In addition, how occupancy of these sites affects conformational movements has not yet been investigated. Here we have examined the role of the ADMIDAS site in ligand binding by α5β1. 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 α1 helix of βA. The ADMIDAS is also important for the transduction of cation-induced conformational changes from βA to the underlying hybrid domain. Monoclonal Antibodies and Proteins—Rat mAbs 16 and 13 recognizing the human α5 and β1 subunits, respectively, were gifts from Dr. K. Yamada (NIDCR, National Institutes of Health). Mouse anti-human α5 mAb P1D6 was a gift from Dr. E. Wayner (Fred Hutchinson Cancer Research Center, Seattle, WA). Mouse anti-human α5 mAb SNAKA52 and mouse anti-human β1 mAbs 12G10 and 8E3 were produced as described (14Mould A.P. Garratt A.N. Askari J.A. Akiyama S.K. Humphries M.J. FEBS Lett. 1995; 363: 118-122Crossref PubMed Scopus (125) Google Scholar, 4Burrows L. Clark K. Mould A.P. Humphries M.J. Biochem. J. 1999; 344: 527-533Crossref PubMed Scopus (51) Google Scholar). A Fab fragment of 12G10 was prepared as previously described (9Mould A.P. Barton S.J. Askari J.A. McEwan P.A. Buckley P.A. Craig S.E. Humphries M.J. J. Biol. Chem. 2003; 278: 17028-17035Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Mouse anti-human β1 mAb TS2/16 was a gift from F. Sánchez (Hospital de la Princesa, Madrid, Spain). Mouse anti-human β1 mAbs JB1A and N29 were gifts from J. Wilkins (University of Manitoba, Winnipeg, Canada). Mouse anti-human β1 mAb 15/7 was a gift from T. Yednock (Elan Pharmaceuticals, South San Francisco, CA). Mouse anti-human β1 mAbs 4B4 and HUTS-4 were purchased from Beckman Coulter (High Wycombe, United Kingdom) and Chemicon (Harrow, UK), respectively. Mouse anti-human β1 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 (15Mould A.P. Askari J.A. Aota S. Yamada K.M. Irie A. Takada Y. Mardon H.J. Humphries M.J. J. Biol. Chem. 1997; 272: 17283-17292Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). For solid phase assays 3Fn6–10 was biotinylated as before (6Mould A.P. Askari J.A. Barton S. Kline A.D. McEwan P.A. Craig S.E. Humphries M.J. J. Biol. Chem. 2002; 277: 19800-19806Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) using sulfo-LC-NHS biotin (Perbio, Chester, UK). Expression Vector Construction and Mutagenesis—C-terminal truncated human α5 and β1 constructs encoding α5 residues 1–613 and β1 residues 1–455 fused to the hinge regions and CH2 and CH3 domains of human IgGγ1 (α5-(1–613)-Fc and β1-(–455)-Fc)) were generated as previously described (16Coe A.P. Askari J.A. Kline A.D. Robinson M.K. Kirby H. Stephens P.E. Humphries M.J. J. Biol. Chem. 2001; 276: 35854-35866Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). C-terminal truncated constructs containing the full-length extracellular domains (α5-(1–951)-Fc and β1-(1–708)-Fc)) were produced as before (16Coe A.P. Askari J.A. Kline A.D. Robinson M.K. Kirby H. Stephens P.E. Humphries M.J. J. Biol. Chem. 2001; 276: 35854-35866Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). To aid the formation of heterodimers, the CH3 domain of the α5 construct contained a "hole" mutation, whereas the CH3 domain of the β1 constructs carried a "knob" mutation as described (16Coe A.P. Askari J.A. Kline A.D. Robinson M.K. Kirby H. Stephens P.E. Humphries M.J. J. Biol. Chem. 2001; 276: 35854-35866Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 17Ridgway J.B. Presta L.G. Carter P. Protein Eng. 1996; 9: 617-621Crossref PubMed Scopus (524) Google Scholar). The mutations in the β1 subunit were carried out using oligonucleotide-directed PCR mutagenesis, as described (9Mould A.P. Barton S.J. Askari J.A. McEwan P.A. Buckley P.A. Craig S.E. Humphries M.J. J. Biol. Chem. 2003; 278: 17028-17035Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). 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 (16Coe A.P. Askari J.A. Kline A.D. Robinson M.K. Kirby H. Stephens P.E. Humphries M.J. J. Biol. Chem. 2001; 276: 35854-35866Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) 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 β1-(–455)-Fc with 1 μg of wild-type α5-(–613)-Fc DNA/well, or 1 μg of wild-type or mutant β1-(1–708)-Fc with 1 μg of wild-type α5-(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 × g for 5 min. For comparison of purified wild-type heterodimers with heterodimers containing the ADMIDAS or LIMBS mutations in β1, 75-cm2 flasks of subconfluent CHOL761h cells were transfected with 5 μg of wild-type or mutant β1 construct, and 5 μg of wild-type α5 construct as described above. After 4 days, culture supernatants were harvested by centrifugation at 1000 × g for 5 min. Wild-type or mutant heterodimers were purified using Protein A-Sepharose essentially as described before (16Coe A.P. Askari J.A. Kline A.D. Robinson M.K. Kirby H. Stephens P.E. Humphries M.J. J. Biol. Chem. 2001; 276: 35854-35866Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Concentration measurements of wild-type or mutant heterodimers were performed using a purified α5β1-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 (6Mould A.P. Askari J.A. Barton S. Kline A.D. McEwan P.A. Craig S.E. Humphries M.J. J. Biol. Chem. 2002; 277: 19800-19806Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 9Mould A.P. Barton S.J. Askari J.A. McEwan P.A. Buckley P.A. Craig S.E. Humphries M.J. J. Biol. Chem. 2003; 278: 17028-17035Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 13Mould A.P. Akiyama S.K. Humphries M.J. J. Biol. Chem. 1995; 270: 26270-26277Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 16Coe A.P. Askari J.A. Kline A.D. Robinson M.K. Kirby H. Stephens P.E. Humphries M.J. J. Biol. Chem. 2001; 276: 35854-35866Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). 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 α5β1-Fc or α5β1-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 ½-area EIA/RIA, Corning Science Products, High Wycombe, UK) were coated with goat anti-human γ1 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 α5-(–613) β1-(–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-α5 or anti-β1 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 = (AM15/7 – Am15/7) × ((AWT8E3 – Am8E3)/(AM8E3 – Am8E3)), where A15/7M = mean absorbance of wells coated with mutant integrin, Am15/7 = mean absorbance of wells coated with mock supernatant, AWT8E3 = mean absorbance of 8E3 binding to wells coated with wild-type integrin, Am8E3 = mean absorbance of 8E3 binding to wells coated with mock supernatant, and AM8E3 = mean absorbance of 8E3 binding to wells coated with mutant integrin. 8E3 recognizes a non-functional epitope in the N-terminal region of the β1 subunit (16Coe A.P. Askari J.A. Kline A.D. Robinson M.K. Kirby H. Stephens P.E. Humphries M.J. J. Biol. Chem. 2001; 276: 35854-35866Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). 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. 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 α5β1 (16Coe A.P. Askari J.A. Kline A.D. Robinson M.K. Kirby H. Stephens P.E. Humphries M.J. J. Biol. Chem. 2001; 276: 35854-35866Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). We mainly used a truncated version of α5β1, α5-(–613) β1-(–455), fused to the Fc region of human IgGγ1 (hereafter referred to as trα5β1-Fc). This heterodimer contains the α subunit β-propeller and thigh domain with the β subunit A, PSI, and hybrid domains (7Xiong J.-P. Stehle T. Diefenbach B. Zhang R. Dunker R. Scott D.L. Joachimiak A. Goodman S. Arnaout M.A. Science. 2001; 294: 339-345Crossref PubMed Scopus (1118) Google Scholar), and retains the ligand-binding properties of the full-length receptor (16Coe A.P. Askari J.A. Kline A.D. Robinson M.K. Kirby H. Stephens P.E. Humphries M.J. J. Biol. Chem. 2001; 276: 35854-35866Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The advantages of this system have been described previously (6Mould A.P. Askari J.A. Barton S. Kline A.D. McEwan P.A. Craig S.E. Humphries M.J. J. Biol. Chem. 2002; 277: 19800-19806Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 9Mould A.P. Barton S.J. Askari J.A. McEwan P.A. Buckley P.A. Craig S.E. Humphries M.J. J. Biol. Chem. 2003; 278: 17028-17035Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). 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 (16Coe A.P. Askari J.A. Kline A.D. Robinson M.K. Kirby H. Stephens P.E. Humphries M.J. J. Biol. Chem. 2001; 276: 35854-35866Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Wild-type trα5β1-Fc had low activity when Fc captured but had high activity after direct adsorption (Fig. 1, A and B); the activating mAb 12G10 (15Mould A.P. Askari J.A. Aota S. Yamada K.M. Irie A. Takada Y. Mardon H.J. Humphries M.J. J. Biol. Chem. 1997; 272: 17283-17292Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) 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). The D137A and D138A mutations were also made in a construct containing the full-length extracellular domains of α5 and β1 (hereafter referred to as α5β1-Fc). The mutations had little or no effect on the expression of α and β subunit epitopes, apart from low expression of the 15/7 activation epitope in the D138A mutant (data not shown). After Fc capture, the wild-type α5β1-Fc had constitutively high ligand binding activity but the mutants displayed little or no activity (Fig. 2). Activating mAbs TS2/16 and 12G10 had little effect on the ligand binding activity of the wild-type receptor but partially (D137A) or completely (D138A) restored the activity of the ADMIDAS mutants (Fig. 2). These results show that under conditions where the wild-type receptor is fully active (either constitutively or after mAb- or coating-induced activation), the ADMIDAS mutants have low activity. Because activity can be rescued (at least in part) by activating mAbs, the ADMIDAS mutants are not defective in ligand binding but instead the mutations appear to inhibit ligand binding by inactivating the receptor (i.e. shifting the equilibrium toward the inactive state). Consistent with this interpretation, the D137A and D138A trα5β1-Fc mutants showed decreased expression of activation epitopes, such as that recognized by mAb 12G10 (Table II). Interestingly, Asp137 and Asp138 lie at the top of the α1 helix of βA and we have previously shown that a movement of this helix is important for activation (6Mould A.P. Askari J.A. Barton S. Kline A.D. McEwan P.A. Craig S.E. Humphries M.J. J. Biol. Chem. 2002; 277: 19800-19806Abst
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