Integrin Activation Involves a Conformational Change in the α1 Helix of the β Subunit A-domain
2002; Elsevier BV; Volume: 277; Issue: 22 Linguagem: Inglês
10.1074/jbc.m201571200
ISSN1083-351X
AutoresA. Paul Mould, Janet A. Askari, Stephanie Barton, Adam Kline, Paul McEwan, Susan E. Craig, Martin J. Humphries,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoThe ligand-binding region of integrin β subunits contains a von Willebrand factor type A-domain: an α/β "Rossmann" fold containing a metal ion-dependent adhesion site (MIDAS) on its top face. Although there is evidence to suggest that the βA-domain undergoes changes in tertiary structure during receptor activation, the identity of the secondary structure elements that change position is unknown. The mAb 12G10 recognizes a unique cation-regulated epitope on the β1 A-domain, induction of which parallels the activation state of the integrin (i.e. competency for ligand recognition). The ability of Mn2+ and Mg2+ to stimulate 12G10 binding is abrogated by mutation of the MIDAS motif, demonstrating that the MIDAS is a Mn2+/Mg2+ binding site and that occupancy of this site induces conformational changes in the A-domain. The cation-regulated region of the 12G10 epitope maps to Arg154/Arg155 in the α1 helix. Our results demonstrate that the α1 helix undergoes conformational alterations during integrin activation and suggest that Mn2+ acts as a potent activator of β1integrins because it can promote a shift in the position of this helix. The mechanism of β subunit A-domain activation appears to be distinct from that of the A-domains found in some integrin α subunits. The ligand-binding region of integrin β subunits contains a von Willebrand factor type A-domain: an α/β "Rossmann" fold containing a metal ion-dependent adhesion site (MIDAS) on its top face. Although there is evidence to suggest that the βA-domain undergoes changes in tertiary structure during receptor activation, the identity of the secondary structure elements that change position is unknown. The mAb 12G10 recognizes a unique cation-regulated epitope on the β1 A-domain, induction of which parallels the activation state of the integrin (i.e. competency for ligand recognition). The ability of Mn2+ and Mg2+ to stimulate 12G10 binding is abrogated by mutation of the MIDAS motif, demonstrating that the MIDAS is a Mn2+/Mg2+ binding site and that occupancy of this site induces conformational changes in the A-domain. The cation-regulated region of the 12G10 epitope maps to Arg154/Arg155 in the α1 helix. Our results demonstrate that the α1 helix undergoes conformational alterations during integrin activation and suggest that Mn2+ acts as a potent activator of β1integrins because it can promote a shift in the position of this helix. The mechanism of β subunit A-domain activation appears to be distinct from that of the A-domains found in some integrin α subunits. Integrins are α/β heterodimeric transmembrane receptors that have widespread essential functions in development, tissue organization, and the immune system (1Sheppard D. Matrix Biol. 2000; 19: 203-209Crossref PubMed Scopus (120) Google Scholar). Integrins recognize a variety of extracellular matrix and cell-surface ligands; however, ligand recognition is frequently not constitutive but is instead under strict cellular control by "inside-out" signaling. Acquisition of the active state has also been shown to require divalent cations. For β1 integrins, ligand binding is promoted by Mg2+ or Mn2+ but only weakly by Ca2+ (2Mould A.P. J. Cell Sci. 1996; 109: 2613-2618Crossref PubMed Google Scholar). A well known but unexplained property of Mn2+ is its ability to mimic the process of inside-out signaling to strongly up-regulate integrin function (3Dransfield I. Cabanas C. Craig A. Hogg N. J. Cell Biol. 1992; 116: 219-226Crossref PubMed Scopus (399) Google Scholar, 4Lu C. Shimoka M. Zang Q. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2393-2398Crossref PubMed Scopus (170) Google Scholar). The molecular basis of integrin-ligand interactions has been greatly elucidated by the recent x-ray crystal structure of αVβ3 (5Xiong 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 (1100) Google Scholar). The ligand binding "head" of the integrin is seen to contain a seven-bladed β-propeller fold in the α subunit and a von Willebrand factor type A-domain in the β subunit (βA-domain). 1The abbreviations used are: βA-domainβ subunit von Willebrand factor A-domainαA-domainα subunit von Willebrand factor A-domainMIDASmetal ion-dependent adhesion sitemAbmonoclonal antibodyPBSphosphate-buffered salineBSAbovine serum albuminELISAenzyme-linked immunosorbent assaytrα5β1-Fcrecombinant soluble integrin heterodimer containing C-terminally truncated α5and β1 subunits (α5 residues 1–613 and β1 residues 1–455) fused to the Fc region of human IgGγ1CHOChinese hamster ovary1The abbreviations used are: βA-domainβ subunit von Willebrand factor A-domainαA-domainα subunit von Willebrand factor A-domainMIDASmetal ion-dependent adhesion sitemAbmonoclonal antibodyPBSphosphate-buffered salineBSAbovine serum albuminELISAenzyme-linked immunosorbent assaytrα5β1-Fcrecombinant soluble integrin heterodimer containing C-terminally truncated α5and β1 subunits (α5 residues 1–613 and β1 residues 1–455) fused to the Fc region of human IgGγ1CHOChinese hamster ovary Cation-binding sites are present on the lower face of the β-propeller domain and the upper face of the βA-domain (5Xiong 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 (1100) Google Scholar). The key regions involved in ligand recognition are loops on the upper surface of the β-propeller and the upper face of the βA-domain, which contains a metal ion-dependent adhesion site (MIDAS) (5Xiong 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 (1100) Google Scholar, 6Lee O.-J. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (798) Google Scholar, 7Humphries M.J. Biochem. Soc. Trans. 2000; 28: 311-339Crossref PubMed Google Scholar). Nevertheless, as the crystal structure only provides a "snapshot" of one integrin conformation, attention is now focused on understanding the conformational changes that occur during the transition from the inactive to active state (8Humphries M.J. Mould A.P. Science. 2001; 294: 316-317Crossref PubMed Scopus (18) Google Scholar). These changes are thought to include shape shifting in the βA-domain (4Lu C. Shimoka M. Zang Q. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2393-2398Crossref PubMed Scopus (170) Google Scholar, 7Humphries M.J. Biochem. Soc. Trans. 2000; 28: 311-339Crossref PubMed Google Scholar). β subunit von Willebrand factor A-domain α subunit von Willebrand factor A-domain metal ion-dependent adhesion site monoclonal antibody phosphate-buffered saline bovine serum albumin enzyme-linked immunosorbent assay recombinant soluble integrin heterodimer containing C-terminally truncated α5and β1 subunits (α5 residues 1–613 and β1 residues 1–455) fused to the Fc region of human IgGγ1 Chinese hamster ovary β subunit von Willebrand factor A-domain α subunit von Willebrand factor A-domain metal ion-dependent adhesion site monoclonal antibody phosphate-buffered saline bovine serum albumin enzyme-linked immunosorbent assay recombinant soluble integrin heterodimer containing C-terminally truncated α5and β1 subunits (α5 residues 1–613 and β1 residues 1–455) fused to the Fc region of human IgGγ1 Chinese hamster ovary The A-domain contains a central hydrophobic β sheet encircled by seven α helices (α1–α7) (5Xiong 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 (1100) Google Scholar). Some α subunits also contain an A-domain and a key feature of the activation of these domains has been shown to be a large movement of the α7 helix (9Lee O.-J. Bankston L.A. Arnaout M.A. Liddington R. Structure. 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). Here we investigate conformational changes in the βA-domain using the anti-β1 mAb 12G10, which recognizes a cation- and ligand-induced epitope (10Mould A.P. Garratt A.N. Askari J.A. Akiyama S.K. Humphries M.J. FEBS Lett. 1995; 363: 118-122Crossref PubMed Scopus (123) Google Scholar, 11Mould A.P. Garratt A.N. Puzon-McLaughlin W. Takada Y. Humphries M.J. Biochem. J. 1998; 331: 821-828Crossref PubMed Scopus (93) Google Scholar). We show that movements in the α1 helix of the βA-domain parallel changes in the activation state of the integrin. Our results provide insights into the mechanisms of Mn2+ and Ca2+-induced shape changes in the β1 subunit, and therefore into the opposing roles played by these divalent ions in regulating integrin function. Our findings also imply that the mechanism of βA-domain activation is different to that of αA-domains. Rat mAbs 16 and 13 recognizing the human α5 and β1 subunits, respectively, were gifts from Dr. K. Yamada (NIDCR, National Institutes of Health, Bethesda, MD). 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 mAb 12G10 were produced as described (10Mould A.P. Garratt A.N. Askari J.A. Akiyama S.K. Humphries M.J. FEBS Lett. 1995; 363: 118-122Crossref PubMed Scopus (123) Google Scholar, 12Burrows L. Clark K. Mould A.P. Humphries M.J. Biochem. J. 1999; 344: 527-533Crossref PubMed Scopus (51) Google Scholar). Mouse anti-human mAb TS2/16 was a gift from F. Sánchez-Madrid (Hospital de la Princesa, Madrid, Spain). Mouse anti-human mAbs 4B4 and P4C10 were purchased from Beckman Coulter (High Wycombe, UK) and Invitrogen (Paisley, Scotland, UK), respectively. All mAbs were used as purified IgG except P4C10 (as ascites). C-terminally 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 were generated as previously described (13Coe A.P.F. 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 heterodimerization, the CH3 domain of the α5 construct contained a "hole" mutation, whereas the CH3 domain of the β1 construct carried a "knob" mutation as described (13Coe A.P.F. 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, 14Ridgway J.B. Presta L.G. Carter P. Protein Eng. 1996; 9: 617-621Crossref PubMed Scopus (492) Google Scholar). Mutations in the A-domain of the β1 subunit were carried out using oligonucleotide-directed PCR mutagenesis, as described (13Coe A.P.F. 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). Oligonucleotides were purchased from MWG Biotech (Milton Keynes, UK). The presence of the mutations was verified by DNA sequencing. Chinese hamster ovary cells L761h variant (13Coe A.P.F. 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% nonessential amino acids (growth medium). Cells were detached using 0.05% (w/v) trypsin, 0.02% (w/v) EDTA in PBS, and plated overnight into six-well culture plates (Costar). 1 μg of wild-type or mutant β1-(1–455)-Fc, and 1 μg of α5-(1–613)-Fc DNA/well was used to transfect the cells using LipofectAMINE Plus reagent (Invitrogen) according to the manufacturer's instructions. After 4 days, medium was harvested by centrifugation at 1000 × g for 5 min. For comparison of wild-type trα5β1-Fc with trα5β1-Fc containing the D130A or R154R/AS mutations in β1, 75-cm2 flasks of subconfluent CHOL761h cells were transfected with 5 μg of wild-type or mutant β1-(1–455)-Fc and 5 μg of α5-(1–613)-Fc DNA 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 previously (13Coe A.P.F. 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). A recombinant fragment of fibronectin containing type III repeats 6–10 (III6–10) 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). III6–10 and mAb 12G10 were biotinylated as before (11Mould A.P. Garratt A.N. Puzon-McLaughlin W. Takada Y. Humphries M.J. Biochem. J. 1998; 331: 821-828Crossref PubMed Scopus (93) Google Scholar, 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), except that sulfo-LC-NHS biotin (Perbio, Chester, UK) was used in place of sulfo-NHS biotin. Purified integrin was diluted to approximately 1 μg/ml in Dulbecco's PBS and added to the wells of a half-area enzyme immunoassay/radio immunoassay plate (Costar, Corning Science Products, High Wycombe, UK; 25 μl/well) for 16 h at room temperature. Wells were blocked for 1–3 h with 200 μl of 5% (w/v) BSA, 150 mm NaCl, 0.05% (w/v) NaN3, 25 mmTris-Cl, pH 7.4 (blocking buffer). Wells were then washed three times with 200 μl of 150 mm NaCl, 25 mm Tris-Cl, pH 7.4, containing 1 mg/ml BSA (buffer A). Buffer A was treated with Chelex beads (Bio-Rad, Hemel Hempstead, UK) to remove any small contaminating amounts of endogenous Ca2+ and Mg2+ ions. 12G10 (0.1 μg/ml) in buffer A with varying concentrations of Mn2+, Mg2+, or 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 1:500 dilution of ExtrAvidin® peroxidase conjugate (Sigma, Poole, UK) in buffer A for 20 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). Background binding to BSA was subtracted from all measurements. Measurements obtained were the mean ± S.D. of four replicate wells. For comparison of the effects of divalent cations on 12G10 binding to wild-type trα5β1-Fc and the R154R/AS mutant, the assay was performed using 12G10 concentrations that gave approximately half-maximal antibody binding in 1 mmMn2+ (0.1 μg/ml for wild-type trα5β1-Fc, 10 μg/ml for R154R/AS mutant). Binding was measured in 2 mm EDTA, Mn2+, Mg2+, or Ca2+. Measurements obtained were the mean ± S.D. of four replicate wells. Measurement of the binding of III6–10 to purified wild-type or mutant trα5β1-Fc was performed exactly as described for biotinylated 12G10 (see above), except that biotinylated III6–10 was incubated with integrin for 3 h at 30 °C. All assays were performed using a concentration of biotinylated III6–10 that gave approximately half-maximal ligand binding in 1 mmMn2+ (0.1 μg/ml). A 96-well plate (Costar half-area enzyme immunoassay/radio immunoassay) was coated with goat anti-human γ1 Fc (Jackson Immunochemicals, Stratech Scientific, Luton, UK) at a concentration of 2.6 μg/ml in Dulbecco's PBS (50 μl/well) for 16 h. The coating solution was replaced with blocking buffer for 1 h. The blocking solution was removed, and cell culture supernatants were added (25 μl/well) for 1 h. All supernatants were assayed in triplicate, and supernatant from mock-transfected cells was used as a negative control. The plate was washed three times in buffer A containing 1 mm MnCl2 (buffer B; 200 μl/well), and anti-α5 or anti-β1 mAbs (10 μg/ml, or 1 μg/ml for SNAKA52) were added (50 μl/well). The plate was incubated for 2 h and then washed three 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 washed four times in buffer B, and color was developed using 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) substrate (50 μl/well). All steps were performed at room temperature. Each experiment shown is representative of at least three separate experiments. To investigate the mechanisms of integrin activation, we employed a recently described system for expression of recombinant soluble α5β1 (13Coe A.P.F. 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). For these particular studies, we have used a truncated version of α5β1, α5-(1–613)β1-(1–455), fused to the Fc region of human IgGγ1 (hereafter referred to as trα5β1-Fc). This heterodimer contains the ligand-binding head and thigh domains of the integrin (5Xiong 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 (1100) Google Scholar) and has been shown to retain the properties of the full-length receptor (13Coe A.P.F. 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 contrast to previous mutagenesis-based analyses of integrin function, which have largely employed cell-expressed integrins, this system is ideal (a) because it permits the rapid analysis of the effects of mutations and (b) because the effects of mutations that normally preclude expression at the plasma membrane can be studied. 12G10 is a previously characterized activating mAb directed against the β1 A-domain, whose binding to α5β1 is modulated by divalent cations and ligand (10Mould A.P. Garratt A.N. Askari J.A. Akiyama S.K. Humphries M.J. FEBS Lett. 1995; 363: 118-122Crossref PubMed Scopus (123) Google Scholar, 11Mould A.P. Garratt A.N. Puzon-McLaughlin W. Takada Y. Humphries M.J. Biochem. J. 1998; 331: 821-828Crossref PubMed Scopus (93) Google Scholar). The binding of 12G10 to trα5β1-Fc was promoted by Mn2+and to a lesser degree by Mg2+, whereas Ca2+was inhibitory (Fig. 1A). The effects of these cations on 12G10 binding closely paralleled their effects on ligand binding (Fig. 1B). Importantly, as for the native integrin, ligand binding is strongly activated by Mn2+ and more weakly by Mg2+, whereas Ca2+ is a very poor activator (16Mould A.P. Akiyama S.K. Humphries M.J. J. Biol. Chem. 1995; 270: 26270-26277Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). These results show that the βA-domain undergoes conformational changes in response to cation binding (reported by modulation of the 12G10 epitope) that correspond with changes in the activation state of the integrin. The identity of the cation-binding site(s) involved in activation of β1integrins by Mn2+ and Mg2+ is unknown. The MIDAS is a strong candidate for this site, but this has been difficult to test experimentally because mutation of the MIDAS residues completely abrogates ligand recognition (although expression is unaffected; Refs. 17Takada Y. Ylanne J. Mandelman D. Puzon W. Ginsberg M.H. J. Cell Biol. 1992; 119: 913-921Crossref PubMed Scopus (98) Google Scholar, 18Kamata T. Puzon W. Takada Y. Biochem. J. 1995; 305: 945-954Crossref PubMed Scopus (124) Google Scholar, 19Puzon-McLaughlin W. Takada Y. J. Biol. Chem. 1996; 271: 20438-20443Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). In agreement with these previous studies, trα5β1-Fc with MIDAS mutations did not bind ligand under any cation conditions, even though such mutants (e.g. D130A) retained all the epitopes of conformation-sensitive α5 and β1 mAbs (Table I). Because mAb binding was retained, we tested the effect of the D130A mutation on the ability of divalent cations to regulate 12G10 binding to trα5β1-Fc (Fig. 2). The binding of 12G10 to the D130A mutant in the absence of divalent cations was similar to the wild-type integrin (comparing Fig. 1A with Fig. 2); however, the ability of Mn2+ and Mg2+ to stimulate 12G10 binding was totally lost in the MIDAS mutant. Interestingly, the inhibition of 12G10 binding by Ca2+ seen for the wild-type integrin was enhanced in the MIDAS mutant. Similar results were obtained with a "double" MIDAS mutation D130A/S132A (data not shown). Conversely, mutation of cation-binding sites in the α5 subunit β-propeller did not affect the capacity of Mn2+ or Mg2+ to modulate 12G10 binding (20Kline A.D. Analysis of Integrin Activation and Divalent Cation Binding by Site-directed Mutagenesis. Ph.D. thesis. University of Manchester, Manchester, United Kingdom2001Google Scholar). Hence, these data demonstrate that the MIDAS is a Mn2+/Mg2+-binding site and that occupancy of this site induces conformational movements that are detected by changes in 12G10 binding.Table ISummary of mAb reactivity with β1 A-domain mutantsβ1mutantAnti-α5Anti-β1P1D616SNAKA5213TS2/16P4C104B412G10D130A+++++++++++++++++++++++N151A++++++++++++++++++++++++M153A++++++++++++++++++++++++R154A++++++++++++++++++++++R155A+++++++++++++++++++++++I156A++++++++++++++++++++++++R154R/AS++++++++++++++++++++++/−CHO L761h cells were transfected with α5-(1–613)-Fc and wild-type or mutant β1-(1–455)-Fc. Cell culture supernatants were analyzed for reactivity with anti-α5 and anti-β1 mAbs by sandwich ELISA. The anti-α5 mAbs recognize the β-propeller domain (12Burrows L. Clark K. Mould A.P. Humphries M.J. Biochem. J. 1999; 344: 527-533Crossref PubMed Scopus (51) Google Scholar), and the anti-β1 mAbs are all directed against the βA-domain (11Mould A.P. Garratt A.N. Puzon-McLaughlin W. Takada Y. Humphries M.J. Biochem. J. 1998; 331: 821-828Crossref PubMed Scopus (93) Google Scholar, 21Takada Y. Puzon W. J. Biol. Chem. 1993; 268: 17597-17601Abstract Full Text PDF PubMed Google Scholar). +++, reactivity 70–100% of wild-type integrin; ++, reactivity 50–70% of wild-type integrin; +, reactivity 20–50% of wild-type integrin; +/−, reactivity < 20% of wild-type integrin. None of the mutations (except D130A) affected recognition of the III6–10 fragment of fibronectin (data not shown). Open table in a new tab CHO L761h cells were transfected with α5-(1–613)-Fc and wild-type or mutant β1-(1–455)-Fc. Cell culture supernatants were analyzed for reactivity with anti-α5 and anti-β1 mAbs by sandwich ELISA. The anti-α5 mAbs recognize the β-propeller domain (12Burrows L. Clark K. Mould A.P. Humphries M.J. Biochem. J. 1999; 344: 527-533Crossref PubMed Scopus (51) Google Scholar), and the anti-β1 mAbs are all directed against the βA-domain (11Mould A.P. Garratt A.N. Puzon-McLaughlin W. Takada Y. Humphries M.J. Biochem. J. 1998; 331: 821-828Crossref PubMed Scopus (93) Google Scholar, 21Takada Y. Puzon W. J. Biol. Chem. 1993; 268: 17597-17601Abstract Full Text PDF PubMed Google Scholar). +++, reactivity 70–100% of wild-type integrin; ++, reactivity 50–70% of wild-type integrin; +, reactivity 20–50% of wild-type integrin; +/−, reactivity < 20% of wild-type integrin. None of the mutations (except D130A) affected recognition of the III6–10 fragment of fibronectin (data not shown). The epitopes of all function-altering mAbs that map to the β1 A-domain include one or more residues in the sequence Asn207–Lys218 (21Takada Y. Puzon W. J. Biol. Chem. 1993; 268: 17597-17601Abstract Full Text PDF PubMed Google Scholar), which based on homology to β3 is predicted to form the α2 helix (5Xiong 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 (1100) Google Scholar,22Tuckwell D.S. Humphries M.J. FEBS Lett. 1997; 400: 297-303Crossref PubMed Scopus (99) Google Scholar). The epitope of 12G10 also maps to this region and includes Lys218 as part of its epitope (11Mould A.P. Garratt A.N. Puzon-McLaughlin W. Takada Y. Humphries M.J. Biochem. J. 1998; 331: 821-828Crossref PubMed Scopus (93) Google Scholar); however, among these regulatory mAbs, 12G10 has the unique property of showing strong cation modulation of binding (11Mould A.P. Garratt A.N. Puzon-McLaughlin W. Takada Y. Humphries M.J. Biochem. J. 1998; 331: 821-828Crossref PubMed Scopus (93) Google Scholar). Therefore, part of the 12G10 epitope may be distinct from that of the other βA-domain mAbs. While investigating the mechanism of integrin activation using alanine-scanning mutagenesis, 2A. P. Mould, J. A. Askari, S. Barton, and M. J. Humphries, manuscript in preparation. we found two mutations that selectively perturbed 12G10 binding (Table I). Mutation of Arg154 or Arg155 to Ala reduced 12G10 binding by ∼65 and ∼35%, respectively, whereas mutation of surrounding residues (Asn151, Met153, Ile156) had no effect. A double mutation R154R/AS reduced 12G10 binding by >80% but did not perturb the binding of other function-modulating mAbs against the β1 A-domain (Table I), and also had no effect on the apparent affinity of ligand binding (data not shown). Therefore, Arg154 and Arg155appear to form part of the 12G10 epitope. 3In further support of this proposal, 12G10 reacts only weakly with a chimeric β1 containing Asn207–Lys218 of human β1 in a backbone of chicken β1 (CH mutant; Ref. 21Takada Y. Puzon W. J. Biol. Chem. 1993; 268: 17597-17601Abstract Full Text PDF PubMed Google Scholar), in contrast to the other function-modulating mAbs, which show good reactivity. In chicken β1 Arg154 and Arg155 are both altered to Glu and Lys, respectively, whereas other residues in this region are unchanged (W. Puzon-McLaughlin, Y. Takada, A. P. Mould, and M. J. Humphries, unpublished observations). By homology to the structure of the β3 A-domain (5Xiong 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 (1100) Google Scholar), Arg154/Arg155 lie at the base of the α1 helix and these residues would be predicted to be in sufficiently close proximity to Lys218 in the α2 helix for all three residues to contribute to the 12G10 epitope (23Davies D.R. Cohen G.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7-12Crossref PubMed Scopus (480) Google Scholar). The above data suggest that Arg154 and Arg155form part of the 12G10 epitope, but these residues do not contribute to other A-domain epitopes. Hence, do Arg154/Arg155 form the cation-regulated region of the 12G10 epitope? To test this proposal, we compared the effects of divalent cations on 12G10 binding to the R154R/AS mutant and wild-type trα5β1-Fc. The results (Fig. 3A) showed that the abilities of Mn2+, Mg2+, and Ca2+ to modulate 12G10 binding were strongly attenuated by the R154R/AS mutation. The mutation did not affect the cation regulation of ligand binding (Fig. 3B) or of α5 epitopes (Ref. 11Mould A.P. Garratt A.N. Puzon-McLaughlin W. Takada Y. Humphries M.J. Biochem. J. 1998; 331: 821-828Crossref PubMed Scopus (93) Google Scholar; data not shown), suggesting that the mutation does not itself affect cation-induced conformational changes but rather that the ability of 12G10 to detect these changes is specifically compromised by the mutation. Therefore, the portion of the 12G10 epitope that is responsive to cation binding lies in the α1 helix, indicating that the position of this helix is different in the active and inactive states. Using the anti-β1 mAb 12G10 as a probe of βA-domain conformation, we have shown that: (i) the βA-domain undergoes shape changes that correlate with changes in the activation state of the integrin, (ii) occupancy of the MIDAS site in the βA-domain by Mn2+ or Mg2+ induces these changes, and (iii) βA-domain activation involves movement of the α1 helix. Taking these results together, we propose that the βA-domain can exist in at least two conformational states: an "active" conformation with the α1 helix in a position characterized by high 12G10 binding and an "inactive" conformation with the α1 helix in a different position, characterized by low 12G10 binding. Movement of the α1 helix appears to form an essential part of the activation mechanism of the βA-domain because α1 movement closely parallels the activation state and a lack of α1 movement (in the Ca2+-occupied integrin) corresponds to low activity. Furthermore, the epitopes of function-blocking anti-chicken β1 mAbs have been shown to include residues in the α1 helix (24Shi D.T. Boetigger D. Buck C.A. J. Cell Sci. 1997; 110: 2619-2628PubMed Google Scholar), and the epitopes of function-altering anti-human β1 mAbs include residues in the α2 helix, which lies adjacent to α1 (5Xiong 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 (1100) Google Scholar, 21Takada Y. Puzon W. J. Biol. Chem. 1993; 268: 17597-17601Abstract Full Text PDF PubMed Google Scholar). Based on previous analyses of the mode of action of regulatory anti-integrin mAbs (2Mould A.P. J. Cell Sci. 1996; 109: 2613-2618Crossref PubMed Google Scholar, 25Mould A.P. Akiyama S.K. Humphries M.J. J. Biol. Chem. 1996; 271: 20365-20374Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), it appears that they are likely to function allosterically by stabilizing the position of α1 in either the active or inactive conformation. Additionally, it has been shown that mutation of residues in the α1 helix can activate ligand binding (26Bajt M.L. Loftus J.C. Gawaz M.P. Ginsberg M.H. J. Biol. Chem. 1992; 267: 22211-22216Abstract Full Text PDF PubMed Google Scholar). Our data provide evidence that the MIDAS is primarily a Mn2+/Mg2+ binding site and suggest an explanation for the opposing effects of Mn2+ and Ca2+ on β1 integrin function. Mn2+ can induce a large shift in the equilibrium between active and inactive states because of its ability to promote α1 helix movement upon binding to the MIDAS site. On the other hand, Ca2+ is unable to cause the same conformational change. It is likely that Ca2+ can occupy the MIDAS site because Ca2+ can support low affinity ligand binding to α5β1 and high affinity binding of activation-independent ligands to α4β1(34Chen L.L. Whitty A. Scott D. Lee W.C. Cornebise M. Adams S.P. Petter R.C. Lobb R.R. Pepinsky R.B. J. Biol. Chem. 2001; 276: 36520-36529Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). However, Ca2+ binding to sites other than the MIDAS appears to shift the equilibrium toward the inactive conformation (low 12G10 binding), as shown by the strong inhibition of 12G10 binding by Ca2+ in the D130A mutant. Some integrins contain an A-domain in their α subunits (e.g. the β2 family). These domains can exist in inactive ("closed") or active ("open") states dependent upon movement of the C-terminal helix (α7). The open form can be induced in the presence of a ligand or pseudo-ligand, or by locking the position of α7 (9Lee O.-J. Bankston L.A. Arnaout M.A. Liddington R. Structure. 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 27Emsley J. Knight G.C. Farndale R. Barnes M.J. Liddington R.C. Cell. 2000; 100: 47-56Abstract Full Text Full Text PDF Scopus (831) Google Scholar, 28Xiong J.-P., Li, R. Essafi M. Stehle T. Arnaout M.A. J. Biol. Chem. 2000; 275: 38762-38767Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 29Shimoaka M., Lu C. Palframan R.T. von Andrian U.H. McCormack A. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6009-6014Crossref PubMed Scopus (189) Google Scholar). There appear to be some differences between the activation mechanism of αA-domains and the βA-domain. First, the nature of the metal ion at the MIDAS does not directly influence the equilibrium between inactive and active states in αA-domains (4Lu C. Shimoka M. Zang Q. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2393-2398Crossref PubMed Scopus (170) Google Scholar, 28Xiong J.-P., Li, R. Essafi M. Stehle T. Arnaout M.A. J. Biol. Chem. 2000; 275: 38762-38767Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), whereas, based on data reported here for the βA-domain, the nature of the divalent ion can markedly affect this equilibrium. Second, in contrast to the βA-domain, there is no evidence for allosteric regulation of activity by mAbs to αA-domains whose epitopes include residues in the α1 helix (30Oxvig C., Lu, C. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2215-2220Crossref PubMed Scopus (122) Google Scholar). Third, although in αA-domains the open form can be induced by mutation of residues that form a hydrophobic pocket surrounding α7 (31Huth J.R. Olejniczak E.T. Mendoza R. Liang H. Harris E.A.S. Lupher M.L., Jr. Wilson A.E. Fesik S.W. Staunton D.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5231-5236Crossref PubMed Scopus (142) Google Scholar), mutation of the equivalent residues in β1 does not alter integrin activity.2 Fourth, the crystal structure of the β3 A-domain indicates that the α7 helix is unlikely to undergo large conformational movements (5Xiong 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 (1100) Google Scholar). All these findings suggest that the βA-domain is regulated differently to the αA-domains in that movement of the α1 helix (rather than α7) is a key feature of βA-domain activation. Nevertheless, comparison of the open and closed forms of αA-domains shows that there is an inward shift of the α1 helix in the open form (9Lee O.-J. Bankston L.A. Arnaout M.A. Liddington R. Structure. 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar), and a similar movement could take place in the βA-domain (Fig. 4). In integrins that contain an A-domain in the α subunit, the βA-domain does not participate directly in ligand binding (4Lu C. Shimoka M. Zang Q. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2393-2398Crossref PubMed Scopus (170) Google Scholar). Nevertheless, Mn2+ and mAbs to the βA-domain can strongly modulate the activity these integrins (3Dransfield I. Cabanas C. Craig A. Hogg N. J. Cell Biol. 1992; 116: 219-226Crossref PubMed Scopus (399) Google Scholar, 4Lu C. Shimoka M. Zang Q. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2393-2398Crossref PubMed Scopus (170) Google Scholar). The epitopes of activating and inhibitory anti-β2 mAbs have also been shown to contain residues in the α1 helix of the β2A-domain (4Lu C. Shimoka M. Zang Q. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2393-2398Crossref PubMed Scopus (170) Google Scholar, 32Huang C. Zang Q. Takagi J. Springer T.A. J. Biol. Chem. 2000; 275: 21514-21524Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 33Xiong Y.-M. Zhang L. J. Biol. Chem. 2001; 276: 19340-19349Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Therefore, movement of the α1 helix may also regulate the activation state of this class of integrin. Is α1 helix movement involved in the activation of integrins by inside-out signaling? It has been shown that the expression of the 12G10 epitope correlates with the activity of cell-surface β1 integrins, whereas expression of other β1 A-domain epitopes is constitutive (35Belkin A.M Retta S.F. Pletjushkina O.Y. Balzac F. Silengo L. Fassler R. Koteliansky V.E. Burridge K. Tarone G. J. Cell Biol. 1997; 139: 1583-1595Crossref PubMed Scopus (109) Google Scholar, 36Mastrangelo A.M. Homan S.M. Humphries M.J. LaFlamme S. J. Cell Sci. 1998; 112: 217-229Google Scholar). Because 12G10 differs from the other A-domain mAbs in having part of its epitope in the α1 helix, these data imply that inside-out signaling alters the position of α1. Inside-out signaling may also cause a shift in the conformation of the α1 helix in β2integrins. For αLβ2 and αMβ2, activating cytoplasmic domain mutations led to the induction of the mAb 24 epitope, which includes Arg122 in the α1 helix of the β2 A-domain (4Lu C. Shimoka M. Zang Q. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2393-2398Crossref PubMed Scopus (170) Google Scholar, 37Lu C. Takagi J. Springer T.A. J. Biol. Chem. 2001; 276: 14642-14648Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). It has often been questioned whether Mn2+-induced integrin activation accurately mimics physiologic activation. However, a common feature of both types of activation appears to be movement of the α1 helix; hence, their molecular mechanisms may be very similar. Finally, how might α1 helix movement be important for activation? The top (MIDAS) face of the βA-domain interacts closely with the upper surface of the α subunit β-propeller domain (5Xiong 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 (1100) Google Scholar). In particular, loops on the top face of the A-domain close to the α1 helix (notably the α2-α3 loop) contact loops on the β-propeller domain that participate in ligand recognition. Hence, α1 helix movement is likely to affect the α subunit/β subunit interface, potentially leading to changes in exposure of the ligand binding loops. In support of this hypothesis, we have shown that divalent cations affect the binding of inhibitory mAbs on the α subunit (11Mould A.P. Garratt A.N. Puzon-McLaughlin W. Takada Y. Humphries M.J. Biochem. J. 1998; 331: 821-828Crossref PubMed Scopus (93) Google Scholar); the epitopes of these mAbs include residues in the same loops that are important for ligand recognition (12Burrows L. Clark K. Mould A.P. Humphries M.J. Biochem. J. 1999; 344: 527-533Crossref PubMed Scopus (51) Google Scholar, 38Mould A.P. Askari J.A. Humphries M.J. J. Biol. Chem. 2000; 275: 20324-20336Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 39Humphries J.D. Askari J.A. Zhang X.-P. Takada Y. Humphries M.J. Mould A.P. J. Biol. Chem. 2000; 275: 20337-20345Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In integrins with an A-domain in the α subunit, there is evidence that the MIDAS face of the βA-domain is in contact with the lower face of the αA-domain (40Zang Q., Lu, C. Huang C. Takagi J. Springer T.A. J. Biol. Chem. 2001; 275: 22202-22212Abstract Full Text Full Text PDF Scopus (30) Google Scholar); hence, conformational changes in the βA-domain could affect the position of the α7 helix in the αA-domain and thereby alter the activation state of this domain. In summary, we have shown that a conformational shift in the α1 helix of the βA-domain is involved the regulation of integrin activity. Integrins are important therapeutic targets in many inflammatory and vascular disorders (41Curley G.P. Blum H. Humphries M.J. Cell. Mol. Life Sci. 1999; 56: 427-441Crossref PubMed Scopus (107) Google Scholar), and our findings suggest a novel way in which highly specific regulators of integrin activity could be developed. A more complete understanding of the activation mechanism will require crystallization of an integrin in both active and inactive states. After this manuscript was accepted for publication, the crystal structure of integrin αvβ3 in complex with an RGD ligand was reported (Xiong, K.-P., Stehle, T., Zhang, R., Joachimiak, A., Frech, M., Goodman, S., and Arnaout, M. A. (2002) Science 296, 151–155). The pivotal conformational change between the unliganded (5Xiong 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 (1100) Google Scholar) and liganded structures is an inward movement of the α1 helix in the βA-domain. This conformational change appears to be causally linked to occupancy of the MIDAS site by a Mn2+ion in the liganded structure. We thank A. Coe, P. Stephens, and M. Robinson for the α5 and β1 constructs. We are grateful to K. Yamada, E. Wayner, and F. Sánchez-Madrid for mAbs, K. Yamada and S. Aota for the III6–10 construct, A. Arnaout for the coordinates of the αvβ3structure, and J. Bella for advice on molecular modeling.
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