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Conformational Changes in the Integrin औA Domain Provide a Mechanism for Signal Transduction via Hybrid Domain Movement

2003; Elsevier BV; Volume: 278; Issue: 19 Linguagem: Inglês

10.1074/jbc.m213139200

ISSN

1083-351X

Autores

A. Paul Mould, Stephanie Barton, Janet A. Askari, Paul McEwan, Patrick A. Buckley, Susan E. Craig, Martin J. Humphries,

Tópico(s)

Monoclonal and Polyclonal Antibodies Research

Resumo

The ligand-binding head region of integrin औ subunits contains a von Willebrand factor type A domain (औA). Ligand binding activity is regulated through conformational changes in औA, and ligand recognition also causes conformational changes that are transduced from this domain. The molecular basis of signal transduction to and from औA is uncertain. The epitopes of mAbs 15/7 and HUTS-4 lie in the औ1 subunit hybrid domain, which is connected to the lower face of औA. Changes in the expression of these epitopes are induced by conformational changes in औA caused by divalent cations, function perturbing mAbs, or ligand recognition. Recombinant truncated α5औ1with a mutation L358A in the α7 helix of औA has constitutively high expression of the 15/7 and HUTS-4 epitopes, mimics the conformation of the ligand-occupied receptor, and has high constitutive ligand binding activity. The epitopes of 15/7 and HUTS-4 map to a region of the hybrid domain that lies close to an interface with the α subunit. Taken together, these data suggest that the transduction of conformational changes through औA involves shape shifting in the α7 helix region, which is linked to a swing of the hybrid domain away from the α subunit. The ligand-binding head region of integrin औ subunits contains a von Willebrand factor type A domain (औA). Ligand binding activity is regulated through conformational changes in औA, and ligand recognition also causes conformational changes that are transduced from this domain. The molecular basis of signal transduction to and from औA is uncertain. The epitopes of mAbs 15/7 and HUTS-4 lie in the औ1 subunit hybrid domain, which is connected to the lower face of औA. Changes in the expression of these epitopes are induced by conformational changes in औA caused by divalent cations, function perturbing mAbs, or ligand recognition. Recombinant truncated α5औ1with a mutation L358A in the α7 helix of औA has constitutively high expression of the 15/7 and HUTS-4 epitopes, mimics the conformation of the ligand-occupied receptor, and has high constitutive ligand binding activity. The epitopes of 15/7 and HUTS-4 map to a region of the hybrid domain that lies close to an interface with the α subunit. Taken together, these data suggest that the transduction of conformational changes through औA involves shape shifting in the α7 helix region, which is linked to a swing of the hybrid domain away from the α subunit. monoclonal antibody औ subunit von Willebrand factor type A domain α subunit von Willebrand factor type A domain metal-ion dependent adhesion site bovine serum albumin recombinant soluble integrin heterodimer containing C-terminally truncated α5 and औ1 subunits (α5 residues 1–613 and औ1 residues 1–455) fused to the Fc region of human IgGγ1 ligand-induced binding site ligand-attenuated binding site Integrins mediate a wide variety of essential cell-matrix and cell-cell interactions and also participate in many common disease processes (1Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6684) Google Scholar, 2Sheppard D. Matrix Biol. 2000; 19: 203-209Crossref PubMed Scopus (118) Google Scholar). Integrins are heterodimers containing non-covalently associated α and औ subunits; each subunit has a large extracellular domain linked to a transmembrane segment and a short cytoplasmic tail. Integrins participate in bi-directional signaling; ligand recognition is dynamically regulated by "inside-out" signaling, and ligand occupancy leads to "outside-in" signals that affect cell migration, growth, differentiation, and survival (3Hughes P.E. Pfaff M. Trends Cell Biol. 1998; 8: 359-364Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 4Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3754) Google Scholar, 5Vinogradova 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 (438) Google Scholar). Modulation of integrin activity is essential in such processes as leukocyte migration to sites of tissue injury and the aggregation of platelets to form a hemostatic plug. Integrin activation can be mimicked in vitro by divalent cations such as Mn2+ or Mg2+ (6Dransfield I. Cabanas C. Craig A. Hogg N. J. Cell Biol. 1992; 116: 219-226Crossref PubMed Scopus (398) Google Scholar). Three major conformational states of integrins can be distinguished using monoclonal antibodies (mAbs)1: an inactive (resting or low affinity) state, an active (or high affinity) state, and a ligand-occupied state (7Mould A.P. J. Cell Sci. 1996; 109: 2613-2618Crossref PubMed Google Scholar). The conformations of the inactive and active states are discriminated by low and high expression, respectively, of activation epitopes (such as those recognized by 12G10, 15/7, and 9EG7 for the औ1 subunit, see Refs. 8Mould 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 (111) Google Scholar, 9Yednock T.A. Cannon C. Vandevert C. Goldbach E.G. Shaw G. Ellis D.K. Liaw C. Fritz L.C. Tanner L.I. J. Biol. Chem. 1995; 270: 28740-28750Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 10Bazzoni G. Shih D.-T. Buck C.A. Hemler M.E. J. Biol. Chem. 1995; 270: 25570-25577Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). The ligand-occupied conformer expresses high levels of ligand-induced binding site (LIBS) epitopes (which are generally also activation epitopes) and shows decreased expression of ligand-attenuated binding site (LABS) epitopes (such as mAb 13 for the औ1 subunit, see Ref. 11Mould A.P. Akiyama S.K. Humphries M.J. J. Biol. Chem. 1996; 271: 20365-20374Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). The conformational states are in equilibrium; therefore, antibodies that recognize activation epitopes or LIBS tend to cause activation and stabilize the ligand-occupied state. Conversely, antibodies that recognize LABS appear to block ligand binding by preventing conformational changes involved in ligand recognition (7Mould A.P. J. Cell Sci. 1996; 109: 2613-2618Crossref PubMed Google Scholar, 11Mould A.P. Akiyama S.K. Humphries M.J. J. Biol. Chem. 1996; 271: 20365-20374Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 12Mould 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 (144) Google Scholar). The molecular basis of integrin function has been powerfully elucidated by the recent x-ray crystal structures of the extracellular domains of αVऔ3 in both an unliganded state (13Xiong 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 (1088) Google Scholar) and in complex with a small peptide ligand (14Xiong J.-P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1368) Google Scholar). Overall, the integrin structure resembles that of a "head" on two "legs." The ligand-binding head region of the integrin contains a seven-bladed औ-propeller in the α subunit, the top face of which is in close juxtaposition with a von Willebrand factor type A domain in the औ subunit (औA). औA consists of seven α helices encircling a central औ-sheet and is connected at its N and C termini to an immunoglobulin-like "hybrid" domain and forms an extensive interface with it. The key regions involved in ligand recognition are loops on the upper surface of the औ-propeller and the upper face of the औA, which contains a metal ion-dependent adhesion site (MIDAS) and an adjacent MIDAS cation-binding site (13Xiong 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 (1088) Google Scholar, 14Xiong J.-P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1368) Google Scholar, 15Humphries M.J. Biochem. Soc. Trans. 2000; 28: 311-339Crossref PubMed Google Scholar). A small number of subtle conformational changes between the unliganded and liganded states were observed. The most important of these appeared to be a shift of the α1 helix in औA, and a slight closing up of the interface between the upper surface of the औ-propeller and the upper face of the औA. A surprising feature of the crystal structures was that the two legs are severely bent at the "knees," such that the head is in close contact with lower legs. Because the peptide ligand was soaked into the crystals of unliganded αVऔ3, it is unclear whether the small conformational changes observed between the unliganded and liganded structures (13Xiong 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 (1088) Google Scholar, 14Xiong J.-P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1368) Google Scholar) are representative of those that take place upon ligand occupancy of the native integrin. Importantly, no pathway for the transduction of conformational changes from the head to the legs (or from legs to head) was evident. Hence, the molecular basis of both outside-in and inside-out signaling remains to be clarified. Recently, evidence (16Belgova N. Blacklow S.C. Takagi J. Springer T.A. Nat. Struct. Biol. 2002; 9: 282-287Crossref PubMed Scopus (256) Google Scholar, 17Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (907) Google Scholar) has been presented that the bent form of the integrin is in the inactive state and that this may undergo a switchblade-like straightening to attain the active conformation. Nevertheless, precisely how this straightening is linked to activation of ligand binding in the head domain is uncertain. A key regulator of integrin activity is known to be the conformation of the औA domain (15Humphries M.J. Biochem. Soc. Trans. 2000; 28: 311-339Crossref PubMed Google Scholar, 18Lu C. Shimoka M. Zang Q. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2393-2398Crossref PubMed Scopus (168) Google Scholar), and we have shown that a movement of the α1 helix activates this domain (8Mould 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 (111) Google Scholar). We hypothesized that the α1 helix could occupy two different positions: position 1 characterized by high binding of the mAb 12G10 (the active conformation), and position 2 characterized by low binding of 12G10 (the inactive conformation). The inward movement of α1 helix observed in the liganded αVऔ3structure (14Xiong J.-P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1368) Google Scholar) supports this proposal. Hence, position 1 appears to correspond to the "in" state and position 2 to the "out" state of the α1 helix. About half of the integrin α subunits contain a similar domain (αA or I), and in these domains an inward movement of the α1 helix is linked to rearrangement of cation-coordinating residues at the MIDAS and a dramatic downward shift of the C-terminal α7 helix and its preceding loop (19Lee O.-J. Bankston L.A. Arnaout M.A. Liddington R. Structure. 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). However, no change in the position of the α7 helix of औA was observed between the two x-ray structures, and it was suggested that activation of औA does not involve α7 movement (13Xiong 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 (1088) Google Scholar, 14Xiong J.-P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1368) Google Scholar, 20Arnaout M.A. Immunol. Rev. 2002; 186: 125-1406Crossref PubMed Scopus (92) Google Scholar). Here we provide evidence that changes in the expression of activation epitopes in the hybrid domain are linked to shape-shifting in the α7 helix region of औA. This movement appears to participate in the conformational changes involved in both activation and ligand binding. Our data suggest that an outward swing of the hybrid domain is coupled to α7 helix motion, and hence lend support to a recent model of integrin activation (21Takagi J. Springer T.A. Immunol. Rev. 2002; 186: 141-163Crossref PubMed Scopus (315) Google Scholar). There are both strong similarities and some differences between औA and αA domain activation. Rat mAbs 16 and 13 recognizing the human α5 and औ1 subunits, respectively, were gifts from Dr. K. Yamada (NIDCR, National Institutes of Health, Bethesda). 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 औ1 mAbs 12G10 and 8E3 were produced as described (22Mould A.P. Garratt A.N. Askari J.A. Akiyama S.K. Humphries M.J. FEBS Lett. 1995; 363: 118-122Crossref PubMed Scopus (121) Google Scholar,23Burrows L. Clark K. Mould A.P. Humphries M.J. Biochem. J. 1999; 344: 527-533Crossref PubMed Scopus (50) 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 JB1A and N29 were gifts from J. Wilkins (University of Manitoba, Winnipeg, Canada). Mouse anti-human mAb 15/7 was a gift from T. Yednock (Elan Pharmaceuticals, South San Francisco, CA). Mouse anti-human mAbs 4B4 and HUTS-4 were purchased from Beckman Coulter (High Wycombe, UK) and Chemicon (Harrow, UK), respectively. All mAbs were used as purified IgG. C-terminally truncated human α5 and औ1 constructs encoding α5 residues 1–613 and औ1 residues 1–455 fused to the hinge regions and CH2 and CH3 domains of human IgGγ1 (α5-(1–613)-Fc and औ1-(1–455)-Fc) were generated as described previously (24Coe 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 the formation of heterodimers, the CH3 domain of the α5 construct contained a "hole" mutation, whereas the CH3 domain of the औ1 constructs carried a "knob" mutation as described (24Coe 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, 25Ridgway J.B. Presta L.G. Carter P. Protein Eng. 1996; 9: 617-621Crossref PubMed Scopus (474) Google Scholar). The L358A and S359A mutations in the औ1subunit were carried out using oligonucleotide-directed PCR mutagenesis, as described (24Coe 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 (Southampton, UK). The presence of the mutations (and the lack of any other changes to the wild-type sequence) was verified by DNA sequencing. Chinese hamster ovary cells L761h variant (24Coe 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 107 fetal calf serum, 2 mm glutamine, and 17 non-essential amino acids (growth medium). Cells were detached using 0.057 (w/v) trypsin, 0.027 (w/v) EDTA in phosphate-buffered saline, and plated overnight into 6-well culture plates (Costar). Approximately 1 ॖg of wild-type or mutant औ1-(1–455)-Fc or औ1-(121–455)-Fc and 1 ॖg of wild-type 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, supernatants were harvested by centrifugation at 1000 × g for 5 min. For comparison of purified wild-type heterodimers with heterodimers containing the L358A or S359A mutations in औ1, 75-cm2 flasks of sub-confluent CHOL761h cells were transfected with 5 ॖg of wild-type or mutant औ1-(1–455)-Fc and 5 ॖg 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 before (24Coe 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 described previously (12Mould 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 (144) Google Scholar). A mutant fragment in which the RGD integrin-binding sequence is replaced by the inactive sequence KGE (III6–10KGE, see Ref. 26Danen E.J.H. Aota S. van Kraats A.A. Yamada K.M. Ruiter D.J. van Muijen G.N.P. J. Biol. Chem. 1995; 270: 21612-21618Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) was produced and purified in the same manner. III6–10 was biotinylated as before (8Mould 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 (111) Google Scholar) using sulfo-LC-NHS biotin (Perbio, Chester, UK). Fab fragments of N29, TS2/16, and 12G10 were prepared by ficin cleavage of purified IgG, followed by removal of Fc-containing fragments using protein A-Sepharose, according to the manufacturer's instructions (Perbio). None of the Fab fragments showed any reactivity with goat anti-mouse IgG (Fc-specific) peroxidase conjugate (Sigma). 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 ॖg/ml in Dulbecco's phosphate-buffered saline (50 ॖl/well) for 16 h. Wells were then blocked for 1−3 h with 200 ॖl of 57 (w/v) BSA, 150 mm NaCl, 0.057 (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-(1–613) and औ1-(1–455)-Fc diluted 1:1 with 150 mm NaCl, 25 mm Tris-Cl, pH 7.4 (25 ॖl/well), was added for 1–2 h at room temperature. Wells were then washed three times with 200 ॖl 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) to remove any small contaminating amounts of endogenous Ca2+ and Mg2+ ions. mAbs (1 ॖg/ml) in buffer A with varying concentrations of Mn2+, Mg2+, or Ca2+ were added to the plate (50 ॖl/well). The plate was then incubated at 30 °C for 2 h. Unbound antibody was aspirated, and the wells were 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 ॖl/well). Wells were then washed four times with buffer A, and color was developed using 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) substrate (50 ॖl/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. For comparison of the effects of divalent cations on 15/7 and HUTS-4 binding to wild-type heterodimer and the L358A and S359A mutants, plates were coated with anti-human Fc, blocked as described above, and then incubated with supernatant from cells transfected with wild-type or mutant heterodimers. mAb binding was measured as described above in 2 mm EDTA, 2 mm Mn2+, 2 mm Mg2+, or 2 mm Ca2+. Measurements obtained were the mean ± S.D. of four replicate wells. Plates were coated with anti-human Fc and blocked as described above. Wells were then incubated with supernatant from cells transfected with wild-type or mutant heterodimers for 1–2 h at room temperature as above. Wells were washed three times with 200 ॖl of 150 mm NaCl, 1 mm MnCl2, 25 mm Tris-Cl, pH 7.4, containing 1 mg/ml BSA (buffer B). 15/7 or HUTS-4 (l ॖg/ml in buffer B) was added to the plates (50 ॖl/well) either alone or in the presence of Fab fragments of N29, TS2/16, or 12G10 (5 ॖg/ml), mAb 13 IgG (10 ॖg/ml), or III6–10 (20 ॖg/ml). The plates were then incubated at 30 °C for 2 h. Unbound antibody was aspirated, and the wells were washed three times with buffer B. Bound 15/7 or HUTS-4 was quantitated by addition of 1:2000 dilution of goat anti-mouse IgG (Fc-specific, precleared with rat serum proteins) peroxidase conjugate (Sigma) in buffer B for 30 min at room temperature (50 ॖl/well). Wells were then washed four times with buffer A, and color was developed as above. Background binding of 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. Plates were coated with anti-human Fc and blocked as described above. The blocking solution was removed, and cell culture supernatants were added (25 ॖl/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 B (200 ॖl/well), and anti-α5 or anti-औ1 mAb (5 ॖg/ml) was added (50 ॖl/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 ॖl/well) for 30 min, and the plate was washed four times in buffer B, and color was developed as above. All steps were performed at room temperature. Results shown are the mean ± S.D. of three separate experiments. Plates were coated with anti-human Fc and blocked as described above. Wells were then incubated with protein A-purified heterodimers diluted to ∼1 ॖg/ml with 150 mm NaCl, 25 mm Tris-Cl, pH 7.4 (25 ॖl/well), for 1–2 h at room temperature. Wells were washed three times with 200 ॖl of buffer B. Biotinylated III6–10 (0.1 ॖg/ml) in buffer B was added to the plate (50 ॖl/well) alone or in the presence of N29, TS2/16, or 12G10 (5 ॖg/ml). The plate was then incubated at 30 °C for 2 h. Unbound ligand was aspirated, and the wells were washed three times with buffer B. Bound ligand was quantitated by addition of 1:500 dilution of ExtrAvidin® peroxidase conjugate (Sigma) in buffer B for 20 min at room temperature (50 ॖl/well). Wells were then washed four times with buffer B, and color was developed using 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) substrate (50 ॖl/well). Background binding to BSA-coated wells was subtracted from all measurements. Measurements obtained were the mean ± S.D. of four replicate wells. Substitution of human residues with the corresponding residues in murine औ1 within the hybrid domain sequence 361–425 was performed using a PCR-based mutagenesis kit (Gene Tailor, Invitrogen) according to the manufacturer's instructions. CHOL761h cells were transfected with wild-type or mutant constructs and supernatants harvested as described above. Binding of 15/7, HUTS-4, and TS2/16 to mutant heterodimers was performed as described above, relative to the wild-type control. 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 three replicate wells. Results shown are mean ± S.D. of three separate experiments. In each assay involving a comparison between different heterodimers, the binding of mAb 8E3 (5 ॖg/ml) was used to normalize 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)), whereAM15/7 = 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, andAM8E3 = mean absorbance of 8E3 binding to wells coated with mutant integrin. 8E3 recognizes a non-functional epitope in the N-terminal region of the औ1 subunit (24Coe 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). Essentially identical results were obtained from normalization using mAb N29 against the PSI domain (Ref. 27Ni H. Li A. Simonsen N. Wilkins J.A. J. Biol. Chem. 1998; 273: 7981-7987Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, data not shown). 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. A model of the α5-propeller and thigh domains and औ1A and hybrid domains was built based on an alignment against the αVऔ3 crystal structure (13Xiong 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 (1088) Google Scholar), using the same procedures as described previously (8Mould 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 (111) Google Scholar). The PSI domain (residues 1–60 of औ1) was not included in the model. Representation of the structure was produced using PyMol. 2W. L. Delano, The Pymol Molecular Graphics System, Delano Scientific, San Carlos, CA (www.pymol.org). To investigate the mechanisms of integrin activation, we employed a recently described system for expression of recombinant soluble α5औ1 (24Coe 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 α5औ1, α5-(1–613) औ1-(1–455), fused to the Fc region of human IgGγ1 (24Coe 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) (hereafter referred to as trα5औ1-Fc). This heterodimer contains the α subunit औ-propeller and thigh domain, and the औ subunit A, hybrid, and PSI domains (13Xiong 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 (1088) Google Scholar), and has been shown to retain the properties of the full-length receptor (24Coe 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). This system is particularly useful (a) because it permits the rapid analysis of the effects of mutations, and (b) because conformational changes in the head region can be studied in isolation,i.e. in the absence of any complicating effects due to the presence of the lower leg domains (e.g. unbending, see Refs.16Belgova N. Blacklow S.C. Takagi J. Springer T.A. Nat. Struct. Biol. 2002; 9: 282-287Crossref PubMed Scopus (256) Google Scholar and 17Takagi J. Petre B.M. Walz T. Springer T.A. 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