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Amino Acid Residues in the PSI Domain and Cysteine-rich Repeats of the Integrin β2 Subunit That Restrain Activation of the Integrin αxβ2

2001; Elsevier BV; Volume: 276; Issue: 10 Linguagem: Inglês

10.1074/jbc.m005868200

1083-351X

Qun S. Zang, Timothy A. Springer,

Glycosylation and Glycoproteins Research

The leukocyte integrin αXβ2 (p150,95) recognizes the iC3b complement fragment and functions as the complement receptor type 4. αXβ2 is more resistant to activation than other β2 integrins and is inactive in transfected cells. However, when human αX is paired with chicken or mouse β2, αXβ2 is activated for binding to iC3b. Activating substitutions were mapped to individual residues or groups of residues in the N-terminal plexin/semaphorin/integrin (PSI) domain and C-terminal cysteine-rich repeats 2 and 3. These regions are linked by a long range disulfide bond. Substitutions in the PSI domain synergized with substitutions in the cysteine-rich repeats. Substitutions T4P, T22A, Q525S, and V526L gave full activation. Activation of binding to iC3b correlated with exposure of the CBR LFA-1/2 epitope in cysteine-rich repeat 3. The data suggest that the activating substitutions are present in an interface that restrains the human αX/human β2 integrin in the inactive state. The opening of this interface is linked to structural rearrangements in other domains that activate ligand binding. The leukocyte integrin αXβ2 (p150,95) recognizes the iC3b complement fragment and functions as the complement receptor type 4. αXβ2 is more resistant to activation than other β2 integrins and is inactive in transfected cells. However, when human αX is paired with chicken or mouse β2, αXβ2 is activated for binding to iC3b. Activating substitutions were mapped to individual residues or groups of residues in the N-terminal plexin/semaphorin/integrin (PSI) domain and C-terminal cysteine-rich repeats 2 and 3. These regions are linked by a long range disulfide bond. Substitutions in the PSI domain synergized with substitutions in the cysteine-rich repeats. Substitutions T4P, T22A, Q525S, and V526L gave full activation. Activation of binding to iC3b correlated with exposure of the CBR LFA-1/2 epitope in cysteine-rich repeat 3. The data suggest that the activating substitutions are present in an interface that restrains the human αX/human β2 integrin in the inactive state. The opening of this interface is linked to structural rearrangements in other domains that activate ligand binding. monoclonal antibody metal ion-dependent adhesion site fetal bovine serum chicken human mouse The integrin αXβ2 (p150,95, CD11c/CD18) is one of four integrins that are restricted in expression to leukocytes and have different α subunits associated with a common integrin β2 subunit (1Springer T.A. Lachmann P.J. Peters D.K. Rosen F.S. Walport M.J. Clinical Aspects in Immunology. 5th Ed. Blackwell Scientific, Oxford1990: 199-219Google Scholar, 2Van der Vieren M. Trong H.L. Wood C.L. Moore P.F. St. John T. Staunton D.E. Gallatin W.M. Immunity. 1995; 3: 683-690Abstract Full Text PDF PubMed Scopus (232) Google Scholar). αXβ2 is also known as the complement receptor type 4. Integrin αXβ2 is expressed on the surface of macrophages, monocytes, granulocytes, and certain activated and B lymphocyte subpopulations (3Miller L.J. Schwarting R. Springer T.A. J. Immunol. 1986; 137: 2891-2900PubMed Google Scholar, 4Keizer G.D. Borst J. Visser W. Schwarting R. de Vries J.E. Figdor C.G. J. Immunol. 1987; 138: 3130-3136PubMed Google Scholar, 5Postigo A.A. Corbi A.L. Sanchez-Madrid F. De Landazuri M.O. J. Exp. Med. 1991; 174: 1313-1322Crossref PubMed Scopus (83) Google Scholar, 6Hogg N. Takacs L. Palmer D.G. Selvendran Y. Allen C. Eur. J. Immunol. 1986; 16: 240-248Crossref PubMed Scopus (114) Google Scholar). Upon activation, αXβ2 binds to its ligands, complement component iC3b (7Bilsland C.A.G. Diamond M.S. Springer T.A. J. Immunol. 1994; 152: 4582-4589PubMed Google Scholar, 8Myones B.L. Dalzell J.G. Hogg N. Ross G.D. J. Clin. Invest. 1988; 82: 640-651Crossref PubMed Scopus (199) Google Scholar, 9Ross G.D. Reed W. Dalzell J.G. Becker S.E. Hogg N. J. Leukocyte Biol. 1992; 51: 109-117Crossref PubMed Scopus (88) Google Scholar) and fibrinogen (5Postigo A.A. Corbi A.L. Sanchez-Madrid F. De Landazuri M.O. J. Exp. Med. 1991; 174: 1313-1322Crossref PubMed Scopus (83) Google Scholar, 10Loike J.D. Sodeik B. Cao L. Leucona S. Weitz J.I. Detmers P.A. Wright S.D. Silverstein S.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1044-1048Crossref PubMed Scopus (231) Google Scholar), and mediates leukocyte adherence to endothelium and other cells, possibly by binding to intercellular adhesion molecule 1 (ICAM-1) (4Keizer G.D. Borst J. Visser W. Schwarting R. de Vries J.E. Figdor C.G. J. Immunol. 1987; 138: 3130-3136PubMed Google Scholar, 11Keizer G.D. te Velde A.A. Schwarting R. Figdor C.G. de Vries J.E. Eur. J. Immunol. 1987; 17: 1317-1322Crossref PubMed Scopus (111) Google Scholar, 12Stacker S.A. Springer T.A. J. Immunol. 1991; 146: 648-655PubMed Google Scholar, 13te Velde A.A. Keizer G.D. Figdor C.G. Immunology. 1987; 61: 261-267PubMed Google Scholar). Comparisons among leukocyte integrins suggest that αXβ2 has the highest barrier to activation of ligand binding. On cells that coexpress the integrins αXβ2 and Mac-1 (αMβ2), stronger cellular activation is required to activate αXβ2 than αMβ2 for binding to the ligand iC3b (8Myones B.L. Dalzell J.G. Hogg N. Ross G.D. J. Clin. Invest. 1988; 82: 640-651Crossref PubMed Scopus (199) Google Scholar). When transfected into COS or 293T cells, the integrins LFA-1 (αLβ2) and αMβ2are active in binding ligands; however, αXβ2 is not (14Diamond M.S. Garcia-Aguilar J. Bickford J.K. Corbi A.L. Springer T.A. J. Cell Biol. 1993; 120: 1031-1043Crossref PubMed Scopus (471) Google Scholar). Construction of chimeric αM and αX α subunits showed that many reciprocal exchanges activated ligand binding, suggesting that structural perturbations released restraints that otherwise held αXβ2 in an inactive state (14Diamond M.S. Garcia-Aguilar J. Bickford J.K. Corbi A.L. Springer T.A. J. Cell Biol. 1993; 120: 1031-1043Crossref PubMed Scopus (471) Google Scholar). Interestingly, although αXβ2 expressed on COS-7 cells could not bind to the ligand iC3b, an interspecies hybrid, αXβ2, comprised of chicken β2and human αX subunits was constitutively active in binding iC3b (7Bilsland C.A.G. Diamond M.S. Springer T.A. J. Immunol. 1994; 152: 4582-4589PubMed Google Scholar). By contrast, human αM/chicken β2 and human αM/human β2integrin heterodimers bound iC3b equally well. Studies with mAb1 map ligand binding to the I domain of the αXβ2 αXsubunit (7Bilsland C.A.G. Diamond M.S. Springer T.A. J. Immunol. 1994; 152: 4582-4589PubMed Google Scholar). These findings suggest that an intersubunit restraint on αX conformation is loosened with the chicken β2 subunit so that αX can more readily adopt the conformation that binds iC3b. It may be significant in light of this finding and the finding that αXβ2is more difficult to activate than αLβ2 or αMβ2 that the association between αX and β2 is more difficult to disrupt with denaturing conditions than the association between αL and β2 or between αM and β2(15Sanchez-Madrid F. Nagy J. Robbins E. Simon P. Springer T.A. J. Exp. Med. 1983; 158: 1785-1803Crossref PubMed Scopus (612) Google Scholar). A key question of current integrin research is the nature of the structural alterations in "inside-out signaling" that enables ligand binding by the extracellular domain in response to signals impinging on the cytoplasmic/transmembrane domains. Electron microscopy reveals an overall integrin structure of a globular head region connected to the cell membrane by two stalk regions (16Weisel J.W. Nagaswami C. Vilaire G. Bennett J.S. J. Biol. Chem. 1992; 267: 16637-16643Abstract Full Text PDF PubMed Google Scholar). The head region binds ligand and contains domains from the N-terminal portions of both the α and β subunits. Seven 60-amino acid repeats in the N-terminal half of the α subunit have been predicted to fold into a β-propeller domain (17Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 65-72Crossref PubMed Scopus (390) Google Scholar). A subset of integrin α subunits, including the αX subunit, contains an I domain inserted between β-sheets 2 and 3 of the β-propeller domain. The I domain has a structure like small G proteins with a metal ion-dependent adhesion site at the top of the domain where ligand is bound (18Lee J.-O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (806) Google Scholar, 19Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 101: 47-56Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar). A conformational change at the MIDAS that regulates ligand binding is linked structurally to a large movement of the C-terminal α-helix that connects the bottom of the I domain to the β-propeller domain (19Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 101: 47-56Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar, 20Lee J.-O. Bankston L.A. Arnaout M.A. Liddington R.C. Structure. 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 21Oxvig C. Lu C. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2215-2220Crossref PubMed Scopus (123) Google Scholar, 22Li R. Rieu P. Griffith D.L. Scott D. Arnaout M.A. J. Cell Biol. 1998; 143: 1523-1534Crossref PubMed Scopus (123) Google Scholar, 23Shimaoka M. Shifman J.M. Jing H. Takagi J. Mayo S.L. Springer T.A. Nat. Struct. Biol. 2000; 7: 674-678Crossref PubMed Scopus (117) Google Scholar). A domain in the β subunit has a predicted fold that is like the I domain and a MIDAS-like site (18Lee J.-O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (806) Google Scholar, 24Tozer E.C. Liddington R.C. Sutcliffe M.J. Smeeton A.H. Loftus J.C. J. Biol. Chem. 1996; 271: 21978-21984Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 25Tuckwell D.S. Humphries M.J. FEBS Lett. 1997; 400: 297-303Crossref PubMed Scopus (100) Google Scholar, 26Huang 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). This β subunit I-like domain associates with the side of the α subunit β-propeller domain at β-sheets 2 and 3 (27Puzon-McLaughlin W. Kamata T. Takada Y. J. Biol. Chem. 2000; 275: 7795-7802Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 28Zang Q. Lu C. Huang C. Takagi J. Springer T.A. J. Biol. Chem. 2000; 275: 22202-22212Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) and is thus near to the α subunit I domain, which links to β-sheets 2 and 3 at the top of the β-propeller domain. The stalk regions provide the crucial link between the signals impinging on the α and β subunit transmembrane and cytoplasmic domains and the conformational changes that occur in the ligand-binding head region. In the α subunit, the stalk region appears to consist of the region C-terminal to the predicted β-propeller domain. The stalk region is predicted to consist of domains with a two-layer β-sandwich structure (29Lu C. Oxvig C. Springer T.A. J. Biol. Chem. 1998; 273: 15138-15147Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Four subregions of the αM stalk have been defined with mAb epitopes, three of which react with mAbs whether or not the β subunit is coexpressed. In the β subunit, the stalk region appears to consist of the cysteine-rich regions that precede and follow the I-like domain, i.e. residues 1–103 and 342–678 in β2. These cysteine-rich regions are linked by a long range disulfide bond defined in β3 that is predicted to link Cys-3 and Cys-425 in β2 (30Calvete J.J. Henschen A. González-Rodrı́guez J. Biochem. J. 1991; 274: 63-71Crossref PubMed Scopus (158) Google Scholar). The N-terminal cysteine-rich region of residues ∼1–50 shares sequence homology with membrane proteins including plexins, semaphorins, and the c-met receptor (31Bork P. Doerks T. Springer T.A. Snel B. Trends Biochem. Sci. 1999; 24: 261-263Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). This region has two predicted α-helices and has been termed the "PSI domain" forplexins, semaphorins, andintegrins. The segment from residues 425 to 590 has a cysteine content of 20% and is composed of four cysteine-rich repeats. The first repeat is less similar to the others and at its N-terminal end contains the cysteine that disulfide bonds to the PSI domain. Several monoclonal antibodies that activate integrins or report conformational changes have been mapped to the C-terminal region of the β subunit that includes the cysteine-rich repeats (28Zang Q. Lu C. Huang C. Takagi J. Springer T.A. J. Biol. Chem. 2000; 275: 22202-22212Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 32Faull R.J. Wang J. Leavesley D.I. Puzon W. Russ G.R. Vestweber D. Takada Y. J. Biol. Chem. 1996; 271: 25099-25106Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 33Ni H. Li A. Simonsen N. Wilkins J.A. J. Biol. Chem. 1998; 273: 7981-7987Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 34Takagi J. Isobe T. Takada Y. Saito Y. J. Biochem. ( Tokyo ). 1997; 121: 914-921Crossref PubMed Scopus (32) Google Scholar, 35Bazzoni G. Shih D.-T. Buck C.A. Hemler M.A. J. Biol. Chem. 1995; 270: 25570-25577Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 36Shih D.T. Edelman J.M. Horwitz A.F. Grunwald G.B. Buck C.A. J. Cell Biol. 1993; 122: 1361-1371Crossref PubMed Scopus (45) Google Scholar, 37Stephens P. Romer J.T. Spitali M. Shock A. Ortlepp S. Figdor C. Robinson M.K. Cell Adhes. Commun. 1995; 3: 375-384Crossref PubMed Scopus (48) Google Scholar) and to the N-terminal cysteine-rich region (33Ni H. Li A. Simonsen N. Wilkins J.A. J. Biol. Chem. 1998; 273: 7981-7987Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Many of these mAbs recognize epitopes that become exposed after integrin activation. One of these, mAb KIM127 to the β2 subunit, is not dependent on association with the α subunit for reactivity and indeed reacts better with the free β2 subunit than with the integrin αβ heterodimer (38Huang C. Lu C. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3156-3161Crossref PubMed Scopus (60) Google Scholar). Thus, structural changes in the stalk region that include exposing antibody epitopes on the integrin β subunit are associated with integrin activation. Here, we have defined regions of the integrin β2 subunit involved in regulating ligand binding by αXβ2. Ligand binding is activated when the human αX subunit is complexed with the chicken β2 subunit (7Bilsland C.A.G. Diamond M.S. Springer T.A. J. Immunol. 1994; 152: 4582-4589PubMed Google Scholar). We hypothesized that this reflects a release of structural contacts between the human αX and human β2 subunits that normally restrain αXβ2 in a nonligand binding conformation. To map these contacts within the β2 subunit, we have utilized chicken/human β2 chimeras. We map the key differences and provide direct evidence that residues that restrain ligand binding by β2 are present in both the N-terminal cysteine-rich PSI domain and the C-terminal cysteine-rich repeats. 293T cells (human renal epithelial transformed cells) were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS), nonessential amino acids (Life Technologies, Inc.), 2 mm glutamine, and 50 μg/ml gentamicin. The mouse anti-human αX mAb CBRp150/2E1 (7Bilsland C.A.G. Diamond M.S. Springer T.A. J. Immunol. 1994; 152: 4582-4589PubMed Google Scholar) and the anti-human β2 mAbs KIM185 (39Andrew D. Shock A. Ball E. Ortlepp S. Bell J. Robinson M. Eur. J. Immunol. 1993; 23: 2217-2222Crossref PubMed Scopus (111) Google Scholar) and CBR LFA-1/2 (40Petruzzelli L. Maduzia L. Springer T. J. Immunol. 1995; 155: 854-866PubMed Google Scholar) have been previously described. Human or chicken β2 cDNA were inserted in vector AprM8 (41Huang C. Springer T.A. J. Biol. Chem. 1995; 270: 19008-19016Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Chimeras and substitution mutants were generated by polymerase chain reaction overlap extension (42Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene ( Amst. ). 1989; 77: 51-59Crossref PubMed Scopus (6851) Google Scholar). Briefly, 5′ and 3′ end primers were designed to include unique restriction sites. Mutations were introduced by a pair of inner complementary primers. After a second round of polymerase chain reaction, the products were digested and ligated with the corresponding predigested plasmids. All constructs were verified by DNA sequencing. Plasmids for transfection were purified by QIAprep Spin Kit or Maxi Kit (Qiagen, Chatsworth, CA). 293T cells were transiently transfected with human αX and wild-type or mutant β2 constructs using calcium phosphate (43Heinzel S.S. Krysan P.J. Calos M.P. DuBridge R.B. J. Virol. 1988; 62: 3738-3746Crossref PubMed Google Scholar, 44DuBridge R.B. Tang P. Hsia H.C. Leong P.M. Miller J.H. Calos M.P. Mol. Cell. Biol. 1987; 7: 379-387Crossref PubMed Scopus (918) Google Scholar). Medium was changed after 7–11 h. Cells were harvested for analysis 48 h after transfection. Cells were washed twice with L15 medium supplemented with 2.5% fetal bovine serum (L15/FBS). Cells (106) were incubated with 50 μl of primary antibody (20 μg/ml purified mAb, or 1:100 dilution of ascites) on ice for 30 min. Cells were then washed three times with L15/FBS, followed by incubation with 50 μl of a 1:20 dilution of fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Zymed Laboratories Inc., San Francisco, CA) for 30 min on ice. After washing three times with L15/FBS, cells were resuspended in 200 μl of cold phosphate-buffered saline and analyzed on a FACScan (Becton Dickinson, San Jose, CA). Antigen expression is presented as mean fluorescence intensity of cells. As described previously (7Bilsland C.A.G. Diamond M.S. Springer T.A. J. Immunol. 1994; 152: 4582-4589PubMed Google Scholar, 14Diamond M.S. Garcia-Aguilar J. Bickford J.K. Corbi A.L. Springer T.A. J. Cell Biol. 1993; 120: 1031-1043Crossref PubMed Scopus (471) Google Scholar)), sheep erythrocytes (Colorado Serum Co., Denver, CO) were washed, resuspended to 6 × 108 cells/10 ml in buffer 1 (Hanks' balanced salt solution, 15 mm HEPES, pH 7.3, and 1 mm MgCl2), and sensitized with 80 μl of IgM anti-Forssman mAb M1/87 culture supernatant for 1 h at room temperature (E-IgM). The cells were then washed and resuspended in 1.8 ml of buffer 2 (Hanks' balanced salt solution, 15 mmHEPES, pH 7.3, 1 mm MgCl2, and 1 mmCaCl2), supplemented with 200 μl of C5-deficient human serum (Sigma). After incubation at 37 °C for 1 h, the resulting E-IgM-iC3b were washed twice and resuspended in 6 ml of buffer 2. To assay the binding of αXβ2 to iC3b, 293T cells transfected with recombinant αXβ2were plated on 12-well polylysine-coated plates for at least 4 h prior to the experiment. After washing with buffer 2, the cells were incubated together with 200 μl of E-IgM-iC3b for 30 min at 37 °C. Unbound erythrocytes were removed by washing three times, and rosettes (>10 erythrocytes/293T cell, >100 cells examined) were scored with microscopy. To locate regions in the integrin β2 subunit that restrain activation of αXβ2, interspecies human/chicken chimeric β2 subunits were made. Chimeras were named according to the species origin of their segments. For example, h103c indicates that residues 1 to 103 are human and residues 104 to the C-terminal end are chicken. Each construct was cotransfected with the human αX subunit into 293T cells. Proper expression was confirmed by immunostaining with antibody CBRp150/2E1 to the αX subunit. All human/chicken β2chimeras studied here were expressed as well as human β2in αXβ2 complexes. The percentage of 293T transfectants expressing αXβ2 ranged from 68 to 85% for chimeras and wild type in all experiments described below. Transfectants were assayed for activation of ligand binding by rosetting with erythrocytes sensitized with iC3b (E-IgM-iC3b). The percentage of rosetting cells was normalized to the percentage of αXβ2+ cells for each construct. Transfectants expressing hybrid αXβ2 (human αX/chicken β2) but not transfectants expressing human αXβ2 formed rosettes with E-IgM-iC3b, confirming previous observations with COS-7 cell transfectants (7Bilsland C.A.G. Diamond M.S. Springer T.A. J. Immunol. 1994; 152: 4582-4589PubMed Google Scholar). The chimeras mapped activation of ligand binding by chicken β2 to two regions, residues 1–71 and residues 421–610 (Fig. 1). The importance of residues 421–610 was shown by activation of iC3b rosetting by chimeras h103c and h421c but not by chimeras h610c, h103c421h, and c71h610c. Residues 1–71 were not sufficient by themselves to activate iC3b binding as shown with chimera c71h; however, they augmented rosetting when present in combination with residues 421–610 (Fig. 1). Thus, with residues 421–610 of chicken origin in chimeras h103c and h421c, about 40% of transfectants rosetted with E-IgM-iC3b. With both residues 1–71 and 421–610 of chicken origin in chimeras c678h and c71h421c, about 80% of transfectants rosetted. This was the same level as with the wild-type chicken β2 subunit. Thus, activation was a synergistic effect of the N- and C-terminal cysteine-rich regions of the chicken β2 subunit. We found that αXβ2 heterodimers containing human αX and mouse β2 subunits were activated for binding to iC3b almost as well as those containing human αX and chicken β2 subunits (Fig.2 A). Human/mouse β2 chimeras showed that the region containing residues 344–612 was activating (Fig. 2 A). The h98m chimera was less activating than mouse β2, suggesting that the N-terminal cysteine-rich region contributed to activation. Furthermore, the m122h, m163h, m254h, m302h, and m344h chimeras showed that the N-terminal cysteine-rich region was not sufficient for activation, similar to the results with the chicken β2 subunit. The activating region in the C-terminal cysteine-rich region in the mouse β2 subunit was defined with a further series of chimeras (Fig. 2 B). These narrowed activation by the C-terminal cysteine-rich region to residues 470–538 since chimera m122h470m was activating, whereas m122h538m was not (Fig.2B). Furthermore, residues in two different segments, 470–502 and 502–538, were activating because chimera m122h502m was partially activating, whereas m122h470m was fully activating, and m122h538m was not activating. Mapping of the C-terminal cysteine-rich repeat region of chicken β2 was refined with five further chicken/human chimeric β2 constructs. Each construct contained N-terminal residues 1–71 and various lengths of the C-terminal cysteine-rich repeats from chicken β2(Fig. 3 A). Rosetting of chimeras c71h446c, c71h470c, and c71h498c with E-IgM-iC3b was similar to that of wild-type chicken β2. However, chimeras c71h527c and c71h562c did not bind to iC3b. Therefore, residues within region 498–527 can activate binding to the ligand iC3b. Within the activating region defined in chicken β2 of 498–527, 11 residues differ between human and chicken. Groups of one to three chicken amino acid residues in this region were introduced into the human β2 subunit and their effect on binding to iC3b was examined (Fig. 3 B). In combination with chicken residues 1–71, four groups of amino acid substitutions were activating: Q510T/Y511F/E513D in repeat 2 and T516N/I517M, R521F/Y522H, and Q525S/V526L in repeat 3. Chimera c71h/Q525S/V526L was as active as chicken β2. The four activating groups of residues were also tested in the absence of any other chicken residues. In this situation, only the mutation Q525S/V526L was activating, and its activity was reduced compared with c71h/Q525S/V526L (Fig.3B). Mapping of the N-terminal cysteine-rich region was refined with three chicken/human chimeras that included different portions of the N-terminal region in combination with the synergistic C-terminal segment (Fig.4 A). All three β2 chimeras, c71h421c, c50h421c, and c29h421c, activated binding to E-IgM-iC3b to the same extent as chicken β2. Thus, residues within the first 29 amino acids of the β2subunit are sufficient to synergistically activate αXβ2. In region 1–29 of the β2 subunit, 11 residues differ between the human and chicken. Groups of these residues were substituted with chicken sequence in combination with the mutation Q525S/V526L in the C-terminal cysteine-rich region in each construct (Fig. 4 B). Most of the mutants rosetted E-IgM-iC3b no better than the parent Q525S/V526L mutant. However, mutants Q1A/T4P/Q525S/V526L and T4P/Q525S/V526L but not Q1A/Q525S/V526L were more active than Q525S/V526L, implicating the substitution T4P in activation. Similarly, mutants T22A/Q25K/Q525S/V526L and T22A/Q525S/V526L but not Q25K/Q525S/V526L were more active than Q525S/V526L, implicating T22A. Moreover, the combination of mutations T4P and T22A was even more active, and the mutant T4P/T22A/Q525S/V526L was as active as chicken β2 (Fig. 4 B). Therefore, four chicken residues, two each in the N-terminal and C-terminal cysteine-rich regions of β2, are sufficient to maximally activate iC3b rosetting by αXβ2. Several mAbs that activate β2 integrins map to the C-terminal cysteine-rich region of the β2 subunit (28Zang Q. Lu C. Huang C. Takagi J. Springer T.A. J. Biol. Chem. 2000; 275: 22202-22212Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 37Stephens P. Romer J.T. Spitali M. Shock A. Ortlepp S. Figdor C. Robinson M.K. Cell Adhes. Commun. 1995; 3: 375-384Crossref PubMed Scopus (48) Google Scholar). The mouse/human substitutions recognized by these mAbs map very near to the substitutions Q525S/V526L that activate αXβ2. Specifically, mAb KIM185 recognizes residues 581–604 and mAb CBR LFA-1/2 recognizes residues 534 and 536. 2C. Lu, M. Ferzly, J. Takagi, and T. A. Springer, manuscript in preparation. Recognition by mAb CBR LFA-1/2 correlates with the activation status of β2integrins; αLβ2 and αMβ2, which are active in 293T cell transfectants, are recognized well by CBR LFA-1/2, whereas αXβ2, which is inactive in 293T cells, is recognized poorly2 (Fig. 5). We examined the effect of activating mutations on expression of CBR LFA-1/2 and KIM185 epitopes (Fig. 5). The KIM185 epitope was expressed equally well by wild-type and mutant αXβ2. By contrast, activating mutations induced exposure of the CBR LFA-1/2 epitope (Fig. 5). The Q525S/V526L mutation partially exposed the CBR LFA-1/2 epitope, whereas the T4P/T22A/Q525S/V526L mutation maximally exposed the epitope, i.e. to the same level as seen with KIM185 mAb. Exposure of the CBR LFA-1/2 epitope correlated with activation of binding to iC3b (Fig. 5). Therefore, the mutations cause structural rearrangements in the stalk region that lead to exposure of the CBR LFA-1/2 epitope and are linked to activation of ligand binding. Furthermore, binding of the CBR LFA-1/2 and KIM185 mAbs demonstrates that the mutations do not disrupt the structure of the cysteine-rich repeats. Among the β2 integrins, αXβ2 is the most resistant to activation and to dissociation of its α and β subunits. Here, we have identified specific amino acid residues that restrain αXβ2 in a conformation in which it does not bind its ligand, iC3b. We extended previous observations with the chicken β2 subunit (7Bilsland C.A.G. Diamond M.S. Springer T.A. J. Immunol. 1994; 152: 4582-4589PubMed Google Scholar) by showing that pairing of human αX with β2 from another species, the mouse, also activates binding to iC3b. Interspecies β2 subunit chimeras associated with human αX subunits demonstrated that the C-terminal cysteine-rich repeats from mouse or chicken were sufficient for partial activation and that the N-terminal cysteine-rich PSI domain was insufficient for activation but synergized with the C-terminal cysteine-rich repeats. Activating substitutions in the N-terminal region were localized within the PSI domain (Fig. 6). Human/chicken substitutions T4P and T22A each synergized with the C-terminal region and, when present together, gave augmented synergy. PSI domains in integrins contain six cysteines that form intradomain disulfide bonds and one cysteine that forms a long range interdomain disulfide (30Calvete J.J. Henschen A. González-Rodrı́guez J. Biochem. J. 1991; 274: 63-71Crossref PubMed Scopus (158) Google Scholar,31Bork P. Doerks T. Springer T.A. Snel B. Trends Biochem. Sci. 1999; 24: 261-263Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Each of the activating substitutions neighbors a cysteine residue (Fig. 6). The substitution T4P neighbors Cys-3, which forms the long range disulfide bond to Cys-425, which is at the beginning of the C-terminal cysteine-rich repeats (Fig. 6). Thus, the two regions in which activating substitutions are found, the PSI domain and cysteine-rich repeats, are linked by a disulfide bond and must be neighboring domains in the three-dimensional structure of the integrin β subunit. Activating substitutions within the C-terminal region localized to cysteine-rich repeats 2 and 3 (Fig. 6). One segment containing activating mouse substitutions localized wholly within repeat 2, whereas another included portions of repeats 2 and 3 (Fig. 6). Fine mapping of three groups of chicken substitutions that activated αXβ2 in synergy with chicken residues in the PSI domain showed that one group mapped to repeat 2 and two groups mapped to repeat 3 (Fig. 6). One pair of substitutions that was sufficient for activation of αXβ2, Q525S/V526L, mapped to repeat 3. We cannot exclude the presence of activating substitutions in repeat 1 because all chimeras in which repeat 1 was mouse or chicken also contained repeats 2 and 3 from mouse or chicken, which were activating by themselves. However, the segments following the PSI domain and preceding repeat 1 were not activating. Furthermore, repeat 4 and more C-terminal segments were not activating. The species-specific differences in repeat 4 are greater than in repeats 2 and 3 (Table I); therefore, an insufficiency of species-specific differences cannot explain the lack of activation by repeat 4.Table IC-terminal cysteine-rich repeats: variation between species and activationRepeatLengthHuman-chicken differencesHuman-mouse differencesActivating substitutionsresidues%%1364722ND1-aND, not determined.2532317+3393826+4394636−1-a ND, not determined. Open table in a new tab What is the mechanism of integrin activation by species-specific substitutions in the PSI domain and cysteine-rich repeats 2 and 3? Many other observations suggest that integrins are restrained in their resting state in a conformation that does not bind ligand and that a wide variety of perturbations can activate ligand binding. Our results suggest that the PSI domain and cysteine-rich repeats 2 and 3 have an important function in restraining integrins in their resting, inactive state. These restraints are overcome when the α and β subunits are from different species; therefore, it appears that there are direct interactions between these β subunit domains and the α subunit that constrain integrins in the inactive configuration. The substitutions are unlikely to disrupt the overall conformation of these domains because they are naturally occurring variations between species. Furthermore, we demonstrated that mAb CBR LFA-1/2, which binds to species-specific residues in repeat 3,2 binds well when the activating mutations Q525S and V526L are present in repeat 3. Therefore, it appears that the activating mutations we have defined are within or near an interface between the β2 and αX subunits. The findings suggest that in resting integrins, there are contacts of the PSI domain and cysteine-rich repeats 2 and 3 with the α subunit and that these contacts help restrain ligand binding. It appears that certain species-specific substitutions disrupt this interaction and, thereby, lower the activation energy required for activation of ligand binding. Binding of iC3b by αXβ2 maps to the αX I domain (7Bilsland C.A.G. Diamond M.S. Springer T.A. J. Immunol. 1994; 152: 4582-4589PubMed Google Scholar). Conformational shifts around the MIDAS in I domains regulate ligand binding and are linked to a large movement of the C-terminal α-helix of the I domain that connects to other integrin subunits (19Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 101: 47-56Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar, 20Lee J.-O. Bankston L.A. Arnaout M.A. Liddington R.C. Structure. 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 21Oxvig C. Lu C. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2215-2220Crossref PubMed Scopus (123) Google Scholar, 22Li R. Rieu P. Griffith D.L. Scott D. Arnaout M.A. J. Cell Biol. 1998; 143: 1523-1534Crossref PubMed Scopus (123) Google Scholar, 23Shimaoka M. Shifman J.M. Jing H. Takagi J. Mayo S.L. Springer T.A. Nat. Struct. Biol. 2000; 7: 674-678Crossref PubMed Scopus (117) Google Scholar). Therefore, it appears that an alteration in contacts in the stalk region between the α subunit and the PSI domain and the cysteine-rich repeats in the β subunit are linked to conformational rearrangements in the ligand-binding domains in the headpiece of integrins. The loss of the restraints that keep αXβ2in an inactive state appears to reflect an opening up of the αβ interface in the stalk region based on exposure of the epitope for the mAb CBR LFA-1/2. This mAb can activate integrins αLβ2 and αMβ2(40Petruzzelli L. Maduzia L. Springer T. J. Immunol. 1995; 155: 854-866PubMed Google Scholar). It showed little reactivity with wild-type αXβ2; however, introduction of activating amino acid substitutions Q525S/V526L in cysteine-rich repeat 3 exposed the CBR LFA-1/2 epitope, and addition of the T4P/T22A substitutions fully exposed the epitope. Exposure correlated with iC3b binding. The CBR LFA-1/2 mAb maps to residues 534 and 5362, and nearby residues 525 and 526, to which activating mutations map in repeat 3. It is unlikely that there is a significant conformational change in this repeat because its structure is constrained by four disulfide bonds. Therefore, we envision a movement apart or change in orientation of the α and β subunits that exposes the CBR LFA-1/2 epitope in repeat 3. Other studies also imply a structural restraint on integrin activation that is localized in the cysteine-rich regions of the β subunit. Activation of the integrin LFA-1 (αLβ2) expressed on COS cells was induced if the C-terminal cysteine-rich repeat region of the β2 subunit was replaced by that of β1 (45Douglass W.A. Hyland R.H. Buckley C.D. Al-Shamkhani A. Shaw J.M. Scarth S.L. Simmons D.L. Law S.K.A. FEBS Lett. 1998; 440: 414-418Crossref PubMed Scopus (26) Google Scholar). A point mutation that introduces aN-glycosylation site into the beginning of cysteine-rich repeat 4 of the β3 subunit activated integrins αIIbβ3 and αvβ3(46Kashiwagi H. Tomiyama Y. Tadokoro S. Honda S. Shiraga M. Mizutani H. Handa M. Kurata Y. Matsuzawa Y. Shattil S.J. Blood. 1999; 93: 2559-2568Crossref PubMed Google Scholar). Furthermore, disruption of the long range disulfide bond between the PSI domain and the cysteine-rich repeats resulted in increased ligand binding affinity of αIIbβ3 (47Liu C.Y. Sun Q.H. Wang R. Paddock C.M. Newman P.J. Blood. 1997; 90 (abstr.): 573Crossref Google Scholar). Moreover, treatment with reducing agents, such as dithiothreitol, induced the active conformation of β1 integrin (33Ni H. Li A. Simonsen N. Wilkins J.A. J. Biol. Chem. 1998; 273: 7981-7987Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) and increased platelet aggregation through the αIIbβ3 integrin (48Zucker M.B. Masiello N.C. Thromb. Haemostasis. 1984; 51: 119-124Crossref PubMed Scopus (75) Google Scholar). Recently, an anti-β1 antibody with an activation-dependent epitope has been mapped to the N-terminal cysteine-rich region, suggesting a role of this region as a regulatory site for integrin activation (33Ni H. Li A. Simonsen N. Wilkins J.A. J. Biol. Chem. 1998; 273: 7981-7987Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). In addition, several monoclonal antibodies against the C-terminal cysteine-rich regions of β1 (32Faull R.J. Wang J. Leavesley D.I. Puzon W. Russ G.R. Vestweber D. Takada Y. J. Biol. Chem. 1996; 271: 25099-25106Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 34Takagi J. Isobe T. Takada Y. Saito Y. J. Biochem. ( Tokyo ). 1997; 121: 914-921Crossref PubMed Scopus (32) Google Scholar), β2 (37Stephens P. Romer J.T. Spitali M. Shock A. Ortlepp S. Figdor C. Robinson M.K. Cell Adhes. Commun. 1995; 3: 375-384Crossref PubMed Scopus (48) Google Scholar), and β3 (49Du X. Gu M. Weisel J.W. Nagaswami C. Bennett J.S. Bowditch R. Ginsberg M.H. J. Biol. Chem. 1993; 268: 23087-23092Abstract Full Text PDF PubMed Google Scholar) integrins have been described as activating mAbs with respect to their ability to promote ligand binding. A plausible explanation is that these mAbs selectively bind to the open conformation of the stalk region and thus stabilize integrins in this conformation and induce linked rearrangements in the ligand-binding domains. Indeed, activating mAbs to both the β1 and β2 cysteine-rich regions have been found to bind better to isolated β subunits than αβ complexes, implying that they favor an open conformation (26Huang 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, 37Stephens P. Romer J.T. Spitali M. Shock A. Ortlepp S. Figdor C. Robinson M.K. Cell Adhes. Commun. 1995; 3: 375-384Crossref PubMed Scopus (48) Google Scholar, 38Huang C. Lu C. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3156-3161Crossref PubMed Scopus (60) Google Scholar, 50Luque A. Gomez M. Puzon W. Takada Y. Sanchez-Madrid F. Cabanas C. J. Biol. Chem. 1996; 271: 11067-11075Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). In contrast to domains in the globular headpiece of integrins, the stalk regions do not appear to directly bind ligand but instead appear to regulate ligand binding and to relay activation signals impinging on the cytoplasmic and transmembrane domains of the integrin α and β subunits. We have identified specific amino acid residues in the PSI domain and cysteine-rich repeats 2 and 3 of the β subunit that form part of the interface between the α and β subunits in the stalk region that restrains conformational movements in the ligand-binding headpiece. It would be very interesting to learn which regions of the α subunit participate in this interface and the molecular details of how structural alterations are communicated from one domain to another in integrins. We thank Mark Ryan for assistance with fluorescence-activated cell sorter analysis.