Epitope Mapping of Monoclonal Antibody to Integrin αL β2 Hybrid Domain Suggests Different Requirements of Affinity States for Intercellular Adhesion Molecules (ICAM)-1 and ICAM-3 Binding
2005; Elsevier BV; Volume: 280; Issue: 32 Linguagem: Inglês
10.1074/jbc.m503239200
ISSN1083-351X
AutoresRenhong Tang, Emilia Tng, S.K. Alex Law, Suet‐Mien Tan,
Tópico(s)Platelet Disorders and Treatments
ResumoIntegrin undergoes different activation states by changing its quaternary conformation. The integrin β hybrid domain acts as a lever for the transmission of activation signal. The displacement of the hybrid domain can serve to report different integrin activation states. The monoclonal antibody (mAb) MEM148 is a reporter antibody that recognizes Mg/EGTA-activated but not resting integrin αL β2. Herein, we mapped its epitope to the critical residue Pro374 located on the inner face of the β2 hybrid domain. Integrin αL β2 binds to its ligands ICAM-1 and ICAM-3 with different affinities. Integrin is proposed to have at least three affinity states, and the position of the hybrid domain differs in each. We made use of the property of mAb MEM148 to analyze and correlate these affinity states in regard to αL β2/intercellular adhesion molecule (ICAM) binding. Our study showed that Mg/EGTA-activated αLβ2 can adopt a different conformation from that activated by activating mAbs KIM185 or MEM48. Unlike ICAM-1 binding, which required only one activating agent, αL β2/ICAM-3 binding required both Mg/EGTA and an activating mAb. This suggests that αLβ2 with intermediate affinity is sufficient to bind ICAM-1 but not ICAM-3, which requires a high affinity state. Furthermore, we showed that the conformation adopted by αLβ2 in the presence of Mg/EGTA, depicting an intermediate activation state, could be reverted to its resting conformation. Integrin undergoes different activation states by changing its quaternary conformation. The integrin β hybrid domain acts as a lever for the transmission of activation signal. The displacement of the hybrid domain can serve to report different integrin activation states. The monoclonal antibody (mAb) MEM148 is a reporter antibody that recognizes Mg/EGTA-activated but not resting integrin αL β2. Herein, we mapped its epitope to the critical residue Pro374 located on the inner face of the β2 hybrid domain. Integrin αL β2 binds to its ligands ICAM-1 and ICAM-3 with different affinities. Integrin is proposed to have at least three affinity states, and the position of the hybrid domain differs in each. We made use of the property of mAb MEM148 to analyze and correlate these affinity states in regard to αL β2/intercellular adhesion molecule (ICAM) binding. Our study showed that Mg/EGTA-activated αLβ2 can adopt a different conformation from that activated by activating mAbs KIM185 or MEM48. Unlike ICAM-1 binding, which required only one activating agent, αL β2/ICAM-3 binding required both Mg/EGTA and an activating mAb. This suggests that αLβ2 with intermediate affinity is sufficient to bind ICAM-1 but not ICAM-3, which requires a high affinity state. Furthermore, we showed that the conformation adopted by αLβ2 in the presence of Mg/EGTA, depicting an intermediate activation state, could be reverted to its resting conformation. Integrins represent a large family of type I heterodimeric (α and β subunits) membrane proteins capable of bidirectional signal transduction serving cell growth, differentiation, and apoptosis (1Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6687) Google Scholar). The β I-like domain is flanked by the hybrid and PSI 1The abbreviations used are: PSI, plexins, semaphorins, and integrins; I-EGF, integrin epidermal growth factor; ICAM, intercellular adhesion molecule; sICAM, soluble ICAM; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; wt, wild type; mAb, monoclonal antibody; IgSF, immunoglobulin superfamily; EI, expression index. 1The abbreviations used are: PSI, plexins, semaphorins, and integrins; I-EGF, integrin epidermal growth factor; ICAM, intercellular adhesion molecule; sICAM, soluble ICAM; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; wt, wild type; mAb, monoclonal antibody; IgSF, immunoglobulin superfamily; EI, expression index. (for plexins, semaphorins, and integrins) domains (Fig. 1A) (2Tan S.M. Robinson M.K. Drbal K. van Kooyk Y. Shaw J.M. Law S.K. J. Biol. Chem. 2001; 276: 36370-36376Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 3Xiong J.P. Stehle T. Diefenbach B. Zhang R. Dunker R. Scott D.L. Joachimiak A. Goodman S.L. Arnaout M.A. Science. 2001; 294: 339-345Crossref PubMed Scopus (1088) Google Scholar). The hybrid domain has been shown to be important in the propagation of the activation signal from one end of the β subunit to the other (2Tan S.M. Robinson M.K. Drbal K. van Kooyk Y. Shaw J.M. Law S.K. J. Biol. Chem. 2001; 276: 36370-36376Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 4Tng E. Tan S.M. Ranganathan S. Cheng M. Law S.K. J. Biol. Chem. 2004; 279: 54334-54339Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 5Mould A.P. Barton S.J. Askari J.A. McEwan P.A. Buckley P.A. Craig S.E. Humphries M.J. J. Biol. Chem. 2003; 278: 17028-17035Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 6Luo B.H. Strokovich K. Walz T. Springer T.A. Takagi J. J. Biol. Chem. 2004; 279: 27466-27471Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Recently, superimposed structural coordinates of liganded-open αIIbβ3 headpiece with that of unliganded-closed αVβ3 revealed an ∼10-Å shift and a concomitant rotation of the hybrid domain relative to the last helix of the β I-like domain upon ligand binding (7Xiao T. Takagi J. Coller B.S. Wang J.H. Springer T.A. Nature. 2004; 432: 59-67Crossref PubMed Scopus (656) Google Scholar). mAbs that attenuate this swing-out motion of the integrin α5β1 hybrid domain prevent effective allosteric activation of the β I-like domain (5Mould A.P. Barton S.J. Askari J.A. McEwan P.A. Buckley P.A. Craig S.E. Humphries M.J. J. Biol. Chem. 2003; 278: 17028-17035Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 6Luo B.H. Strokovich K. Walz T. Springer T.A. Takagi J. J. Biol. Chem. 2004; 279: 27466-27471Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Collective observations from electron microscopy images and crystal structures of integrin αIIbβ3, αVβ3, and α5β1 suggest that integrin may undergo at least three activation states depicted by different quaternary conformations (3Xiong J.P. Stehle T. Diefenbach B. Zhang R. Dunker R. Scott D.L. Joachimiak A. Goodman S.L. Arnaout M.A. Science. 2001; 294: 339-345Crossref PubMed Scopus (1088) Google Scholar, 7Xiao T. Takagi J. Coller B.S. Wang J.H. Springer T.A. Nature. 2004; 432: 59-67Crossref PubMed Scopus (656) Google Scholar, 8Nermut M.V. Green N.M. Eason P. Yamada S.S. Yamada K.M. EMBO J. 1988; 7: 4093-4099Crossref PubMed Scopus (192) Google Scholar, 9Weisel J.W. Nagaswami C. Vilaire G. Bennett J.S. J. Biol. Chem. 1992; 267: 16637-16643Abstract Full Text PDF PubMed Google Scholar, 10Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (907) Google Scholar, 11Takagi J. Strokovich K. Springer T.A. Walz T. EMBO J. 2003; 22: 4607-4615Crossref PubMed Scopus (282) Google Scholar, 12Xiong J.P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S.L. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1369) Google Scholar, 13Mould A.P. Humphries M.J. Curr. Opin. Cell Biol. 2004; 16: 544-551Crossref PubMed Scopus (86) Google Scholar). A bent integrin with the hybrid domain in close proximity of the integrin epidermal growth factor (I-EGF) 3 and 4 domains represents the resting state. The extended integrin with the hybrid domain distally separated from I-EGF 3 and 4 but orientated toward the α subunit β-propeller depicts a low affinity state, whereas the extended integrin with a swing-out hybrid domain away from the α subunit β-propeller represents a high affinity state. The conceptualization of different integrin affinity states also derives from earlier functional studies. Observations were made on integrins having different ligand binding properties under different cellular or extracellular conditions. Resting platelet integrin αIIbβ3 binds immobilized fibrinogen, but binding to soluble fibrinogen, fibronectin, or von Willebrand factor requires platelet activation by agonist (14Savage B. Ruggeri Z.M. J. Biol. Chem. 1991; 266: 11227-11233Abstract Full Text PDF PubMed Google Scholar, 15Phillips D.R. Charo I.F. Scarborough R.M. Cell. 1991; 65: 359-362Abstract Full Text PDF PubMed Scopus (479) Google Scholar, 16Savage B. Shattil S.J. Ruggeri Z.M. J. Biol. Chem. 1992; 267: 11300-11306Abstract Full Text PDF PubMed Google Scholar). Divalent cation Mn2+ activates integrin α4β1 to bind VCAM-1, whereas adhesion to fibronectin-derived CS-1 peptide requires additional activating mAb (17Masumoto A. Hemler M.E. J. Biol. Chem. 1993; 268: 228-234Abstract Full Text PDF PubMed Google Scholar). Real time analysis of integrin α4β1 binding to fluorescent-conjugated ligand mimetic via chemokine receptor activation on leukocytes also suggests integrin acquiring multiple affinity states (18Chigaev A. Blenc A.M. Braaten J.V. Kumaraswamy N. Kepley C.L. Andrews R.P. Oliver J.M. Edwards B.S. Prossnitz E.R. Larson R.S. Sklar L.A. J. Biol. Chem. 2001; 276: 48670-48678Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). For the integrin αLβ2, ligand ICAM-1 exhibits higher affinity for purified αLβ2 from T cells than ICAM-3 (19de Fougerolles A.R. Springer T.A. J. Exp. Med. 1992; 175: 185-190Crossref PubMed Scopus (393) Google Scholar). Prior exposure of αLβ2 to ICAM-1 increased its binding to ICAM-3 (20Buckley C.D. Ferguson E.D. Littler A.J. Bossy D. Simmons D.L. Eur. J. Immunol. 1997; 27: 957-962Crossref PubMed Scopus (10) Google Scholar). Soluble ICAM (sICAM)-3 binds to αLβ2 with 9-fold lower affinity than sICAM-1 (21Woska Jr., J.R. Morelock M.M. Jeanfavre D.D. Caviness G.O. Bormann B.J. Rothlein R. J. Biol. Chem. 1998; 273: 4725-4733Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). We also reported the requirement of two αLβ2-activating mAbs, KIM185 and KIM127, for their adhesion to ICAM-3 as compared with ICAM-1 (22Al-Shamkhani A. Law S.K. Eur. J. Immunol. 1998; 28: 3291-3300Crossref PubMed Scopus (15) Google Scholar). Crystal structures of engineered intermediate affinity αL I-domain (L161C/F299C) and high affinity αL I-domain (K287C/K294C) in complex with ICAM-1 showed differences in their interactions (23Shimaoka M. Xiao T. Liu J.H. Yang Y. Dong Y. Jun C.D. McCormack A. Zhang R. Joachimiak A. Takagi J. Wang J.H. Springer T.A. Cell. 2003; 112: 99-111Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar). Together, it is apparent that distinct αLβ2 affinity states could serve binding to different ICAM ligands. Structural data of intact αLβ2 inter-acting with ICAM are lacking. Hence, structural data derived from the studies of β3 and β1 integrin are useful as a hypothetical activation model to correlate αLβ2 functional states to its conformation. In this article we described the reporter mAb MEM148, which recognizes the free integrin β2 subunit or Mg2+/EGTA-activated αLβ2 but not resting αLβ2 (2Tan S.M. Robinson M.K. Drbal K. van Kooyk Y. Shaw J.M. Law S.K. J. Biol. Chem. 2001; 276: 36370-36376Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 24Drbal K. Angelisova P. Cerny J. Hilgert I. Horejsi V. Immunobiology. 2001; 203: 687-698Crossref PubMed Scopus (27) Google Scholar). We mapped the epitope of MEM148 to the hybrid domain, and the location of the epitope when modeled with the bent structure of αVβ3 faces the α subunit and could, therefore, be masked. Activation as a result of integrin extension could lead to the exposure of the epitope. In addition, we make use of the activation-reporter property of MEM148 to analyze the different αLβ2 affinity states required for ICAM-1 and ICAM-3 binding. Reagents and Antibodies—The following mAbs were gifts from different sources: MHM24 (anti-αL) and MHM23 (anti-β2, heterodimer dependent) were obtained from Prof. A. J. McMichael (John Radcliffe Hospital, Oxford, UK); KIM185 (anti-β2, activating mAb) was from Dr. M. Robinson (CellTech, Slough, UK); MEM48 (anti-β2, activating mAb) and MEM148 (anti-β2) were from Prof. V. Horejsi (Prague, Czech Republic). ICAM-1/Fc and ICAM-3/Fc were prepared as described previously (25Simmons D. Hartley D. Cellular Interactions in Development: A Practical Approach. IRL Press at Oxford University Press, Oxford1993: 93-128Google Scholar). cDNA Expression Constructs—The αL and β2 cDNAs in the expression vector pcDNA3 (Invitrogen) were described previously (26Douglass W.A. Hyland R.H. Buckley C.D. Al-Shamkhani A. Shaw J.M. Scarth S.L. Simmons D.L. Law S.K. FEBS Lett. 1998; 440: 414-418Crossref PubMed Scopus (26) Google Scholar, 27Wright A.H. Douglass W.A. Taylor G.M. Lau Y.L. Higgins D. Davies K.A. Law S.K. Eur. J. Immunol. 1995; 25: 717-722Crossref PubMed Scopus (38) Google Scholar). Previously, we had numbered the N-terminal Met of β2 as "1" (4Tng E. Tan S.M. Ranganathan S. Cheng M. Law S.K. J. Biol. Chem. 2004; 279: 54334-54339Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) and relevant references therein. For the purpose of clarity and ease of reference with other β2 and different β integrins functional and structural studies, herein β2 Met1 is re-numbered as Met-22 (28Barclay A.N. Brown M.H. Law S.K. McKnight A.J. Tomlinson M.G. van der Merwe P.A. The Leucocyte Antigen Facts Book. 2nd Ed. Academic Press, Ltd., London1997: 177-178Google Scholar). Construction of β2 Hu/Mo A in which Met-22-Asn562 of human integrin β2 was replaced with the corresponding region from mouse β2 was described previously (4Tng E. Tan S.M. Ranganathan S. Cheng M. Law S.K. J. Biol. Chem. 2004; 279: 54334-54339Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Amino acid substitutions on the mouse or human β2 constructs were made using the QuikChange™ site-directed mutagenesis kit (Stratagene). All constructs were verified by sequencing (DNA Sequencing Facility, Department of Biochemistry, University of Oxford, Oxford, UK and Research Biolabs sequencing service, Singapore). Cell Culture and Transfection—COS-7 (monkey kidney fibroblast) and MOLT-4 (human T lymphoblast) (ATCC, Manassas, VA) were cultured in complete media RPMI 1640 containing l-glutamine (JRH Biosciences Inc., Australia), 10% (v/v) heat-inactivated fetal bovine serum (Sigma), 100 IU/ml penicillin and 100 μg/ml streptomycin. For epitope mapping studies, COS-7 cells were transfected with wild-type β2 or variant cDNAs by the DEAE-dextran method (25Simmons D. Hartley D. Cellular Interactions in Development: A Practical Approach. IRL Press at Oxford University Press, Oxford1993: 93-128Google Scholar). Flow Cytometry Analysis—Flow cytometry analysis of integrin cell surface expression was performed as described previously (29Tan S.M. Hyland R.H. Al-Shamkhani A. Douglass W.A. Shaw J.M. Law S.K. J. Immunol. 2000; 165: 2574-2581Crossref PubMed Scopus (54) Google Scholar). Briefly, cells were incubated with 20 μg/ml primary mAb in RPMI 1640 for 1 h at 4 °C. Thereafter, cells were washed and incubated with FITC-conjugated sheep anti-mouse F(ab′)2 secondary Ab (1:400 dilution; Sigma) for 45 min at 4 °C. Stained cells were washed once and fixed in 1% (v/v) formaldehyde in PBS. Cells were analyzed on a FACSCalibur (BD Biosciences). Data were analyzed using CellQuest software (BD Biosciences). The expression index (EI) was calculated by % cells gated positive × geo-mean fluorescence intensity. Analysis of mAbs MEM148 and KIM127 Epitope Exposure—MOLT-4 cells were incubated in Mg/EGTA (5 mm MgCl2 and 1.5 mm EGTA) at 37 °C for 0.5 h together with MEM148 or KIM127. Cells were washed once followed by incubation with FITC-conjugated secondary antibody for 45 min at 4 °C. Treatment with EDTA was performed by subsequent incubation of Mg/EGTA-treated cells in 5 mm EDTA at 37 °C for 15 min followed by staining with the respective mAbs. Fluorescence staining was detected by flow cytometry. Washing experiments were performed by subjecting Mg/EGTA-treated cells to two or three washes in RPMI before incubating with antibodies. Cell Surface Labeling and Immunoprecipitation—-Briefly, MOLT-4 cells were washed twice in PBS followed by incubation in sulfo-NHS-biotin (Pierce) at 0.5 mg/ml in PBS for 20 min on ice. The reaction was quenched by washing surface-labeled cells once in PBS containing 10 mm Tris-HCl (pH 8.0) and 0.1% bovine serum albumin (29Tan S.M. Hyland R.H. Al-Shamkhani A. Douglass W.A. Shaw J.M. Law S.K. J. Immunol. 2000; 165: 2574-2581Crossref PubMed Scopus (54) Google Scholar). Thereafter, cells were incubated in Mg/EGTA (5 mm MgCl2 and 1.5 mm EGTA) or phorbol 12-myristate 13-acetate (PMA) (100 ng/ml) with mAbs (irrelevant mAb, MHM23, or MEM148) in RPMI media containing 5% (v/v) heat-inactivated fetal bovine serum and 10 mm HEPES (pH 7.4) for 30 min at 37 °C. Cells were washed twice with PBS and lysed in lysis buffer (10 mm Tris-HCl (pH 8.0), 150 mm NaCl, 1% (v/v) Nonidet P-40) with protease inhibitors followed by immunoprecipitation with rabbit anti-mouse IgG coupled to protein A-Sepharose beads (Sigma). Bound proteins were resolved by SDS-PAGE under reducing conditions and transferred onto Immobilon P membranes (Millipore, Bedford, MA), and protein bands were detected by ECL (GE Healthcare Amersham Biosciences) (29Tan S.M. Hyland R.H. Al-Shamkhani A. Douglass W.A. Shaw J.M. Law S.K. J. Immunol. 2000; 165: 2574-2581Crossref PubMed Scopus (54) Google Scholar). Immobilized Ligand Binding Assay—Adhesion of MOLT-4 cells to ICAM-1 or ICAM-3 coated on Polysorb microtiter wells (Nunc, Rosklide, Denmark) were performed as reported previously (29Tan S.M. Hyland R.H. Al-Shamkhani A. Douglass W.A. Shaw J.M. Law S.K. J. Immunol. 2000; 165: 2574-2581Crossref PubMed Scopus (54) Google Scholar). Briefly, each microtiter well was coated with 0.5 μg of goat anti-human IgG (Fcspecific) in 50 mm bicarbonate buffer (pH 9.2). Nonspecific binding sites were blocked with 0.5% (w/v) bovine serum albumin in PBS for 30 min at 37 °C. ICAM-Fc at 1 ng/μl or other specified concentrations in PBS containing 0.1% (w/v) bovine serum albumin was added to each well and incubated for 2 h at room temperature. Wells were washed twice in RPMI wash buffer (RPMI media containing 5% (v/v) heat-inactivated fetal bovine serum and 10 mm HEPES (pH 7.4)) before assay. Cells labeled with 3.0 mm 2′7′-bis-(2-carboxyethyl)-5-(and-6) carboxyfluorescein fluorescent dye (Molecular Probes, Eugene, OR) were incubated in RPMI wash buffer containing Mg/EGTA (5 mm MgCl2 and 1.5 mm EGTA) and/or activating mAb (10 μg/ml) to activate αLβ2-mediated adhesion. The activating mAbs are MEM48 and KIM185. αL-Specific function-blocking mAb MHM24 (10 μg/ml) was included to demonstrate binding specificity. Cell fluorescence, which indicates the number of cells adhering to ligand-coated wells, is measured using FL600 fluorescent plate reader (Bio-Tek Instruments, Winooski, VT). Soluble Ligand Binding Assay—sICAM-1 or sICAM-3 binding assay was performed as described with slight modifications (30Shimaoka M. Salas A. Yang W. Weitz-Schmidt G. Springer T.A. Immunity. 2003; 19: 391-402Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 31Yang W. Shimaoka M. Salas A. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2906-2911Crossref PubMed Scopus (77) Google Scholar). Soluble ICAMs were prepared by incubating 20 μg/ml ICAM-Fc with 100 μg/ml FITC-conjugated rabbit anti-human IgG Fc antibody (Pierce) in 50 μl of RPMI 1640 media containing 5% (v/v) heat-inactivated fetal bovine serum and 10 mm HEPES (pH 7.4) for 30 min at room temperature. Thereafter, purified mouse IgG was added to a final concentration of 100 μg/ml mAb for 20 min at room temperature. This was performed to quench cross-reactivity to mouse IgG, which was included in the experiment as activating or function-blocking mAb. Cells (2 × 105) were resuspended in this sICAM mixture with or without αLβ2-activating agents for 30 min at 37 °C. Cells were washed with RPMI media and fixed in 1% (v/v) formaldehyde in PBS followed by flow cytometry analyses. For activating studies, Mg/EGTA (5 mm MgCl2 and 1.5 mm EGTA) and/or activating mAbs (MEM48 or KIM185) (10 μg/ml) were used. For blocking studies, function-blocking αL-specific mAb MHM24 (10 μg/ml) was included in the samples. Mapping of mAb MEM148 Epitope—The epitope of mAb MEM148 resides in the integrin β2 hybrid domain and is not expressed in resting integrin αLβ2 but is exposed upon Mg/EGTA treatment (2Tan S.M. Robinson M.K. Drbal K. van Kooyk Y. Shaw J.M. Law S.K. J. Biol. Chem. 2001; 276: 36370-36376Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Because it may serve as a useful reporter mAb for subsequent analyses of integrin affinity states, we further characterize its epitope using a panel of integrin β2 human/mouse "knock-out" mutants generated by site-directed mutagenesis (4Tng E. Tan S.M. Ranganathan S. Cheng M. Law S.K. J. Biol. Chem. 2004; 279: 54334-54339Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). These human integrin β2 mutants have their residues replaced by corresponding mouse β2 residues at positions where they differ in the mid-region (Fig. 1B). COS-7 cells, which can express β2 integrin in the absence of the α subunit (2Tan S.M. Robinson M.K. Drbal K. van Kooyk Y. Shaw J.M. Law S.K. J. Biol. Chem. 2001; 276: 36370-36376Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), were transfected with the β2 human/mouse knock-out mutant cDNAs followed by immunofluorescence staining with mAb MEM148 and flow cytometry analyses. Expression of MEM148 epitope on transfectants was determined (Fig. 2A). The mAb MEM48, which maps to residues Leu534, Phe536, and His543 of human integrin β2 I-EGF 3 (32Lu C. Ferzly M. Takagi J. Springer T.A. J. Immunol. 2001; 166: 5629-5637Crossref PubMed Scopus (139) Google Scholar), was included as the receptor expression control. An approximately 3-fold reduction in MEM148 epitope expression was observed for transfectants expressing β2Hu(H370S/R371I) or β2Hu(N372G/Q373K) as compared with β2Hu(wt). The β2Hu(P374S) variant showed significant (>90%) abrogation of MEM148 epitope expression. We next employed an integrin β2 human/mouse chimera (β2Hu/MoA) (Fig. 1A) in which Met-22-Asn562 of human integrin β2 is replaced by the corresponding region from mouse integrin β2 (4Tng E. Tan S.M. Ranganathan S. Cheng M. Law S.K. J. Biol. Chem. 2004; 279: 54334-54339Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) to generate two "knock-in" mutants. The β2Hu/MoA(S374P) has the Ser374 of the mouse mid-region substituted by the corresponding human Pro residue. The β2Hu/MoA(A368V-S374P) has the segment Ala368-Ser374 of the mouse mid-region replaced by the corresponding human segment Val368-Pro374. In this case, mAb KIM185, which maps to integrin β2 I-EGF 4 and β-tail domain (32Lu C. Ferzly M. Takagi J. Springer T.A. J. Immunol. 2001; 166: 5629-5637Crossref PubMed Scopus (139) Google Scholar), was included as the receptor expression control because the epitope of MEM48 is absent in β2Hu/MoA. The expression of MEM148 epitope on β2Hu/MoA(S374P) was low as compared with β2Hu(wt) (Fig. 2B). However, transfectant bearing β2Hu/MoA(A368V-S374P) fully restored the epitope of MEM148. Thus, we may conclude that although human integrin β2 Pro374 is a critical residue, other residues, His370, Arg371, Asn372, Gln373, are also required for the effective presentation of MEM148 epitope. Model of αLβ2 Illustrating the MEM148 Epitope—Fine mapping of MEM148 epitope allows us to pinpoint its location in the quaternary structure of integrin. A model of integrin αLβ2 was generated by MODELLER using αVβ3 structural coordinates as the template (3Xiong J.P. Stehle T. Diefenbach B. Zhang R. Dunker R. Scott D.L. Joachimiak A. Goodman S.L. Arnaout M.A. Science. 2001; 294: 339-345Crossref PubMed Scopus (1088) Google Scholar) (Fig. 3A). The structure of an intact I-domain-containing integrin is not solved; hence, the αL I-domain was excluded from the model. For clarity, the αL Calf-2, β2 PSI, I-EGFs, and β-tail domains were not included. The critical residue Pro374 resides on the surface of the hybrid domain facing the αL subunit, in contrast to the epitope of mAb 7E4 (Val385) (4Tng E. Tan S.M. Ranganathan S. Cheng M. Law S.K. J. Biol. Chem. 2004; 279: 54334-54339Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), which is located away from the αL subunit (Fig. 3B). This could explain why mAb MEM148 fails to bind resting αLβ2; presumably it assumes a severely bent conformation similar to the resting αVβ3 (3Xiong J.P. Stehle T. Diefenbach B. Zhang R. Dunker R. Scott D.L. Joachimiak A. Goodman S.L. Arnaout M.A. Science. 2001; 294: 339-345Crossref PubMed Scopus (1088) Google Scholar) because its epitope is shielded in this conformation (Fig. 3C). Along the same line of reasoning, the binding of mAb MEM148 to activated αLβ2 adopting an extended conformation would, therefore, be favorable in conjunction with previous observations (2Tan S.M. Robinson M.K. Drbal K. van Kooyk Y. Shaw J.M. Law S.K. J. Biol. Chem. 2001; 276: 36370-36376Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 24Drbal K. Angelisova P. Cerny J. Hilgert I. Horejsi V. Immunobiology. 2001; 203: 687-698Crossref PubMed Scopus (27) Google Scholar). The Transition of αLβ2 from One Affinity State to Another Is Reversible—The mAb MEM148 does not bind to αLβ2 on MOLT-4 cells unless the cells were treated with Mg/EGTA (2Tan S.M. Robinson M.K. Drbal K. van Kooyk Y. Shaw J.M. Law S.K. J. Biol. Chem. 2001; 276: 36370-36376Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Divalent cations have a major influence on the α I-domain and β I-like domain, and it is widely accepted that the activated integrin should adopt an opened and/or extended conformation. However, defining the precise mechanism for the transition of integrin from one affinity state to the next in the presence of activating divalent cations remains challenging. Recently, several quaternary integrin conformations were proposed to depict such transitions (7Xiao T. Takagi J. Coller B.S. Wang J.H. Springer T.A. Nature. 2004; 432: 59-67Crossref PubMed Scopus (656) Google Scholar). Of note, the reversion from one conformation to another may be physiologically relevant to maintain a dynamic integrin population responding to different cellular activation milieu. To this end we determine whether Mg/EGTA-treated αLβ2 can revert to its resting state using MEM148 as the reporter mAb. MOLT-4 cells were incubated in Mg/EGTA-containing RPMI and either MEM148 or KIM127 at 37 °C. The epitope of KIM127 resides in β2 I-EGF 2, and like MEM148, its epitope expression depends on integrin activation (32Lu C. Ferzly M. Takagi J. Springer T.A. J. Immunol. 2001; 166: 5629-5637Crossref PubMed Scopus (139) Google Scholar, 33Robinson M.K. Andrew D. Rosen H. Brown D. Ortlepp S. Stephens P. Butcher E.C. J. Immunol. 1992; 148: 1080-1085PubMed Google Scholar). Significant staining was detected for both mAbs only in the presence of Mg/EGTA (Fig. 4A). The integrin αL-specific mAb MHM24 was included as a control. Next, cells in the presence of Mg/EGTA were subsequently treated with EDTA to deplete existing Mg2+ before incubation with respective mAbs. Epitope expressions of MEM148 and KIM127 were significantly reduced in these samples, whereas control mAb MHM24 staining was not affected. We next tested whether Mg/EGTA-treated cells after washing in media could still express the epitopes of MEM148 and KIM127 (Fig. 4B). Mg/EGTA-treated cells were subjected to three washes in RPMI without additives followed by staining for either of the two mAbs. Low levels of MEM148 or KIM127 epitope expression were detected as compared with Mg/EGTA-treated cells without washing. Cell binding assay to ICAM-1 was investigated to determine whether Mg/EGTA-treated cells followed by washing could still adhere to ICAM-1 (Fig. 4C). In the presence of Mg/EGTA, cells adhered significantly to ICAM-1. However, adhesion was minimal when cells were treated with Mg/EGTA followed by two or three washes in RPMI wash buffer before allowing adherence to ICAM-1. Binding specificity was shown by including αL-specific function-blocking mAb MHM24. Together, our data suggest that reversion of Mg/EGTA-activated αLβ2 to its resting conformation is possible when the activating cation is depleted. We furthered our analyses by changing the order of MOLT-4 treatment with different agents to test whether MEM148 remains bound to Mg/EGTA-treated cells even in the presence of EDTA. Cells were first treated with Mg/EGTA in the presence of MEM148 with subsequent treatment with EDTA or additional washes in media (Fig. 4D). MEM148 staining was detected in both cases. This suggests that the αLβ2·MEM148 complex is stable even when divalent cations were depleted or by extensive washing in media. PMA is known to promote αLβ2 ligand binding (4Tng E. Tan S.M. Ranganathan S. Cheng M. Law S.K. J. Biol. Chem. 2004; 279: 54334-54339Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 34Rothlein R. Springer T.A. J. Exp. Med. 1986; 163: 1132-1149Crossref PubMed Scopus (400) Google Scholar). PMA also induced the expression of MEM148 epitope on myeloid cells, and this was contributed by a proteolytically cleaved fragment of β2 unassociated with the αL, αM, or αX subunit rather than the respective heterodimers (35Drbal K. Angelisova P. Hilgert I. Cerny J. Novak P. Horejsi V. Blood. 2001; 98: 1561-1566Crossref PubMed Scopus (20) Google Scholar). To test whether PMA induced MEM148 epitope expression on MOLT-4, cells were surface-labeled with biotin followed by treatment with Mg/EGTA
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