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• W2122561878 abstract "Integrin αMβ2 (Mac-1, CD11b/CD18) is a noncovalently linked heterodimer of αM and β2 subunits on the surface of leukocytes, where it plays a pivotal role in the adhesion and migration of these cells. Using HEK293 cells expressing αMβ2 or the individual constituent chains on their surface, we analyzed the contributions of the αM or β2 subunits to functional responses mediated by the integrin. In cells expressing only αM or β2, the individual subunits were not associated with the endogenous integrins of the cells, and other partners for the subunits were not detected by surface labeling and immunoprecipitation under a variety of conditions. The αM cells mediated adhesion and spreading on a series of αMβ2 ligands (fibrinogen, Factor X, iC3b, ICAM-1 (intercellular adhesion molecule-1), and denatured ovalbumin) but could not support cell migration to any of these. The spreading of the αM cells suggested an unanticipated linkage of this subunit to the cytoskeleton. The β2 cells supported migration and attachment but not spreading on a subset of the αMβ2 ligands. The heterodimeric receptor and its individual subunits were purified from the cells by affinity chromatography and recapitulated the ligand binding properties of the corresponding cell lines. These data indicate that each subunit of αMβ2 contributes distinct properties to αMβ2 and that, in most but not all cases, the response of the integrin is a composite of the functions of its individual subunits. Integrin αMβ2 (Mac-1, CD11b/CD18) is a noncovalently linked heterodimer of αM and β2 subunits on the surface of leukocytes, where it plays a pivotal role in the adhesion and migration of these cells. Using HEK293 cells expressing αMβ2 or the individual constituent chains on their surface, we analyzed the contributions of the αM or β2 subunits to functional responses mediated by the integrin. In cells expressing only αM or β2, the individual subunits were not associated with the endogenous integrins of the cells, and other partners for the subunits were not detected by surface labeling and immunoprecipitation under a variety of conditions. The αM cells mediated adhesion and spreading on a series of αMβ2 ligands (fibrinogen, Factor X, iC3b, ICAM-1 (intercellular adhesion molecule-1), and denatured ovalbumin) but could not support cell migration to any of these. The spreading of the αM cells suggested an unanticipated linkage of this subunit to the cytoskeleton. The β2 cells supported migration and attachment but not spreading on a subset of the αMβ2 ligands. The heterodimeric receptor and its individual subunits were purified from the cells by affinity chromatography and recapitulated the ligand binding properties of the corresponding cell lines. These data indicate that each subunit of αMβ2 contributes distinct properties to αMβ2 and that, in most but not all cases, the response of the integrin is a composite of the functions of its individual subunits. Integrins are a large family of heterodimeric cell adhesion receptors that mediate a wide spectrum of biological functions (reviewed in Ref. 1Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6892) Google Scholar). The β2 subfamily, often referred to as the leukocyte integrins, is composed of four members that share a common β2 subunit that associates noncovalently with one of four distinct but structurally homologous α subunits to form integrins αMβ2 (Mac-1, CD11b/CD18, CR3), αLβ2 (lymphocyte function-associated antigen-1, CD11a/CD18), p150/95 (αXβ2, CD11c/CD18), and αDβ2 (CD11d/CD18) (reviewed in Refs. 2Larson R.S. Springer T.A. Immunol. Rev. 1990; 114: 181-217Crossref PubMed Scopus (518) Google Scholar and 3Harris E.S. McIntyre T.M. Prescott S.M. Zimmerman G.A. J. Biol. Chem. 2000; 275: 23409-23412Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). αMβ2 is expressed in monocytes, granulocytes, macrophages, and natural killer cells and has been implicated in diverse responses of these cells, including phagocytosis, cell-mediated killing, chemotaxis, and cellular activation. These complex responses depend upon the capacity of αMβ2 to mediate leukocyte adhesion and migration; and consequently, αMβ2 plays a central role in inflammation. The characterization of αMβ2-deficient mice has confirmed these findings by showing that a variety of leukocyte-dependent responses are compromised in these animals (e.g. Refs. 4Ding Z.M. Babensee J.E. Simon S.I. Lu H.F. Perrard J.L. Bullard D.C. Dai X.Y. Bromley S.K. Dustin M.L. Entman M.L. Smith C.W. Ballantyne C.M. J. Immunol. 1999; 163: 5029-5038Crossref PubMed Google Scholar and 5Coxon A. Rieu P. Barkalow F.J. Askari S. Sharpe A.H. Von Andrian U.H. Arnaout M.A. Mayadas T.N. Immunity. 1996; 5: 653-666Abstract Full Text PDF PubMed Scopus (544) Google Scholar). The capacity of αMβ2 to support adhesion and migration depends upon its ability to recognize and mediate responses to a diverse set of structurally unrelated ligands, including human fibrinogen (Fg) 1The abbreviations used are: Fg, fibrinogen; FX, Factor X; MIDAS, metal ion-dependent adhesion site; mAb, monoclonal antibody; HEK293, human epithelial kidney 293; HPLC, high pressure liquid chromatography; FACS, fluorescence-activated cell sorting; HBSS, Hanks' balanced salt solution; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; dOva, denatured ovalbumin; PVP, polyvinylpyrrolidone; SDL, specificity-determining loop. (6Altieri D.C. Mannucci P.M. Capitanio A.M. J. Clin. Investig. 1986; 78: 968-976Crossref PubMed Scopus (101) Google Scholar, 7Wright S.D. Weitz J.I. Huang A.J. Levin S.M. Silverstein S.C. Loike J.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7734-7738Crossref PubMed Scopus (367) Google Scholar), complement fragment iC3b (inactivated complement component 3b) (8Beller D.E. Springer T.A. Schreiber R.D. J. Exp. Med. 1982; 156: 1000-1010Crossref PubMed Scopus (405) Google Scholar, 9Arnaout M.A. Todd R.F. II I Dana N. Melamed J. Schlossman S.F. Colten H.R. J. Clin. Investig. 1983; 72: 171-179Crossref PubMed Scopus (182) Google Scholar), ICAM-1 (intercellular adhesion molecule-1; CD54) (10Diamond M.S. Staunton D.E. de Fougerolles A.R. Stacker S.A. Garcia-Aguilar J. Hibbs M.L. Springer T.A. J. Cell Biol. 1990; 111: 3129-3139Crossref PubMed Scopus (778) Google Scholar), hookworm neutrophil inhibitory factor (11Muchowski P.J. Zhang L. Chang E.R. Soule H.R. Plow E.F. Moyle M. J. Biol. Chem. 1994; 269: 26419-26423Abstract Full Text PDF PubMed Google Scholar, 12Rieu P. Ueda T. Haruta I. Sharma C.P. Arnaout M.A. J. Cell Biol. 1994; 127: 2081-2091Crossref PubMed Scopus (99) Google Scholar), blood coagulation Factor X (FX) (13Altieri D.C. Edgington T.S. J. Biol. Chem. 1988; 263: 7007-7015Abstract Full Text PDF PubMed Google Scholar), and denatured proteins (14Davis G.E. Biochem. Biophys. Res. Commun. 1992; 182: 1025-1031Crossref PubMed Scopus (299) Google Scholar), as well as numerous bacterial and fungal products (e.g. Refs. 15Wright S.D. Jong M.T. J. Exp. Med. 1986; 164: 1876-1888Crossref PubMed Scopus (299) Google Scholar, 16Dabrosin C. Gyorffy S. Margetts P. Ross C. Gauldie J. Am. J. Pathol. 2002; 161: 909-918Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 17Vetvicka V. Thornton B.P. Ross G.D. J. Clin. Investig. 1996; 98: 50-61Crossref PubMed Scopus (361) Google Scholar). iC3b and Fg are two particularly important ligands of αMβ2 in mediating the inflammatory response to invasive agents or foreign materials (18Altieri D.C. Agbanyo F.R. Plescia J. Ginsberg M.H. Edgington T.S. Plow E.F. J. Biol. Chem. 1990; 265: 12119-12122Abstract Full Text PDF PubMed Google Scholar, 19Sanchez-Madrid F. Nagy J.A. Robbins E. Simon P. Springer T.A. J. Exp. Med. 1983; 158: 1785-1803Crossref PubMed Scopus (611) Google Scholar, 20Forsyth C.B. Solovjov D.A. Ugarova T.P. Plow E.F. J. Exp. Med. 2001; 193: 1123-1133Crossref PubMed Scopus (102) Google Scholar). In recognizing Fg, two peptide sequences within its γ chain that interact with αMβ2 have been identified: P1 (γ chain amino acids 190–202) (21Altieri D.C. Plescia J. Plow E.F. J. Biol. Chem. 1993; 268: 1847-1853Abstract Full Text PDF PubMed Google Scholar) and P2 (γ chain amino acids 377–395) (22Ugarova T.P. Solovjov D.A. Zhang L. Loukinov D.I. Yee V.C. Medved L.V. Plow E.F. J. Biol. Chem. 1998; 273: 22519-22527Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Two segments, one within each of its constituent chains, have been implicated in the binding of protein ligands to αMβ2: 1) the αMI (or A) domain, an ∼200-amino acid segment in the N-terminal third of the αM subunit that is structurally very similar to the I domains within the α subunits of the other leukocyte integrins (23Lee J-O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (805) Google Scholar, 24Leitinger B. Hogg N. Biochem. Soc. Trans. 1999; 27: 826-832Crossref PubMed Scopus (19) Google Scholar), and 2Larson R.S. Springer T.A. Immunol. Rev. 1990; 114: 181-217Crossref PubMed Scopus (518) Google Scholar) the β2I-like domain, which is structurally similar to the α subunit I domains as well as the I-like domains found in all β subunits of the integrins (25Tozer 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, 26Xiong 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 (1113) Google Scholar, 27Shimaoka M. Shifman J.M. Jing H. Takagi L. Mayo S.L. Springer T.A. Nat. Struct. Biol. 2000; 7: 674-678Crossref PubMed Scopus (117) Google Scholar). Each of these segments contains a metal ion-dependent adhesion site (MIDAS), which is critical to the ligand binding functions of αMβ2 and other integrins (23Lee J-O. Rieu P. Arnaout M.A. Liddington R. Cell. 1995; 80: 631-638Abstract Full Text PDF PubMed Scopus (805) Google Scholar, 28Qu A. Leahy D.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10277-10281Crossref PubMed Scopus (290) Google Scholar, 29Lee J-O. Bankston L.A. Arnaout M.A. Liddington R.C. Structure. 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 30Calderwood D.A. Tuckwell D.S. Eble J. Kuhn K. Humphries M.J. J. Biol. Chem. 1997; 272: 12311-12317Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). One manifestation of the contribution of the MIDAS to ligand binding is that mutagenesis of the predicted cation coordination residues of the β2 or αM MIDAS destroys the capacity of αMβ2 to bind multiple ligands (e.g. Refs. 31Bajt M.L. Goodman T. McGuire S.L. J. Biol. Chem. 1995; 270: 94-98Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 32Goodman T.G. Bajt M.L. J. Biol. Chem. 1996; 271: 23729-23736Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 33Zhang L. Plow E.F. J. Biol. Chem. 1996; 271: 18211-18216Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 34Seow K. Xiong J. Arnaout M. Welge J. Rippmann F. Goodman S. Biochem. Pharmacol. 2002; 64: 805-812Crossref PubMed Scopus (3) Google Scholar). A second manifestation of the MIDAS contribution is that different divalent cations exert differential effects on ligand binding. Mn2+ enhances ligand binding function, Mg2+ supports it, and Ca2+ can be suppressive (35Dransfield I. Cabanas C. Craig A. Hogg N. J. Cell Biol. 1992; 116: 219-226Crossref PubMed Scopus (400) Google Scholar, 36Leitinger B. McDowall A. Stanley P. Hogg N. Biochim. Biophys. Acta. 2000; 1498: 91-98Crossref PubMed Scopus (136) Google Scholar, 37Li R. Rieu P. Griffith D.L. Scott D. Arnaout M.A. J. Cell Biol. 1998; 143: 1523-1534Crossref PubMed Scopus (123) Google Scholar). The crystal structures of the αMI domain or other I domains show that different cations differentially influence structure (29Lee J-O. Bankston L.A. Arnaout M.A. Liddington R.C. Structure. 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar). Direct evidence for the involvement of the αM subunit in ligand binding has derived from multiple studies in which its I domain has been expressed and shown to engage ligand (11Muchowski P.J. Zhang L. Chang E.R. Soule H.R. Plow E.F. Moyle M. J. Biol. Chem. 1994; 269: 26419-26423Abstract Full Text PDF PubMed Google Scholar, 38Michishita M. Videm V. Arnaout M.A. Cell. 1993; 72: 857-867Abstract Full Text PDF PubMed Scopus (318) Google Scholar, 39Ustinov V.A. Plow E.F. J. Biol. Chem. 2002; 277: 18769-18776Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Evidence for the involvement of the β subunit is less direct. Certain mutations in the β2 subunit abolish the binding of multiple ligands (31Bajt M.L. Goodman T. McGuire S.L. J. Biol. Chem. 1995; 270: 94-98Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 33Zhang L. Plow E.F. J. Biol. Chem. 1996; 271: 18211-18216Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and certain ligands (iC3b and FX) are still capable of binding to recombinant αMβ2 lacking an αMI domain (40Yalamanchili P. Lu C.F. Oxvig C. Springer T.A. J. Biol. Chem. 2000; 275: 21877-21882Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Rather than a direct involvement of the β subunit, the β2I domain may regulate the function of the αM subunit. Consistent with such a regulatory role, epitopes of several monoclonal antibodies (mAbs) that activate the β2 integrins, KIM185, KIM127, MEM48, and CBR lymphocyte function-associated antigen-1/2, reside in or close to the β2I domain (41Ortlepp S. Stephens P.E. Hogg N. Figdor C.G. Robinson M.K. Eur. J. Immunol. 1995; 25: 637-643Crossref PubMed Scopus (58) Google Scholar, 42Bazil V. Strominger J.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3792-3796Crossref PubMed Scopus (87) Google Scholar, 43Yan S.R. Huang M. Berton G. J. Immunol. 1997; 158: 1902-1910PubMed Google Scholar), and cells expressing only the αM subunit in the absence of β2 appear to bind ligands (such as Fg) that do not bind to αMβ2 in which residues in the β2 MIDAS motif have been mutated (22Ugarova T.P. Solovjov D.A. Zhang L. Loukinov D.I. Yee V.C. Medved L.V. Plow E.F. J. Biol. Chem. 1998; 273: 22519-22527Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Thus, the contribution of the β2 subunit to ligand recognition by αMβ2 remains uncertain. In this study, we took advantage of the capacity of human epithelial kidney 293 (HEK293) cells to express the individual αM or β2 subunits or heterodimeric αMβ2 on their surface (22Ugarova T.P. Solovjov D.A. Zhang L. Loukinov D.I. Yee V.C. Medved L.V. Plow E.F. J. Biol. Chem. 1998; 273: 22519-22527Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 44Xiong Y-M. Zhang L. J. Biol. Chem. 2001; 276: 19340-19349Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The adhesive and migratory properties of the cells were analyzed, and the cells also were used as a source to purify and characterize the individual subunits. These comparisons allowed us demonstrate that both subunits can contribute directly to ligand binding and to assign specific functions to each subunit of the receptor. Monoclonal Antibodies, Proteins, and Synthetic Peptides—The mAbs used in this study were 44a (anti-CD11b), OKM1 (anti-CD11b), LM2/1 (anti-CD11b), 904 (anti-CD11b), IB4 (anti-CD18), TS1/18 (anti-CD18), and W6/32 (anti-major histocompatibility complex class I). The hybridoma cell lines producing these mAbs were obtained from American Type Culture Collection, and the mAbs were purified from mouse ascites using recombinant protein G columns (Zymed Laboratories Inc., South San Francisco, CA). mAb 68-5A5 (anti-CD18) was from Neo Markers (Fremont, CA), and MEM148 was purchased from Serotec (Palo Alto, CA). mAbs to integrins α4 (clone P1H4), αV (P3G8), β1 (12G10), and β3 (25E11) were obtained from Chemicon International, Inc. (Temecula, CA). Recombinant human ICAM-1 was purchased from R&D Systems (Minneapolis, MN). Bovine serum albumin, ovalbumin, and human iC3b were obtained from Calbiochem. Human Fg and FX were from Enzyme Research Laboratories (Bethesda, MD). Hookworm neutrophil inhibitory factor was a gift from Corvas International (San Diego, CA). Peptides P1 (GWTVFQKRLDGSV) and P2-C (KIIPFNRLTIG) were synthesized on an Applied Biosystems Model 430A peptide synthesizer and purified by HPLC as described (22Ugarova T.P. Solovjov D.A. Zhang L. Loukinov D.I. Yee V.C. Medved L.V. Plow E.F. J. Biol. Chem. 1998; 273: 22519-22527Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Development of αMβ2, αLβ2, αM, and β2 Cell Lines—HEK293 cells were stably transfected using Lipofectamine Plus reagent (Invitrogen) with 0.5–5 μg of pcDNA3.1 (Invitrogen) containing the full-length cDNAs for αM and/or β2 or vector alone (mock-transfected) as a control as described previously (33Zhang L. Plow E.F. J. Biol. Chem. 1996; 271: 18211-18216Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 45Zhang L. Plow E.F. J. Biol. Chem. 1996; 271: 29953-29957Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Transfected cells were selected using neomycin sulfate (Invitrogen), and cells expressing the receptors were detected and sorted by flow cytometry (FACS) using a FACStar instrument (BD Biosciences) and mAb 904, IB4 or TS1/18. The cell lines obtained were maintained in Dulbecco's modified Eagle's medium/nutrient mixture F-12 supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, 2 μm l-glutamine, and 2 mg/ml neomycin (all from Invitrogen) (33Zhang L. Plow E.F. J. Biol. Chem. 1996; 271: 18211-18216Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). For selected experiments, a HEK cell line expressing both αMβ2 and the urokinase-type plasminogen activator receptor was used; these cells have been described previously (46Pluskota E. Solovjov D.A. Plow E.F. Blood. 2003; 101: 1582-1590Crossref PubMed Scopus (101) Google Scholar). To verify that the specificity-determining loop was present and that β2I domain was not mutated in the β2 cells, total RNA was isolated from 1.5 × 105 β2 cells, and cDNA was prepared using the QIAamp DNA mini kit (Qiagen Inc., Santa Clara, CA) according to the manufacturer's protocol. The cDNA obtained was used as template for PCR performed with 5′-GATCCTGACTCCATTCGCTGCGACACCCGGCC-3′ (β2238) as the 5′-primer and 5′-TCCATTGCTGCAGAAGGAGTCGTAGG-3′ (β21239) as the 3′-primer. After amplification, the PCR product was isolated by electrophoresis on 1% agarose gels, and only a band of ∼1 kb was detected. This band was extracted from the gels using the QIAquick gel extraction Kit (Qiagen Inc.) according the manufacturer's protocol, and the sample was concentrated by ethanol precipitation and sequenced using β2238 or 5′-GACCAGGCCAGGCAGCAGCGTTCAACGTGACC-3′ (β2395) as the sequencing primer. FACS—Transfected HEK293 cells were harvested with cell dissociation buffer (Invitrogen); washed twice; suspended in staining medium consisting of Hanks' balanced salt solution (HBSS) containing 5 mm CaCl2, 5 mm MgCl2, 10 mm HEPES (pH 7.4), and 0.1% goat normal serum; and incubated at 4 °C for 30 min with the selected primary mAb at 10 μg/ml. In some experiments, the divalent cations were replaced with 1 mm EDTA. After incubation, the cells were washed twice by centrifugation and resuspended in 30 μg/ml fluorescein-conjugated goat anti-mouse IgG (Zymed Laboratories Inc.) for 30 min at 4 °C in the dark. The cells were washed three times with HBSS/HEPES, and cell-bound antibodies were detected using a FACScan. Data were analyzed using the LYSIS program (BD Biosciences). The mean fluorescence intensity of the αMβ2, αLβ2, αM, and β2 cells used was 150–400 units when staining with an appropriate mAb (see “Results”) compared with ∼5 units for mock-transfected cells and <10 units for a non-reactive mAb. Immunoprecipitation of Surface-expressed Proteins—Transfected HEK293 cells were harvested with cell dissociation buffer as described above, washed twice with Dulbecco's phosphate-buffered saline (PBS), and resuspended to 108 cells/ml in Dulbecco's PBS. Surface-expressed proteins were labeled with EZ-Link™ sulfosuccinimidyl 6-(biotinamido)hexanoate (Pierce) according to the manufacturer's protocol. After 30 min, non-reacted sulfo-NHS-LC-biotin was removed by washing three times with HBSS/HEPES; the cells were resuspended in HBSS/HEPES to 108 cells/ml; and mAb 44a (for the αM cells) or IB4 (for all other cells) was added. After incubation for 60 min at 4 °C, non-bound antibody was removed by washing three times with HBSS/HEPES, and 109 cells were pelleted by centrifugation and solubilized at 4 °C with lysis buffer composed of Tris-buffered saline (TBS) (pH 7.4) containing 5 mm CaCl2, 5 mm MgCl2, protease inhibitor mixture for mammalian cells (Sigma) and one of the following detergents: 5% Triton X-100 (Fisher), 5% Tween 20 (Fisher), 1% CHAPS (Sigma), or 20 mmn-octyl β-d-glucopyranoside (Calbiochem). After mixing at 4 °C for 20 min, the samples were clarified by centrifugation, and 15 μl of protein G-agarose (Pierce) pretreated with non-transfected HEK293 cell lysate were added. After overnight incubation at 4 °C, the beads were collected by centrifugation and washed four times with cold cell lysis buffer. After the last wash, 60 μl of SDS electrophoresis endurance loading buffer (ICS BioExpress, Kaysville, UT) were added; samples were boiled for 2 min; and bound proteins were separated by electrophoresis in 4–20% polyacrylamide gel plates (ISC BioExpress) using a Tris/SDS/HEPES buffer system. Separated proteins were transferred from the gel onto polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA), and protein bands were developed using horseradish peroxidase-conjugated streptavidin (Pierce) and Opti-4CN-amplified substrate for horse-radish peroxidase (Bio-Rad). Receptor and Subunit Purification—αMβ2, αM, and β2 were purified from transfected HEK293 cells using a modification of the method of Miller et al. (47Miller L.J. Wiebe J. Springer T.A. J. Immunol. 1987; 138: 2381-2383PubMed Google Scholar). Briefly, 10 g of each cell line were harvested, washed, and lysed with 20 ml of 1% Triton X-100 in TBS containing 0.5 ml of protease inhibitor mixture for mammalian cells and 5 mm EDTA for 30 min at 4 °C. The cell lysate was clarified by centrifugation, diluted 4-fold with TBS, and loaded onto a mAb column (1 × 4 cm) at 4 °C. For αM purification, immobilized mAb LM2/1 (anti-αM) was used; and for αMβ2 and β2 purification, immobilized mAb IB4 (anti-β2) was employed. To prepare the immunoaffinity columns, purified mAbs were coupled to CNBr-activated Sepharose 4B (Amersham Biosciences) according to the manufacturer's protocol to final concentrations of 1.8–2.2 mg of immobilized protein/1 ml of swollen gel. After washing with 100 ml of TBS containing 10 mmn-octyl β-d-glucopyranoside and 1 mm CaCl2, bound protein was eluted with 2 column volumes of 20 mm sodium acetate buffer (pH 4.2) containing 10 mmn-octyl β-d-glucopyranoside. Immediately after elution, the pH of the eluates was adjusted to pH 7.2 with 1 m Tris. In addition to SDS-PAGE (see “Results”), the purified β2 subunit from these cells was characterized by HPLC gel filtration chromatography on a 7.5 mm × 60 cm UltroPac TSK G4000SW column (LKB Bromma, Uppsala, Sweden) in 0.15 m NaCl and 0.05 m Tris (pH 7.4). Ligand Binding Assays—Na125I (specific activity of 15 mCi of 125I/mg of iodine; Amersham Biosciences) was used to radioiodinate purified αMβ2, αM, and β2 by a modified chloramine-T procedure (48Miles L.A. Plow E.F. J. Biol. Chem. 1985; 260: 4303-4311Abstract Full Text PDF PubMed Google Scholar). Upon SDS-PAGE followed by autoradiography, radiolabeled αMβ2 showed only two bands with estimated molecular masses of ∼150 and 90 kDa, and radiolabeled αM and β2 showed only one band each with molecular masses of 150 and 90 kDa, respectively. The patterns and mobilities were indistinguishable from those of the unlabeled forms of the receptor and its subunits. The radiolabeled proteins were stored at -20 °C and used within 1 month. Fg, FX, iC3b, ICAM-1, and denatured ovalbumin (dOva) were biotinylated using EZ-Link sulfosuccinimidyl 6-(biotinamido)hexanoate according to the manufacturer's protocol. Each biotinylated protein (2 nm) was mixed with 0.5 ml of Ultra-Link immobilized streptavidin (Pierce) and incubated for 1 h at 4 °C. The non-reacted streptavidin was blocked with 1% biotin; and the beads were washed three times with 20 ml of TBS, stored at 4 °C, and used within 1 week. To determine binding of the ligands to the radiolabeled receptor or its subunits, 20 μl of the ligand beads were incubated with 10 μg of radioiodinated αMβ2, αM, or β2 for 30 min at 37 °C in the presence of 2 mm MnCl2 and then washed five times with TBS containing 2 mm MnCl2 and 10 mmn-octyl β-d-glucopyranoside. The amount of radioactivity retained by the beads was measured using an Isotec γ counter (ICN Flow Titertec, ICN Biomedicals, Irvine, CA). Each point is the mean ± S.E. of three independent experiments. Cell Attachment/Adhesion Assays—48-Well Costar tissue culture plates were coated with 200 μl of different concentrations (0–100 nm) of iC3b, Fg, FX, ICAM-1, or dOva overnight at 4 °C and then post-coated with 0.5% polyvinylpyrrolidone (PVP) for 1 h at room temperature (33Zhang L. Plow E.F. J. Biol. Chem. 1996; 271: 18211-18216Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Control wells were coated with PVP only. Prior to use, the plates were rinsed three times with PBS. Transfected HEK293 cells were harvested as described above, washed three times with 50 ml of divalent ion-free HBSS/HEPES (pH 7.4), and resuspended in divalent ion-free HBSS/HEPES, and then the selected divalent cations were added. The cells were seeded at 1.5–2 × 105 cells/well onto the assay plates and incubated at 37 °C for 30 min. For inhibition experiments, the cells were pretreated with the selected mAbs or other reagents for 15 min at 37 °C prior to addition to the coated wells. To determine the extent of attachment/adhesion, the plates were washed three times with PBS, and the number of adherent cells in each well was quantified using the Cyquant cell proliferation assay kit (Molecular Probes, Inc., Eugene, OR) according to the manufacturer's instructions. Briefly, after washing, the plates were frozen at -70 °C for 4 h and thawed in the presence of cell lysis buffer containing green fluorescent dye, which can be incorporated into DNA. After 30 min at room temperature in the dark, the fluorescence was measured using a CytoFluor II fluorescence multiwell plate reader (Molecular Devices, Inc., Sunnyvale, CA) using an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The data from cell adhesion and migration assays (see below) are presented as mean fluorescence intensity ± S.D. of three independent experiments. To distinguish attached and spread cells from those attached but not spread, after adhesion, the plates were rinsed three times with PBS, and the adherent cells were immediately photographed (magnification ×200). The spread cells were quantified as a percent of the attached cells, counting a total of 1000 cells. Cell Migration Assays—Cell migration assays were performed in serum-free Dulbecco's modified Eagle's medium/nutrient mixture F-12 using Costar 24-transwell plates with tissue culture-treated 8-μm pore polycarbonate filters (Corning Inc.) as described previously (20Forsyth C.B. Solovjov D.A. Ugarova T.P. Plow E.F. J. Exp. Med. 2001; 193: 1123-1133Crossref PubMed Scopus (102) Google Scholar). The lower chambers contained 600 μl of medium with the selected ligands, and the upper chambers contained final volumes of 200 μl after addition of the cells. To begin the assay, 50 μl of cell suspension (2 × 105 cells/well) in medium were added to the upper chambers, and the plates were placed in a humidified incubator at 37 °C and 5% CO2. Assays were stopped after 16 h by removing the upper wells and wiping the inside of the upper wells three times with a cotton swab to remove non-migrated cells. The migrated cells, present on the undersurface of the membrane as well as in the lower chambers, were quantitated using the Cyquant cell proliferation assay kit as described above. Integrin Clustering and Immunofluorescence—Transfected cell lines were seeded onto CC2™-treated Lab-Tek® Chamber Slides™ (Nalge Nunc International, Naperville, IL) at 5 × 104 cells/well in culture medium and incubated for 16 h at 37 °C and 5% CO2. The cells were washed, and antibody against the integrin α4 or β1 subunit was added at 10 μg/ml in HBSS (pH 7.3) containing 1 mm CaCl2, 1 mm MgCl2, and 1% bovine serum albumin and incubated for 30 min at 4 °C. Cells were washed and incubated with Alexa 568-conjugated F(ab′)2 fragments of goat anti-mouse IgG (Molecular Probes, Inc.) at 10 μg/ml for 30 min at 4 °C. After washing, integrin cluster formation was allowed to occur at 37 °C for 30–45 min. Next, cells were fixed with 4% paraformaldehyde for 20 min at 22 °C and stained with biotinylated mAb to the αM subunit (44a) or the β2 subunit (IB4) for 30 min at 22 °C, followed by incubation with Alexa 488-conjugated avidin (1:500 dilution; Molecular Probes, Inc.) for 30 min at 22 °C. To analyze integrin localization and clustering on resting cells, the cells were first fixed and then stained to visualize the specific subunits as described above. The slides were mounted using Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA) and observed under a fluorescence microscope (Leica Inc., Bannockburn, IL" @default.
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• W2122561878 title "Distinct Roles for the α and β Subunits in the Functions of Integrin αMβ2" @default.
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