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• W2044037317 abstract "The leukocyte integrin αLβ2 mediates cell adhesion and migration during inflammatory and immune responses. Ligand binding of αLβ2 is regulated by or induces conformational changes in the inserted (I) domain. By using a micropipette, we measured the conformational regulation of two-dimensional (2D) binding affinity and the kinetics of cell-bound intercellular adhesion molecule-1 interacting with αLβ2 or isolated I domain expressed on K562 cells. Locking the I domain into open and intermediate conformations with a disulfide bond increased the affinities by ∼8000- and ∼30-fold, respectively, from the locked closed conformation, which has similar affinity as the wild-type I domain. Most surprisingly, the 2D affinity increases were due mostly to the 2D on-rate increases, as the 2D off-rates only decreased by severalfold. The wild-type αLβ2, but not its I domain in isolation, could be up-regulated by Mn2+ or Mg2+ to have high affinities and on-rates. Locking the I domain in any of the three conformations abolished the ability of divalent cations to regulate 2D affinity. These results indicate that a downward displacement of the I domain C-terminal helix, induced by conformational changes of other domains of the αLβ2, is required for affinity and on-rate up-regulation. The leukocyte integrin αLβ2 mediates cell adhesion and migration during inflammatory and immune responses. Ligand binding of αLβ2 is regulated by or induces conformational changes in the inserted (I) domain. By using a micropipette, we measured the conformational regulation of two-dimensional (2D) binding affinity and the kinetics of cell-bound intercellular adhesion molecule-1 interacting with αLβ2 or isolated I domain expressed on K562 cells. Locking the I domain into open and intermediate conformations with a disulfide bond increased the affinities by ∼8000- and ∼30-fold, respectively, from the locked closed conformation, which has similar affinity as the wild-type I domain. Most surprisingly, the 2D affinity increases were due mostly to the 2D on-rate increases, as the 2D off-rates only decreased by severalfold. The wild-type αLβ2, but not its I domain in isolation, could be up-regulated by Mn2+ or Mg2+ to have high affinities and on-rates. Locking the I domain in any of the three conformations abolished the ability of divalent cations to regulate 2D affinity. These results indicate that a downward displacement of the I domain C-terminal helix, induced by conformational changes of other domains of the αLβ2, is required for affinity and on-rate up-regulation. Lymphocyte function-associated antigen-1, or αLβ2 integrin, mediates a variety of leukocyte adhesion and signaling processes, such as firm adhesion to the vascular surface during the initiation of inflammatory reaction or during lymphocyte trafficking, transendothelial migration into inflamed tissues or lymphoid tissues (1Springer T.A. Cell. 1994; 76: 301-314Abstract Full Text PDF PubMed Scopus (6414) Google Scholar), and adhesion to antigen-presenting cells to form the immunological synapse (2Grakoui A. Bromley S.K. Sumen C. Davis M.M. Shaw A.S. Allen P.M. Dustin M.L. Science. 1999; 285: 221-227Crossref PubMed Scopus (2563) Google Scholar). αLβ2 binds to members of the immunoglobin superfamily of cell adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1). 3The abbreviations used are: ICAM-1intercellular adhesion molecule-12Dtwo-dimensional3Dthree-dimensionalDTTdithiothreitolFITCfluorescein isothiocyanateGPIglycosylphosphatidylinositolHBSS-Hanks' balanced salt solution without calcium and without magnesiumI domaininserted domainMIDASmetal ion-dependent adhesion siteMESFmolecules of equivalent soluble fluorophoremAbmonoclonal antibodyRBCred blood cellsSPRsurface plasmon resonanceWTwild-type. Ligand binding of inactive αLβ2 is of too low an affinity to be detected by many conventional methods, but high affinity can be induced by cell activation, a process referred to as inside-out signaling. Conformational changes in the αLβ2 molecule, especially in the inserted (I) domain, are required for such affinity up-regulation. Ligand binding to the I domain can also induce conformational changes that propagate from the integrin head to its tail to enable binding of cytoplasmic molecules, a process referred to as outside-in signaling. Thus, conformational changes are crucial to the regulation of integrin functions (3Shimaoka M. Takagi J. Springer T.A. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 485-516Crossref PubMed Scopus (447) Google Scholar). intercellular adhesion molecule-1 two-dimensional three-dimensional dithiothreitol fluorescein isothiocyanate glycosylphosphatidylinositol Hanks' balanced salt solution without calcium and without magnesium inserted domain metal ion-dependent adhesion site molecules of equivalent soluble fluorophore monoclonal antibody red blood cells surface plasmon resonance wild-type. Although the crystal structures of several integrin domains have been solved, including the whole extracellular segment of the αvβ3 integrin alone (4Xiong 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 (1118) Google Scholar) and in complex with an RGD peptide (5Xiong J.P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S.L. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1410) Google Scholar), the dynamic process for the conformational regulation of integrin binding affinity and kinetics remains elusive. The ligand-binding site of αLβ2 for ICAM-1 is located at the I domain in the αL subunit, which is so named because its ∼200 amino acids are inserted between blades 2 and 3 of the β-propeller domain (6Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 65-72Crossref PubMed Scopus (390) Google Scholar). The I domain is also named αA domain because it assumes a dinucleotide-binding fold with a central β-sheet surrounded by several α helices that is similar to the A domains of von Willebrand factor (7Colombatti A. Bonaldo P. Blood. 1991; 77: 2305-2315Crossref PubMed Google Scholar). The C and N termini on one face connect the I domain to the β-propeller domain. A metal ion-dependent adhesion site (MIDAS) is located on the opposite face, which, together with the surrounding residues, forms the ICAM-1-binding site. Crystallographic (8Shimaoka 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 (424) Google Scholar), nuclear magnetic resonance (9Legge G.B. Kriwacki R.W. Chung J. Hommel U. Ramage P. Case D.A. Dyson H.J. Wright P.E. J. Mol. Biol. 2000; 295: 1251-1264Crossref PubMed Scopus (61) Google Scholar, 10Huth J.R. Olejniczak E.T. Mendoza R. Liang H. Harris E.A. Lupher Jr., M.L. Wilson A.E. Fesik S.W. Staunton D.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5231-5236Crossref PubMed Scopus (142) Google Scholar), and molecular dynamics simulation (11Jin M. Andricioaei I. Springer T.A. Structure. 2004; 12: 2137-2147Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) studies have provided evidence for conformational changes of the αL I domain among three distinct conformations, named open, intermediate, and closed conformers. It has been proposed that three residues of the β6-α7 loop interact with the α1 helix one at a time to hold the α7 helix in three distinct positions that correspond to these three conformations; each position moves the α7 helix downward by one helical turn (8Shimaoka 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 (424) Google Scholar). The movement of the α7 helix is linked to a repacking of the hydrophobic face of this α helix, which is coupled to the structural rearrangement of the MIDAS and divalent cation coordination (Fig. 1A). The regulation of ligand binding affinity and kinetics of αLβ2 by conformational changes has been examined by using mutants that introduce a pair of cysteines to form a disulfide bond to lock the αL I domain in either the closed, intermediate, or open conformation by altering the α7 helix position (8Shimaoka 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 (424) Google Scholar, 12Lu C. Shimaoka M. Ferzly M. Oxvig C. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2387-2392Crossref PubMed Scopus (119) Google Scholar). The isolated locked I domain mutants exhibited low (6.3 × 102 m-1), intermediate (3.3 × 105 m-1), and high (6.7 × 106 m-1) three-dimensional (3D) affinities for ICAM-1 as measured by surface plasmon resonance (SPR) (8Shimaoka 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 (424) Google Scholar, 13Shimaoka M. Lu C. Palframan R.T. von Andrian U.H. McCormack A. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6009-6014Crossref PubMed Scopus (190) Google Scholar). The increase in on-rate (∼50–60-fold) and the decrease in off-rate (∼100–250-fold) both contributed to the increase in binding affinity from the locked closed to the locked open I domains. The isolated locked open I domain was sufficient for full adhesive activity, since it had a 3D binding affinity for ICAM-1 (and mediated ICAM-1-dependent cell adhesion) at a level similar to those of fully activated wild-type (WT) αLβ2 (12Lu C. Shimaoka M. Ferzly M. Oxvig C. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2387-2392Crossref PubMed Scopus (119) Google Scholar, 13Shimaoka M. Lu C. Palframan R.T. von Andrian U.H. McCormack A. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6009-6014Crossref PubMed Scopus (190) Google Scholar, 14Labadia M.E. Jeanfavre D.D. Caviness G.O. Morelock M.M. J. Immunol. 1998; 161: 836-842PubMed Google Scholar). Ligand binding in the I domain can be regulated by and can induce a series of intra- and inter-domain conformational changes, which propagate through other domains from the cytoplasmic tail to the I domain (inside-out signaling) or from the I domain to the cytoplasmic tail (outside-in signaling). It has been proposed that, prior to the initiation of inside-out signaling, the inactive integrin assumes a bent conformation. Binding of an intracellular protein (e.g. talin) to the integrin cytoplasmic domain may result in the separation of the α- and β-cytoplasmic/transmembrane domains, thereby destabilizing the extracellular interface between the two subunits. This may lead to a switchblade-like opening of the bent integrin, resulting in an extended conformation. The disruption of the subunit interface may enable the hybrid domain to swing out from the I-like domain in the β subunit, facilitating the downward movement of the I-like domain C-terminal α helix that may be coupled to a rearrangement in the MIDAS of the I-like domain (Fig. 1B) (15Luo B.H. Strokovich K. Walz T. Springer T.A. Junichi T. J. Biol. Chem. 2004; 279: 27466-27471Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 16Mould 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 (106) Google Scholar, 17Xiao T. Takagi J. Coller B.S. Wang J.H. Springer T.A. Nature. 2004; 432: 59-67Crossref PubMed Scopus (684) Google Scholar). An “intrinsic ligand,” a residue in the I domain α7 helix, may bind the activated I-like domain MIDAS (18Alonso J.L. Essafi M. Xiong J.P. Stehle T. Arnaout M.A. Curr. Biol. 2002; 12: R340-R342Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 19Yang Y. Jun C.D. Liu J.H. Zhang R. Joachimiak A. Springer T.A. Wang J.H. Mol. Cell. 2004; 14: 269-276Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). This binding may pull down the α7 helix of the I domain and convert it to the activated conformation for high affinity ligand binding (20Takagi J. Springer T.A. Immunol. Rev. 2002; 186: 141-163Crossref PubMed Scopus (321) Google Scholar). Although various lines of evidence have been obtained in support of the above models for affinity/kinetic regulation by conformational changes, previous measurements were made by SPR with one of the binding partners in the fluid phase, i.e. 3D kinetics and affinity 4Strictly speaking, 3D binding requires both binding partners in the fluid phase. However, binding in SPR experiments, like binding of soluble ligands to cell surface receptors, is quite similar to and has the same binding affinity and on-rate units as the true 3D binding. (8Shimaoka 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 (424) Google Scholar, 13Shimaoka M. Lu C. Palframan R.T. von Andrian U.H. McCormack A. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6009-6014Crossref PubMed Scopus (190) Google Scholar, 14Labadia M.E. Jeanfavre D.D. Caviness G.O. Morelock M.M. J. Immunol. 1998; 161: 836-842PubMed Google Scholar). By comparison, cell-cell adhesion and cell-substrate adhesion are mediated by binding between receptors and ligands anchored on two apposing surfaces, i.e. two-dimensional (2D) interactions. The binding affinity Ka is the ratio of equilibrium concentration of bonds to those of free receptors and ligands. However, concentration is measured as number of molecules per volume in 3D but number of molecules per area (i.e. surface density) in 2D, resulting in different units for Ka (m-1 in 3D and μm2 in 2D). The kinetic on-rate kon also has different units in different dimensions (m-1 s-1 in 3D and μm2 s-1 in 2D). The 2D kon is the rate of bond formation between unit densities of receptors and ligands that are respectively anchored on two apposing surfaces of unit area. By comparison, the 3D kon is the rate of bond formation between unit concentrations of receptors and ligands in solution of unit volume. There has been increasing recognition that 2D binding parameters cannot be readily converted from their 3D counterparts (21Dustin M.L. Bromley S.K. Davis M.M. Zhu C. Annu. Rev. Cell Dev. Biol. 2001; 17: 133-157Crossref PubMed Scopus (132) Google Scholar). It is therefore important to directly measure in situ the more physiologically relevant 2D binding affinity and kinetic rates. Here we quantified the regulation of 2D binding affinity and kinetics of the αLβ2-ICAM-1 interaction by conformational changes using the micropipette adhesion frequency experiment (22Chesla S.E. Selvaraj P. Zhu C. Biophys. J. 1998; 75: 1553-1572Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). Our results showed that when expressed on the cell surface, isolated locked I domains exhibited binding affinities and kinetics similar to those of whole αLβ2 integrins containing I domains locked in the corresponding conformations. Up-regulation of ligand binding affinity of the αLβ2 was mostly because of the conformational changes in the I domain, which in turn was regulated by other domains by pulling down the I domain C-terminal α7 helix. Our data also revealed a surprising discrepancy between the 2D and 3D off-rate measurements. The 2D off-rates of the locked open and locked closed I domains only differed a few fold, which is in sharp contrast to the 3D results of ∼100-fold difference. Cell Lines, Proteins, and Antibodies—The human erythroleukemia cell line K562 stably transfected with WT, locked open (K287C/K294C), locked intermediate (L161C/F299C), or locked closed (L289C/K294C) αL I domain fused with a platelet-derived growth factor receptor transmembrane domain and the first five amino acids of its cytoplasmic domain or with intact αLβ2 containing the WT or locked I domains have been described previously (8Shimaoka 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 (424) Google Scholar, 13Shimaoka M. Lu C. Palframan R.T. von Andrian U.H. McCormack A. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6009-6014Crossref PubMed Scopus (190) Google Scholar). Human red blood cells (RBCs) were isolated from whole peripheral blood of healthy donors. Approximately 7 ml of whole blood was collected by venipuncture into sterile Vacutainers (BD Biosciences) containing EDTA. This was carefully layered over 3 ml of Histopaque 1119 (Sigma) and centrifuged (30 min, 2000 × g, room temperature). The supernatant was removed, and the pelleted RBCs were washed once in RBC storage solution (EAS45) (23Dumaswala U.J. Wilson M.J. Jose T. Daleke D.L. Blood. 1996; 88: 697-704Crossref PubMed Google Scholar). RBCs were stored aseptically at 4 °C in EAS45 at 20% hematocrit. Mouse and human glycosylphosphatidylinositol (GPI)-anchored ICAM-1 molecules were purified from Chinese hamster ovary cell transfectants by affinity chromatography 5P. Selvaraj, unpublished data. (24Carpen O. Pallai P. Staunton D.E. Springer T.A. J. Cell Biol. 1992; 118: 1223-1234Crossref PubMed Scopus (264) Google Scholar). Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human αL I domain monoclonal antibody (mAb) MEM-25 (IgG1) (Caltag Laboratories, Burlingame, CA) and a nonbinding FITC-conjugated mouse isotype-matched control antibody were used to determine surface expression of the I domain and αLβ2 on K562 cells by immunofluorescence flow cytometry. FITC-conjugated mouse mAb MEM-111 (Caltag Laboratories) and FITC-conjugated rat mAb YN1/1.7.4 (eBioscience, San Diego) were used to determine the site densities of the reconstituted human and mouse ICAM-1, respectively. The mouse anti-human αL mAb 38 (Ancell, Bayport, MN) was used for antibody-antigen adhesion kinetic measurement. FITC-conjugated rat anti-mouse IgG2a mAb (Fc-specific) (Pharmingen) was used to measure the site density of mAb 38 coated on RBCs. The anti-ICAM-1-capturing mouse mAb CA7 was a generous gift of Dr. Robert Rothlein (Boehringer-Ingelheim Pharmaceuticals, Ridgefield, CT). The integrin-activating mAb CBR LFA1/2 has been described previously (25Petruzzelli L. Maduzia L. Springer T.A. J. Immunol. 1995; 155: 854-866PubMed Google Scholar). Coupling of ICAM-1 to RBCs—Mouse or human GPI-ICAM-1 was reconstituted in RBC membrane by a 2.5-h incubation with different concentrations to achieve the desired site densities (26Williams T.E. Nagarajan S. Selvaraj P. Zhu C. J. Biol. Chem. 2001; 276: 13283-13288Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Capture antibody CA7 or anti-human αL I domain mAb 38 was covalently coupled to the membranes of RBCs by the chromium chloride (CrCl3) method described previously (22Chesla S.E. Selvaraj P. Zhu C. Biophys. J. 1998; 75: 1553-1572Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). CA7-coated RBCs were subsequently washed and incubated with human ICAM-1. Coated RBCs were washed and assayed for coating density by flow cytometry and stored in EAS45 at 4 °C. Cell Sorting and Site Density Determination—Sorting of K562 cells to express several narrowly distributed densities (with the mean ranging from 10 to 2000 μm-2) of isolated I domains and whole αLβ2 were done using a BD FACSVantage SE flow cytometer (Immunocytometry Systems). Molecular site densities on the cell surface were determined by flow cytometry. For the isolated I domain and αLβ2 site densities, K562 cells were incubated with FITC MEM-25 (or isotype-matched FITC mouse control antibody) for 30 min at room temperature. For mouse and human ICAM-1 site densities, RBCs with and without (for controls) ICAM-1 coupling were incubated with FITC YN1/1.7.4 and FITC MEM-111, respectively. For mAb 38 site density, RBCs with and without (for controls) mAb 38 coupling were incubated with FITC rat anti-mouse IgG2a mAb. Samples were read on a BD LSR flow cytometer (BD Biosciences) using fluorescence-activated cell sorter DiVa 3.1 software. Standard beads (Quantum™ 25 FITC High Level, Bangs Laboratory, Fishers, IN) were prepared for quantification of molecules of equivalent soluble fluorophore (MESF). A calibration curve was generated by plotting the MESF against the mean fluorescence intensities of each of the five bead populations. The MESF for the cells were calculated by comparing the fluorescence intensities of the cells with those of the standard beads. The site densities on the cell surface of the molecule in question were determined by dividing the MESF value by the apparent cell surface area and the fluorophore/protein ratio. Micropipette Adhesion Frequency Assay—The micropipette adhesion frequency assay has been described previously (22Chesla S.E. Selvaraj P. Zhu C. Biophys. J. 1998; 75: 1553-1572Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). K562 cells were washed with Hanks' balanced salt solution without calcium and without magnesium (HBSS-) three times before the experiments and resuspended in HBSS- with or without 2 mm Mn2+ or 2 mm Mg2+ plus 2 mm of EGTA in the presence or absence of 10 mm dithiothreitol (DTT) and 45–55% double distilled H2O to partially swell the RBCs. In some control experiments, K562 cells were washed with HBSS- with 5 mm EDTA and then with HBSS- before being assayed in HBSS- with or without 5 mm EDTA. In other control experiments, the HBSS-/EDTA-washed cells were incubated with 2 mm of Mn2+, Mg2+ (plus 2 mm EGTA), or Ca2+ and then washed with HBSS- before being assayed in HBSS-. By using two micropipettes, a K562 cell and an RBC were aspirated gently and aligned with a small axial gap between them (Fig. 2). One pipette was connected to a computer-controlled piezoelectric actuator that was programmed to move back and forth at a uniform rate of 1 μm/s. Cells were placed in contact for a prescribed duration and then retracted away from each other. Upon retraction, the unaspirated portion of the RBC was either separated freely from the K562 cell and returned to its original spherical shape, indicating no adhesion (scoring 0), or remained bound with an elongated shape, indicating adhesion (scoring 1), until final detachment by further retraction. This test cycle was repeated 100 times using the same pair of cells, keeping the contact duration (t) and the area (Ac, ∼3 μm2) constant, and averaging the adhesive scores to calculate an adhesion frequency (Pa). More than 5 pairs of cells were tested per contact time to obtain the mean ± S.E. of Pa at that t. The contact duration was varied from 0.5 to 60 s to obtain a Pa versus t binding curve. For each species of I domain or αLβ2, 2–3 curves were generated using a total of ∼100 pairs of cells by varying the site densities of I domain/αLβ2 (mr) or ICAM-1 (ml). The 2D effective binding affinity (AcKa) and off-rate (koff) were calculated by fitting Equation 1 (22Chesla S.E. Selvaraj P. Zhu C. Biophys. J. 1998; 75: 1553-1572Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar)Pa=1-exp{-mrmlAcKa(1-exp(-kofft))}(Eq. 1) to the binding curves by using iteratively reweighted nonlinear regression (27Williams T.E. Selvaraj P. Zhu C. Biophys. J. 2000; 79: 1858-1866Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). For some experiments, the adhesion frequency was determined at sufficiently long contact duration without measuring a full binding curve to simply estimate the effective binding affinity (AcKa) from Equation 2,AcKa=1mrmlln[1-Pa(∞)]-1(Eq. 2) which is derived from the steady-state version (i.e. t → ∞) of Equation 1. Measuring Specific Integrin-ICAM-1 Binding Affinity and Kinetics—Expressed on the cell surface, both isolated I domains and whole αLβ2 heterodimers mediated sufficient levels of adhesion with ICAM-1 reconstituted on RBC to be measured by the micropipette, which is capable of measuring interactions with affinities too low for many conventional adhesion assays to detect (28Williams T.E. Nagarajan S. Selvaraj P. Zhu C. Biophys. J. 2000; 79: 1867-1875Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). By sorting the K562 cells to express appropriate levels of I domain or whole αLβ2 and reconstituting appropriate levels of ICAM-1 on RBCs, the equilibrium frequencies of adhesion of each pair of interacting molecules were adjusted to midrange levels, i.e. 0.2–0.8. To estimate reliably binding parameters, 2–3 curves were generated for each species by varying the site densities of integrin or ICAM-1 or both (Fig. 3). The detected interactions were mostly specific, as the adhesion frequency between the K562 cells and RBCs without ICAM-1 coupling was <0.06, which was deemed as nonspecific binding (Fig. 3 and data not shown). The use of Equations 1 and 2 requires that Pa be a specific adhesion frequency, which can be calculated from the total adhesion frequency (Pt) after removing the nonspecific adhesion frequency (Pn) according to Equation 3 (21Dustin M.L. Bromley S.K. Davis M.M. Zhu C. Annu. Rev. Cell Dev. Biol. 2001; 17: 133-157Crossref PubMed Scopus (132) Google Scholar, 26Williams T.E. Nagarajan S. Selvaraj P. Zhu C. J. Biol. Chem. 2001; 276: 13283-13288Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Pa=(Pt-Pn)/(1-Pn)(Eq. 3) For all integrin molecules tested, measurement of specific ICAM-1 binding affinity and kinetics requires that the I domain MIDAS be occupied by a divalent cation, as binding was reduced to the nonspecific level when measured in HBSS- with EDTA (Fig. 4A). Washing the cells first with HBSS- containing EDTA and then with HBSS- to remove EDTA and resuspending them in HBSS- for adhesion test also diminished binding to the nonspecific level (Fig. 4B). Thus, after stripping the metal ion off the I domain MIDAS with EDTA, no specific adhesion frequency above the noise level could be calculated from Equation 3, thereby preventing estimates of integrin-ICAM-1 binding affinity and kinetics from Equations 1 and 2. However, washing the cells previously exposed to divalent cations with and then performing micropipette assay in HBSS- without EDTA resulted in sufficiently high specific adhesion signals above noise from Equation 3. This indicates that without using EDTA, the metal ion pre-bound to the I domain MIDAS remained bound for hours in HBSS- to enable specific integrin-ICAM-1 binding. To study further the regulation of ICAM-1 binding affinities of the different I domains and whole αLβ2 by divalent cations, we chose the base-line experimental condition to be one in which K562 cells were first allowed to bind divalent cations, washed with HBSS-, and then assayed for ICAM-1 binding in HBSS-. Inclusion of 2 mm Mn2+ or 2 mm Mg2+ in the chamber solution where micropipette adhesion assay was performed was chosen to be the activating experimental condition. Note that in both the base-line and activating conditions, it was the specific adhesion mediated by binding of ICAM-1 to the I domain MIDAS pre-bound with a divalent cation that was measured. Specific ICAM-1 binding affinities and kinetics of WT and mutant αL I domain as well as WT and mutant whole αLβ2 in these two conditions were compared in the present study to elucidate their conformational regulation. The EDTA condition was not used because it did not allow specific ICAM-1 binding affinity and kinetics to be measured by our method. The Locked Open, Intermediate, and Closed I Domains Bind ICAM-1 with High, Intermediate, and Low Affinities—Tested under the base-line condition, locking the I domain into high and intermediate conformations increased the binding affinities by ∼8,000- and ∼30-fold compared with the locked closed I domain, which was on the same order of magnitude as the affinity of the WT I domain (p > 0.06) (Fig. 5A and TABLE ONE). Therefore, the alternation of the C-terminal α7 helix position was sufficient to regulate the ligand binding affinity of the I domain when expressed on cell surface, in agreement with the 3D SPR measurement (8Shimaoka 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 (424) Google Scholar, 13Shimaoka M. Lu C. Palframan R.T. von Andrian U.H. McCormack A. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6009-6014Crossref PubMed Scopus (190) Google Scholar) and consistent with the cell binding measurement (29Salas A. Shimaoka M. Chen S. Carman C.V. Springer T. J. Biol. Chem. 2002; 277: 50255-50262Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Most surprisingly, differences among the off-rates for the four isolated I domains were modest at best, with the locked open I domain having the smallest off-rate of 0.41 s-1 and the locked closed I domain having the largest off-rate of 0.71 s-1 (Fig. 5B and TABLE ONE), in sharp contract to the 3D SPR measurement (8Shimaoka 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 (424) Google Scholar, 13Shimaoka M. Lu C. Palframan R.T. von Andrian U.H. McCormack A. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6009-6014Crossref PubMed Scopus (190) Google Scholar).TABLE ONE2D and 3D binding affinities and off-rates of isolated αL I domain and αLβ2 integrin at different conformational states All 2D data are from this study, and the ligand was mouse GPI-ICAM-1. The two sets of 3D data for I domains interacting with huma" @default.
• W2044037317 created "2016-06-24" @default.
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• W2044037317 creator A5036800314 @default.
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• W2044037317 date "2005-12-01" @default.
• W2044037317 modified "2023-09-28" @default.
• W2044037317 title "Two-dimensional Kinetics Regulation of αLβ2-ICAM-1 Interaction by Conformational Changes of the αL-Inserted Domain" @default.
• W2044037317 cites W1550140963 @default.
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• W2044037317 doi "https://doi.org/10.1074/jbc.m510407200" @default.
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