Locking the β3 Integrin I-like Domain into High and Low Affinity Conformations with Disulfides
2004; Elsevier BV; Volume: 279; Issue: 11 Linguagem: Inglês
10.1074/jbc.m312732200
ISSN1083-351X
AutoresBing Luo, Junichi Takagi, Timothy A. Springer,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoAlthough integrin α subunit I domains exist in multiple conformations, it is controversial whether integrin β subunit I-like domains undergo structurally analogous movements of the α7-helix that are linked to affinity for ligand. Disulfide bonds were introduced into the β3 integrin I-like domain to lock its β6-α7 loop and α7-helix in two distinct conformations. Soluble ligand binding, ligand mimetic mAb binding and cell adhesion studies showed that disulfide-bonded receptor αIIbβ3T329C/A347C was locked in a low affinity state, and dithiothreitol treatment restored the capability of being activated to high affinity binding; by contrast, disulfide-bonded αIIbβ3V332C/M335C was locked in a high affinity state. The results suggest that activation of the β subunit I-like domain is analogous to that of the α subunit I domain, i.e. that axial movement in the C-terminal direction of the α7-helix is linked to rearrangement of the I-like domain metal ion-dependent adhesion site into a high affinity conformation. Although integrin α subunit I domains exist in multiple conformations, it is controversial whether integrin β subunit I-like domains undergo structurally analogous movements of the α7-helix that are linked to affinity for ligand. Disulfide bonds were introduced into the β3 integrin I-like domain to lock its β6-α7 loop and α7-helix in two distinct conformations. Soluble ligand binding, ligand mimetic mAb binding and cell adhesion studies showed that disulfide-bonded receptor αIIbβ3T329C/A347C was locked in a low affinity state, and dithiothreitol treatment restored the capability of being activated to high affinity binding; by contrast, disulfide-bonded αIIbβ3V332C/M335C was locked in a high affinity state. The results suggest that activation of the β subunit I-like domain is analogous to that of the α subunit I domain, i.e. that axial movement in the C-terminal direction of the α7-helix is linked to rearrangement of the I-like domain metal ion-dependent adhesion site into a high affinity conformation. Integrins are large heterodimeric adhesion molecules that convey signals bidirectionally across the plasma membrane (1Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6950) Google Scholar, 2Shimaoka M. Takagi J. Springer T.A. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 485-516Crossref PubMed Scopus (447) Google Scholar). Both integrin subunits are type I transmembrane proteins with large extracellular domains. Priming of the extracellular domain for ligand binding (i.e. increasing its affinity for ligand) is initiated by moving apart the α and β subunit cytoplasmic domains and probably separation of the transmembrane domains as well (3Kim M. Carman C.V. Springer T.A. Science. 2003; 301: 1720-1725Crossref PubMed Scopus (646) Google Scholar). Conversely, binding of ligand can also initiate cytoplasmic domain separation (3Kim M. Carman C.V. Springer T.A. Science. 2003; 301: 1720-1725Crossref PubMed Scopus (646) Google Scholar); the equilibria relating conformational change and ligand binding are linked (4Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar). The low affinity integrin conformation is highly bent, with the headpiece that contains the ligand binding domains in an extensive interface with the tailpiece that contains the α and β subunit legs (4Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar, 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, 6Xiong 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, 7Beglova N. Blacklow S.C. Takagi J. Springer T.A. Nat. Struct. Biol. 2002; 9: 282-287Crossref PubMed Scopus (261) Google Scholar, 8Takagi J. Strokovich K. Springer T.A. Walz T. EMBO J. 2003; 22: 4607-4615Crossref PubMed Scopus (290) Google Scholar) (Fig. 1A). After priming or ligand binding, a switchblade-like opening extends the headpiece away from the membrane (4Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar, 7Beglova N. Blacklow S.C. Takagi J. Springer T.A. Nat. Struct. Biol. 2002; 9: 282-287Crossref PubMed Scopus (261) Google Scholar) (Fig. 1, B and C). In extended integrins, two conformations of the headpiece are seen. The open conformation of the headpiece (Fig. 1C) is present when ligand is bound and differs from the closed conformation (Fig. 1B) in the presence of an obtuse angle between the β-subunit hybrid and I-like domains (4Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar, 8Takagi J. Strokovich K. Springer T.A. Walz T. EMBO J. 2003; 22: 4607-4615Crossref PubMed Scopus (290) Google Scholar). Recently, we mutationally introduced N-glycosylation sites into the interface of the hybrid and I-like domains to stabilize the open headpiece. The wedged-open mutants exhibited constitutively high affinity for ligand and adopted an extended conformation (9Luo B.-H. Springer T.A. Takagi J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2403-2408Crossref PubMed Scopus (124) Google Scholar). We have proposed that the change in affinity at the ligand binding site in the I-like domain around its metal ion-dependent adhesion site (MIDAS) 1The abbreviations used are: MIDAS, metal ion-dependent adhesion site; TBS, Tris-buffered saline; DTT, dithiothreitol; biotin-BMCC, 1-biotinamido-4 (4′-[maleimodoethyl-cyclohexane]-carboxamido)butane; FITC, fluorescein isothiocyanate; HBS, Hepes-buffered saline; BSA, bovine serum albumin; mAb, monoclonal antibody; CHO, Chinese hamster ovary. is communicated to the interface with the hybrid domain on the opposite end of the I-like domain by axial displacement in the C-terminal direction of the I-like domain α7-helix (4Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar, 8Takagi J. Strokovich K. Springer T.A. Walz T. EMBO J. 2003; 22: 4607-4615Crossref PubMed Scopus (290) Google Scholar, 9Luo B.-H. Springer T.A. Takagi J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2403-2408Crossref PubMed Scopus (124) Google Scholar) (Fig. 1, B and C). The β-subunit I-like domain is inserted in the hybrid domain, and thus these domains have two interconnections. A piston-like movement at the I-like α7-helix connection and pivoting about the other connection would yield a swing at the I-like-hybrid domain interface approximating that seen in electron microscopy studies (4Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar, 8Takagi J. Strokovich K. Springer T.A. Walz T. EMBO J. 2003; 22: 4607-4615Crossref PubMed Scopus (290) Google Scholar). One basis for proposing this mechanism for communicating a change in affinity to the I-like domain MIDAS is that the structurally homologous I domain inserted in some integrin α subunits undergoes a similar piston-like movement of its C-terminal α7-helix, which regulates the affinity of its MIDAS for ligand (2Shimaoka M. Takagi J. Springer T.A. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 485-516Crossref PubMed Scopus (447) Google Scholar). There is controversy concerning this proposed mechanism. Soaking of a ligand-mimetic Arg-Gly-Asp (RGD) peptide into integrin αVβ3 crystals, in which αVβ3 was constrained in the bent conformation by lattice contacts, induced β6-α7 loop and α1-helix movements, but not α7-helix displacement (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). It was therefore suggested that α I and β I-like domains are activated by distinct mechanisms. Demonstration of movement of an epitope in the α1-helix was used to support the hypothesis that the mechanism of I-like domain activation differs from that of the I domain (10Mould A.P. Askari J.A. Barton S. Kline A.D. McEwan P.A. Craig S.E. Humphries M.J. J. Biol. Chem. 2002; 277: 19800-19805Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). On the other hand, conformational change at this region would not contradict C-terminal α7-helix movement, and the mutation L358A in the α7-helix of the β1 I-like domain causes activation, supporting some type of conformational change around the α7-helix upon ligand binding (11Mould 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). Furthermore, solution x-ray scattering studies and exposure of epitopes on the inner side of the hybrid domain in the presence of ligand (11Mould 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, 12Mould A.P. Barton S.J. Askari J.A. Craig S.E. Humphries M.J. J. Biol. Chem. 2003; 278: 51622-51629Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) support the direct observations of hybrid domain swing-out (4Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar, 8Takagi J. Strokovich K. Springer T.A. Walz T. EMBO J. 2003; 22: 4607-4615Crossref PubMed Scopus (290) Google Scholar). Here, we directly test the hypothesis that specific rearrangements occur in the β6-α7 loop and α7-helix of β I-like domains that are structurally analogous to those that occur in α I domains and are linked to integrin activation. Disulfide bonds have previously been introduced into α I domains to constrain the β6-α7 loop and α7-helix. The αL (13Lu 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, 14Lu C. Shimaoka M. Zang Q. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2393-2398Crossref PubMed Scopus (170) Google Scholar, 15Shimaoka M. Lu C. Palframan R. von Andrian U.H. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6009-6014Crossref PubMed Scopus (190) Google Scholar, 16Shimaoka M. Xiao T. Liu J.-H. Yang Y. Dong Y. Jun C.-D. McCormack A. Zhang R. Joachimiak A. Takagi J. Wang J. Springer T.A. Cell. 2003; 112: 99-111Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar) and αM (17Shimaoka M. Lu C. Salas A. Xiao T. Takagi J. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16737-16741Crossref PubMed Scopus (57) Google Scholar) I domains have been locked into closed, intermediate, or open conformers with low, intermediate, or high affinity for ligand, respectively. Crystal structure studies on the mutant αL I domains confirmed alterations in the β6-α7 loop corresponding to α7-helix displacements of one and two turns of helix in the intermediate and open conformations, respectively (16Shimaoka M. Xiao T. Liu J.-H. Yang Y. Dong Y. Jun C.-D. McCormack A. Zhang R. Joachimiak A. Takagi J. Wang J. Springer T.A. Cell. 2003; 112: 99-111Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). The disulfide-constrained, high affinity, open conformation of the αL I domain corresponds precisely in the critical β6-α7 loop and MIDAS loops to the open conformation of the wild-type α2 and αM I domains seen when this conformation was stabilized in crystals by ligand or ligand-like lattice contacts (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). The studies reported here on the β I-like domain show that disulfide bonds mutationally introduced into the β6/α7 region lock integrins that lack I domains into two distinct affinity states. The data uniquely support the proposal that downward movement of the α7-helix induces I-like domain activation and demonstrate that α I and β I-like domains are activated by structurally analogous mechanisms. High Affinity I-like Domain Model—The model was built with the Segmod module (20Levitt M. J. Mol. Biol. 1992; 226: 507-533Crossref PubMed Scopus (519) Google Scholar) of GeneMine version 3.5 using residues 108–333 and 347–353 of Protein Data Bank accession number 1JV2 (6Xiong 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) as template and aligning them with residues 108–333 and 340–346 of the model sequence, respectively. This corresponded to a 7-residue, 2-turn displacement of the α7-helix along its helical axis; residues 334–339 were left nontemplated. Plasmid Construction, Transient Transfection, and Immunoprecipitation—Plasmids coding for full-length human αIIb and β3 were subcloned into pEF/V5-HisA or pcDNA3.1/Myc-His(+) as described previously (4Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar). Mutants were made using site-directed mutagenesis with the QuikChange kit (Stratagene, La Jolla, CA), and DNA sequences were confirmed before being transfected into 293T cells using calcium phosphate precipitates (21DuBridge 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). Transfected cells were metabolically labeled with [35S]cysteine/methionine as described (4Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar). Lysates in 20 mm Tris-buffered saline, pH 7.4 (TBS), supplemented with 1 mm Ca2+, 1 mm Mg2+, 1% Triton X-100, and 0.1% Nonidet P-40 were immunoprecipitated with 1 μg of anti-β3 mAb AP3 and protein G-Sepharose at 4 °C for 1 h and subjected to nonreducing SDS 7.5% PAGE and fluorography (22Huang C. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3162-3167Crossref PubMed Scopus (66) Google Scholar). Labeling of Free Cysteines and Western Blotting—Transiently transfected 293T cells, treated with or without 5 mm DTT at 37 °C for 30 min in TBS containing 1 mm Ca2+, followed by washing with TBS plus 1 mm Ca2+ three times, were labeled with 400 μm 1-biotinamido-4 (4′-[maleimodoethyl-cyclohexane]-carboxamido)butane (biotin-BMCC) (Pierce) at room temperature for 30 min, washed with TBS plus 1 mm Ca2+ three times, and lysed with TBS with 1% Triton X-100 and 0.1% Nonidet P-40. αIIbβ3 was immunoprecipitated with AP3 mAb-Sepharose at 4 °C for 1 h and subjected to SDS 7.5% PAGE. Samples were transferred to polyvinylidene difluoride membranes and probed with 2 μg/ml horseradish peroxidase-conjugated avidin at 20 °C for 30 min or with 2 μg/ml anti-myc antibody (Invitrogen) for 30 min at 20 °C, followed by washing and 2 μg/ml horseradish peroxidase-conjugated anti-mouse-IgG (Zymed Laboratories Inc., San Francisco, CA) for 30 min at 20 °C, and then detected by chemiluminescence using the ECL Western blotting kit (Amersham Biosciences). BioMax film (Eastman Kodak Co.) was exposed for about 20 s. The film was scanned using DUOSCAN 1200 (Agfa, Mortsel, Belgium), and data were saved as a tiff file. NIH Image 1.62 (NIMH, National Institutes of Health, Bethesda, MD) was used to determine the intensity of each band within identical rectangular areas. After subtracting the intensity of mock transfectants in the same areas, the ratio of the intensity of BMCC and myc bands was determined. Two-color Ligand Binding Assay on 293T Transfectants—Binding of FITC-labeled human fibrinogen was determined as described (4Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar). Briefly, transiently transfected 293T cells in 20 mm Hepes-buffered saline (pH 7.4) (HBS) supplemented with 5.5 mm glucose and 1% BSA were incubated with 60 μg/ml fluorescein-labeled fibrinogen in the presence of 1 mm EDTA, 5 mm Ca2+, or 1 mm Ca2+ plus 10 μg/ml PT25-2 mAb at 20 °C for 30 min, and then 10 μg/ml Cy3-labeled mAb AP3 was added, and cells were incubated on ice for another 30 min before being subjected to flow cytometry. Fibrinogen binding was expressed as a percentage of the mean fluorescence intensity of FITC-fibrinogen relative to that of staining with AP3 mAb. Expression and Ligand Binding Activity of αIIbβ3 on CHO-K1 Transfectants—The plasmids described above coding for αIIb and β3 were introduced into CHO-K1 cells using calcium phosphate precipitates (21DuBridge 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). Transfectants were selected with 5 mg/ml G418. After 2 weeks, the cells were stained with AP3 mAb and subjected to fluorescence-activated cell sorting to obtain lines expressing the desired level of αIIbβ3. Prior to ligand binding, transfected cells were suspended in HBS supplemented with 5.5 mm glucose and 1% BSA and incubated with 1 mm EDTA, 5 mm Ca2+, or 1 mm Ca2+ plus 10 μg/ml PT25-2, with or without 5 mm DTT, at 20 °C for 30 min. Staining with fluorescein-labeled human fibrinogen and the ligand mimetic PAC-1 mAb (Becton Dickinson, San Jose, CA) was measured as described (4Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar). Cell Adhesion to Immobilized Fibrinogen—Cell adhesion was assayed as described (23Lu C. Springer T.A. J. Immunol. 1997; 159: 268-278PubMed Google Scholar). Briefly, transfected CHO-K1 cells were labeled with 2′,7′-bis-(carboxyethyl)-5(and-6)-carboxyfluorescein (Molecular Probes, Inc., Eugene, OR) and suspended to 106/ml in HBS supplemented with 5.5 mm glucose, 1% BSA, and either 1 mm EDTA, 5 mm Ca2+, or 5 mm DTT plus 5 mm Ca2+. Cell suspensions were incubated in wells that had been coated with different concentrations of fibrinogen followed by blocking with 1% BSA. After incubation at 37 °C for 1 h, unbound cells were washed off after three resuspensions with a multichannel pipette. The fluorescence of input cells and bound cells in each well was quantitated on a fluorescent concentration analyzer (Idexx, Westbrook, ME). Bound cells were expressed as a percentage of total input cells. LIBS Expression—Anti-LIBS mAbs LIBS-1, LIBS-6, and PMI-1 were kind gifts of M. H. Ginsberg (Scripps Research Institute, La Jolla, CA). LIBS expression was measured as described (9Luo B.-H. Springer T.A. Takagi J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2403-2408Crossref PubMed Scopus (124) Google Scholar). In brief, CHO-K1 cells stably expressing wild type or mutant αIIbβ3 in HBS supplemented with 5.5 mm glucose and 1% BSA were incubated under different conditions as indicated in the legend to Fig. 5 for 30 min at 20 °C. LIBS mAbs were added to a final concentration of 10 μg/ml, and cells were incubated on ice for another 30 min before staining with FITC-conjugated anti-mouse IgG and flow cytometry. Design of I-like Domains Locked in Low Affinity and High Affinity Conformations—We hypothesized that in both the unliganded and liganded αVβ3 structures (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, 6Xiong 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), the C-terminal α7-helix of the β3 I-like domain is in a position stabilizing a closed, low affinity conformation; therefore, these structures were used to design low affinity mutants. An open, high affinity conformation was modeled assuming that the α7-helix was displaced in the C-terminal axial direction by two α-helical turns (see “Materials and Methods”). The distance between Cβ atoms of Thr329 and Ala347 in the unliganded and RGD-liganded αVβ3 structures is 4.4 and 4.9 Å, respectively, whereas it is 9.6 Å in the hypothesized high affinity model. Therefore, the mutant β3T329C/A347C was expected to form a disulfide bond in the low but not the high affinity conformation and to be stabilized in the low affinity, closed conformation (Fig. 1, D and F). On the other hand, the distance between the Cβ atoms of Val332 and Met335 in the unliganded and RGD-liganded αVβ3 structures is 10.0 and 8.3 Å, respectively, whereas it is 3.7 Å in the hypothesized high affinity model. Therefore, the mutant β3V332C/M335C was expected to form a disulfide bond in the high but not the low affinity conformation (Fig. 1, E and G) and to be stabilized in the high affinity, open conformation. Expression of Wild Type and Mutant αIIbβ3 Receptors and Formation of Disulfide Bonds—Wild type and mutant β3 subunits were co-transfected with wild type αIIb in 293T cells and subjected to immunostaining flow cytometry (Fig. 2, A and B). The wild type and mutant receptors were recognized equally well by mAb to epitopes constitutively present on the αIIb and β3 subunits, including AP3 (anti-β3), 10E5 (anti-αIIb), HA5 (anti-αIIb), and AP2 (anti-αIIbβ3 complex-specific) (Fig. 2A), suggesting that the two mutant receptors adopted a native fold on the cell surface. However, the mutant β3V332C/M335C receptor was recognized weakly by anti-β3 mAb 7E3 (Fig. 2B). The 7E3 mAb recognizes residues in the specificity-determining, β2-β3 loop near the β3 I-like MIDAS (24Puzon-McLaughlin W. Kamata T. Takada Y. J. Biol. Chem. 2000; 275: 7795-7802Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Since the single cysteine mutants β3V332C and β3M335C were recognized by 7E3 (Fig. 2B), the conformational change induced by the disulfide bond formed between V332C and M335C (see below) appears to diminish the 7E3 epitope. By contrast, mutant β3T329C/A347C was well recognized by 7E3 (data not shown) (see Fig. 4A).Fig. 4Expression and ligand binding activity of mutant αIIbβ3 integrins on CHO-K1 transfectants. A, immunofluorescent staining. Staining of specific transfectants (thick lines) is compared with mock transfectants (thin lines). B and C, soluble fibrinogen (B) and PAC-1 mAb (C) binding. Cells were incubated with ligands in the presence of 5 mm Ca2+, 1 mm Ca2+ plus 10 μg/ml PT25-2 mAb, 5 mm Ca2+ plus 5 mm DTT, or 1 mm Ca2+ plus 10 μg/ml PT25-2 and 5 mm DTT at room temperature for 30 min. Binding was determined as described under “Materials and Methods” as mean fluorescence intensity. D, adhesion of CHO transfectants in the presence of 5 mm Ca2+ or 5 mm DTT plus 5 mm Ca2+ to surfaces coated with fibrinogen at the indicated concentrations. Binding of fluorescently labeled transfectants was determined as described under “Materials and Methods.” Data are representative of three independent experiments, each in quadruplicate. S.D. values were on average 1.3% and always less than 4.6%.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Nonreducing SDS-PAGE of 35S-labeled, immunoprecipitated receptors showed that the αIIb subunits migrated similarly (Fig. 2C), whereas the mutant β3T329C/A347C subunits (Fig. 2C, lanes 6 and 7) migrated slightly faster than wild type β3 (Fig. 2C, lane 5). By contrast, all β3 single cysteine mutants migrated similarly to wild-type β3 (Fig. 2C, lanes 1–4). In general, disulfide bonds increase the mobility of proteins in SDS-PAGE, and these results suggest that the cysteines introduced into the β3T329C/A347C and β3V332C/M335C and β3V332C/M335C mutants form a disulfide bond. To confirm disulfide bond formation, free sulfhydryls were labeled with the maleimide-containing reagent, biotin-BMCC. The idea was that introducing a single cysteine should increase labeling, whereas introducing two cysteines would not increase labeling if they formed a disulfide bond. Transfectants were treated with biotin-BMCC, lysed, immunoprecipitated with AP3 mAb to αIIbβ3, and subjected to SDS-PAGE and blotting with avidin (Fig. 2D). The β3 subunit was fused at its C terminus to a myc tag, and blotting with a myc mAb was used as a control for β3 loading. Whereas wild-type αIIbβ3 showed almost no biotin labeling (Fig. 2D, lane 2), the αIIbβ3 single cysteine mutants V332C and M335C showed marked labeling (Fig. 2D, lanes 4 and 6). The cysteines introduced in the V332C/M335C and T329C/A347C mutants clearly formed disulfides, because labeling was at the same level as the wild type (Fig. 2D, lanes 5 and 7), whereas it would have been twice that of the single cysteine mutants if disulfides had not formed. To estimate the number of free cysteines per β3 subunit, the ratio of the intensity of avidin binding to that of anti-myc binding was determined. As an additional control, wild-type αIIbβ3 on the transfectants was treated with 5 mm DTT for 30 min at 37 °C. The avidin/anti-myc ratios for the wild type, wild type with DTT treatment, β3V332C, β3V332C/M335C, β3M335C, and β3T329C/A347C subunits were 0.05, 0.82, 0.20, 0.04, 0.19, and 0.04, respectively. If β3V332C is assumed to have one additional free cysteine sulfhydryl compared with wild type, then β3M335C has 1.0 additional free cysteine, β3V332C/M335C also and β3T329C/A347 have no additional free β3 cysteines, and wild type αIIbβ3 treated with DTT has 5.2 free cysteines. By contrast, there are a total of 54 cysteines in wild type β3. Ligand Binding Properties of 293T Transfectants with Disulfide-locked Receptors—Binding to soluble fibrinogen was first examined using two-color flow cytometry (4Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar) in transiently transfected 293T cells, in which wild type αIIbβ3 basally has low affinity for ligand. Wild type αIIbβ3 bound fibrinogen when stimulated with the activating mAb PT25-2 but not basally in Ca2+ (Fig. 3A). Each of the four single cysteine mutants behaved similarly to the wild type receptor (Fig. 3A). By contrast, the putative locked closed, double cysteine mutant αIIbβ3T329C/A347C did not bind soluble fibrinogen even in the presence of PT25-2 (Fig. 3A). Furthermore, the putative locked open mutant αIIb β3V332C/M335C bound soluble fibrinogen even in Ca2+, and the addition of PT25-2 mAb did not further increase binding (Fig. 3A). Constitutive binding in Ca2+ by the αIIbβ3V332C/M335C mutant was abolished by two blocking αIIb mAbs, HA5 and 10E5, but neither blocked nor further activated by the activating β3 mAb AP5 (Fig. 3B), confirming that the high affinity binding of the transfected cells was specific. Functional Properties of Mutant Receptors in CHO-K1 Transfectants—To further examine the disulfide-locked receptors, stable CHO-K1 transfectants were established, and clones were selected that expressed similar quantities of wild-type αIIbβ3T329C/A347C, αIIbβ3, and αIIbβ3V332C/M335C. The transfectants were recognized equally well by a panel of mAb to constitutively expressed αIIb, β3, and αIIbβ3 epitopes, with the exception of 7E3 mAb (Fig. 4A). Mutant αIIbβ3V332C/M335C blunted but did not completely abolish the binding of 7E3. CHO-K1 transfectants expressing the wild type receptor did not bind soluble fibrinogen or PAC-1 in Ca2+ but bound when stimulated by activating mAb PT25-2 (Fig. 4, B and C). Treatment with 5 mm DTT at 20 °C for 30 min slightly increased ligand binding to wild type αIIbβ3 in Ca2+, but this binding was much less than that seen with PT25-2 mAb with or without DTT treatment. Mutant αIIbβ3T329C/A347C did not bind fibrinogen or PAC-1 basally, and binding was not stimulatable with PT25-2. However, DTT treatment restored the ability of PT25-2 to stimulate fibrinogen and PAC-1 binding (Fig. 4, B and C), suggesting that the Cys329–Cys347 disulfide bond locked the I-like domain in the closed conformation, and this constraint was released by DTT treatment. By contrast, mutant αIIbβ3V332C/M335C showed high binding to soluble fibrinogen and PAC-1, and binding was not further increased by activation. DTT treatment did not reduce fibrinogen or PAC-1 binding of the αIIbβ3V332C/M335C mutant in Ca2+, probably because the Cys332–Cys335 disulfide bond was stable to reduction under nondenaturing conditions like the vast majority of the native disulfides in β3. The affinity state of disulfide-bonded mutants was further tested in cell adhesion assays on immobilized fibrinogen. High affinity is required for binding to soluble ligand or ligand mimetic mAbs. In contrast, wild type αIIbβ3 can mediate cell adhesion to immobilized fibrinogen in the absence of activation, as long as high coating concentrations above 1 μg/ml of fibrinogen are used (Fig. 4D), consistent with our previous report (9Luo B.-H. Springer T.A. Takagi J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2403-2408Crossref PubMed Scopus (124) Google Scholar). DTT treatment slightly increased the avidity of the wild type receptor, as shown by a shift in the dose-response curve. In contrast, mutant αIIbβ3T329C/A347C did not adhere even at the highest coating concentration of fibrinogen, whereas DTT treatment yielded binding of αIIbβ3T329C/A347C indistinguishable from that of the DTT-treated wild type receptor, suggesting that DTT treatment could release the disulfide bond, which lock
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