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High Affinity Ligand Binding by Integrins Does Not Involve Head Separation

2003; Elsevier BV; Volume: 278; Issue: 19 Linguagem: Inglês

10.1074/jbc.m301516200

1083-351X

Bing Luo, Timothy A. Springer, Junichi Takagi,

Cellular Mechanics and Interactions

Conformational change in the integrin extracellular domain is required for high affinity ligand binding and is also involved in post-ligand binding cellular signaling. Although there is evidence to the contrary, electron microscopic studies showing that ligand binding triggers α- and औ-subunit dissociation in the integrin headpiece have gained popularity and support the hypothesis that head separation activates integrins. To test directly the head separation hypothesis, we enforced head association by introducing disulfide bonds across the interface between the α-subunit औ-propeller domain and the औ-subunit I-like domain. Basal and activation-dependent ligand binding by αIIbऔ3 and αVऔ3 was unaffected. The covalent linkage prevented dissociation of αIIbऔ3 into its subunits on EDTA-treated cells. Whereas EDTA dissociated wild type αIIbऔ3 on the cell surface, a ligand-mimetic Arg-Gly-Asp peptide did not, as judged by binding of complex-specific antibodies. Finally, a high affinity ligand-mimetic compound stabilized noncovalent association between αIIband औ3 headpiece fragments in the presence of SDS, indicating that ligand binding actually stabilized subunit association at the head, as opposed to the suggested subunit separation. The mechanisms of conformational regulation of integrin function should therefore be considered in the context of the associated αऔ headpiece. Conformational change in the integrin extracellular domain is required for high affinity ligand binding and is also involved in post-ligand binding cellular signaling. Although there is evidence to the contrary, electron microscopic studies showing that ligand binding triggers α- and औ-subunit dissociation in the integrin headpiece have gained popularity and support the hypothesis that head separation activates integrins. To test directly the head separation hypothesis, we enforced head association by introducing disulfide bonds across the interface between the α-subunit औ-propeller domain and the औ-subunit I-like domain. Basal and activation-dependent ligand binding by αIIbऔ3 and αVऔ3 was unaffected. The covalent linkage prevented dissociation of αIIbऔ3 into its subunits on EDTA-treated cells. Whereas EDTA dissociated wild type αIIbऔ3 on the cell surface, a ligand-mimetic Arg-Gly-Asp peptide did not, as judged by binding of complex-specific antibodies. Finally, a high affinity ligand-mimetic compound stabilized noncovalent association between αIIband औ3 headpiece fragments in the presence of SDS, indicating that ligand binding actually stabilized subunit association at the head, as opposed to the suggested subunit separation. The mechanisms of conformational regulation of integrin function should therefore be considered in the context of the associated αऔ headpiece. monoclonal antibody fluorescein isothiocyanate Integrins are major metazoan adhesion receptors that play a fundamental role in cellular organization. They mediate cell-extracellular matrix as well as cell-cell adhesion, connect extracellular cues to the cytoskeleton, and activate many intracellular signaling pathways (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9007) Google Scholar, 2Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6868) Google Scholar). One unique aspect of integrins is that the affinity of their extracellular domain for biological ligands can be rapidly up-regulated by signals from within the cell. Rapid and precise control of integrin activation is particularly important for leukocytes and platelets, which circulate in the vascular system, where leukocyte emigration and thrombus formation mediated by integrins must be triggered only at the appropriate location. Integrins compose two noncovalently associated type I transmembrane glycoprotein α- and औ-subunits (3Takagi J. Springer T.A. Immunol. Rev. 2002; 186: 141-163Crossref PubMed Scopus (318) Google Scholar). A crystal structure of the extracellular domain of the integrin αVऔ3 revealed a bent conformation, in which there is an acute angle between the headpiece and tailpiece (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 (1112) Google Scholar), and an extensive headpiece-tailpiece interface (5Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (935) Google Scholar). Recently, we have shown that the bent conformation represents the low affinity receptor and that activation is associated with a switchblade-like motion of the headpiece resulting in a highly extended conformation (5Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (935) Google Scholar). The integrin headpiece contains the ligand-binding site. The headpiece contains the α-subunit औ-propeller and thigh domains and the औ-subunit I-like and hybrid domains (Fig. 1), corresponding approximately to the N-terminal two-thirds of the extracellular domain of each subunit. A crystal structure with a bound ligand-mimetic peptide revealed that ligand binds to an interface formed by the औ-subunit I-like domain and औ-sheets 2–4 of the α-subunit औ-propeller domain (6Xiong J.P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S.L. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1400) Google Scholar) (Fig. 1). Many experiments support the idea that the inter-subunit association at the cytoplasmic region maintains integrins in low affinity state (7Hughes P.E. Diaz-Gonzalez F. Leong L. Wu C. McDonald J.A. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 1996; 271: 6571-6574Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar, 8Takagi J. Erickson H.P. Springer T.A. Nat. Struct. Biol. 2001; 8: 412-416Crossref PubMed Scopus (226) Google Scholar, 9Lu C. Takagi J. Springer T.A. J. Biol. Chem. 2001; 276: 14642-14648Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 10Vinogradova O. Velyvis A. Velyviene A. Hu B. Haas T.A. Plow E.F. Qin J. Cell. 2002; 110: 587-597Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). Originally, this notion led to a “hinge hypothesis,” where association/dissociation of the cytoplasmic tails caused hinging between the two subunits, and ultimately changed the conformation of the ligand-binding extracellular segments (11O'Toole T.E. Katagiri Y. Faull R.J. Peter K. Tamura R. Quaranta V. Loftus J.C. Shattil S.J. Ginsberg M.H. J. Cell Biol. 1994; 124: 1047-1059Crossref PubMed Scopus (580) Google Scholar). However, the nature of the conformational change that regulates ligand binding by integrins has been controversial (2Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6868) Google Scholar, 3Takagi J. Springer T.A. Immunol. Rev. 2002; 186: 141-163Crossref PubMed Scopus (318) Google Scholar, 5Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (935) Google Scholar, 12Plow E.F. Haas T.A. Zhang L. Loftus J. Smith J.W. J. Biol. Chem. 2000; 275: 21785-21788Abstract Full Text Full Text PDF PubMed Scopus (1115) Google Scholar, 13Arnaout M. Goodman S. Xiong J. Curr. Opin. Cell Biol. 2002; 14: 641-652Crossref PubMed Scopus (170) Google Scholar, 14Liddington R.C. Structure. 2002; 10: 605-607Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 15Liddington R.C. Ginsberg M.H. J. Cell Biol. 2002; 158: 833-839Crossref PubMed Scopus (253) Google Scholar). Hantgan et al.(16Hantgan R.R. Paumi C. Rocco M. Weisel J.W. Biochemistry. 1999; 38: 14461-14474Crossref PubMed Scopus (94) Google Scholar, 17Hantgan R.R. Rocco M. Nagaswami C. Weisel J.W. Protein Sci. 2001; 10: 1614-1626Crossref PubMed Scopus (33) Google Scholar), using rotary shadowing EM, reported that binding of RGD peptides induced separation of the headpiece of detergent-solubilized αIIbऔ3. These images suggested a wide separation in the headpiece, with no interaction remaining between the N-terminal halves of the α- and औ-subunits. These observations have been highly influential, in part because they seemed to fit with earlier schematics of integrin activation models where a hinge-like motion at the transmembrane domains is transmitted through rigid α- and औ-subunit tailpiece segments to the headpiece, resulting in movement apart of the α- and औ-subunits in the headpiece (11O'Toole T.E. Katagiri Y. Faull R.J. Peter K. Tamura R. Quaranta V. Loftus J.C. Shattil S.J. Ginsberg M.H. J. Cell Biol. 1994; 124: 1047-1059Crossref PubMed Scopus (580) Google Scholar, 12Plow E.F. Haas T.A. Zhang L. Loftus J. Smith J.W. J. Biol. Chem. 2000; 275: 21785-21788Abstract Full Text Full Text PDF PubMed Scopus (1115) Google Scholar, 18Loftus J.C. Liddington R.C. J. Clin. Invest. 1997; 99: 2302-2306Crossref PubMed Google Scholar). However, recent high resolution negative stain EM studies have shown that the α-subunit leg can exist in two distinct conformations with respect to the headpiece and that the औ-subunit leg is highly flexible (5Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (935) Google Scholar). Existence of multiple modular domains (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 (1112) Google Scholar) also disfavors rigid movement of the entire stalk region. Because the legs are flexible, it is hard to imagine that information can be transmitted to the headpiece as proposed in the hinge model. Furthermore, high resolution EM studies (5Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (935) Google Scholar), as well as many other EM studies (19Erb E.M. Tangemann K. Bohrmann B. Muller B. Engel J. Biochemistry. 1997; 36: 7395-7402Crossref PubMed Scopus (94) Google Scholar, 20Weisel J.W. Nagaswami C. Vilaire G. Bennett J.S. J. Biol. Chem. 1992; 267: 16637-16643Abstract Full Text PDF PubMed Google Scholar, 21Hantgan R.R. Lyles D.S. Mallett T.C. Rocco M. Nagaswami C. Weisel J.W. J. Biol. Chem. 2002; 278: 3417-3426Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 22Du X. Gu M. Weisel J.W. Nagaswami C. Bennett J.S. Bowditch R. Ginsberg M.H. J. Biol. Chem. 1993; 268: 23087-23092Abstract Full Text PDF PubMed Google Scholar), have shown that ligand binding to integrins is not accompanied by head separation. There are other reasons for the popularity of the head separation model. First, it has been suggested that residues that have been implicated in ligand binding are buried in the headpiece and that separation could expose them, resulting in higher affinity binding (2Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6868) Google Scholar). Second, there is the mystery of the synergy site in fibronectin type III module 9 of fibronectin, which is distant from the RGD site in module 10. It has been proposed that separation of the headpiece (2Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6868) Google Scholar,15Liddington R.C. Ginsberg M.H. J. Cell Biol. 2002; 158: 833-839Crossref PubMed Scopus (253) Google Scholar) would facilitate simultaneous binding of the α-subunit to the synergy site and the औ-subunit to the RGD site (23Mould A.P. Askari J.A. Aota S. Yamada K.M. Irie A. Takada Y. Mardon H.J. Humphries M.J. J. Biol. Chem. 1997; 272: 17283-17292Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). However, the liganded αVऔ3 crystal structure shows that the Arg of RGD binds to the α-subunit and the Asp of RGD binds to the औ-subunit (6Xiong J.P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S.L. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1400) Google Scholar), strongly suggesting that headpiece separation would disrupt binding to RGD. Third, there are structural homologies between integrins and G proteins (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 (1112) Google Scholar, 24Lee J.-O. Bankston L.A. Arnaout M.A. Liddington R.C. Structure. 1995; 3: 1333-1340Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar, 25Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 65-72Crossref PubMed Scopus (390) Google Scholar). By taking this analogy further, it has been suggested that upon integrin activation, the औ-propeller and I-like domains might dissociate analogously to the G protein औ- and α-subunits (2Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6868) Google Scholar, 15Liddington R.C. Ginsberg M.H. J. Cell Biol. 2002; 158: 833-839Crossref PubMed Scopus (253) Google Scholar). An alternative model for integrin activation has been proposed that is supported by high resolution EM, physicochemical studies, ligand binding assays, introduction of disulfide bonds that lock in the bent conformation, and localization of epitopes that become exposed after integrin activation (5Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (935) Google Scholar, 26Beglova N. Blacklow S.C. Takagi J. Springer T.A. Nat. Struct. Biol. 2002; 9: 282-287Crossref PubMed Scopus (261) Google Scholar). In this model, activation is regulated by the conformational equilibrium between three states as follows: a bent conformation with low affinity, an extended conformation with a closed headpiece with intermediate affinity, and an extended conformation with an open headpiece with high affinity. Binding of RGD peptide was found not to induce head separation but to induce a dramatic change in angle between the औ-subunit I-like and hybrid domains, leading to the swing-out of the hybrid domain away from the α-subunit (5Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (935) Google Scholar). The prominence of the hybrid domain in the interface between the headpiece and the tailpiece in the bent conformation provides a mechanism for linking the change in angle upon ligand binding to the equilibrium between the bent and extended integrin conformations (5Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (935) Google Scholar). Definitive experimental testing of the head separation model is important. Therefore, we have used mutagenesis to introduce disulfide bonds between the α-subunit औ-propeller domain and the औ-subunit I-like domain, and we have tested the effect of preventing head separation on activation of αIIbऔ3 and αVऔ3 integrins on the cell surface. Furthermore, we test the effect of ligand-mimetic compounds on the association between the α- and औ-subunits in native integrins on the cell surface and in soluble integrin fragments that contain only the headpiece. Mouse monoclonal antibody (mAb)1 PT25-2 recognizing human αIIb subunit (27Tokuhira M. Handa M. Kamata T. Oda A. Katayama M. Tomiyama Y. Murata M. Kawai Y. Watanabe K. Ikeda Y. Thromb. Haemostasis. 1996; 76: 1038-1046Crossref PubMed Scopus (53) Google Scholar) was a gift from Dr. M. Handa (Keio University, Tokyo, Japan). Mouse mAb 10E5 recognizing the human αIIbऔ3 complex (28Coller B.S. J. Clin. Invest. 1985; 76: 101-108Crossref PubMed Scopus (468) Google Scholar) was a gift from Dr. B. S. Coller (Rockefeller University, New York). Mouse anti-औ3 AP3 was from American Type Culture Collection. All other mAbs were obtained from the Fifth International Leukocyte Workshop (29Petruzzelli L. Luk J. Springer T.A. Schlossman S.F. Boumsell L. Gilks W. Harlan J. Kishimoto T. Morimoto T. Ritz J. Shaw S. Silverstein R. Springer T. Tedder T. Todd R. Leucocyte Typing V: White Cell Differentiation Antigens. Oxford University Press, New York1995: 1581-1585Google Scholar). Plasmids coding for full-length human αIIb, αV, and औ3 were subcloned into pcDNA3.1/Myc-His(+) or pEF/V5-HisA as described previously (5Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (935) Google Scholar). Mutants were made using site-directed mutagenesis, and DNA sequences were confirmed before being transfected into 293T cells using calcium phosphate precipitation. Transfected cells were metabolically labeled with [35S]cysteine/methionine as described (5Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (935) Google Scholar). Labeled cell lysates were immunoprecipitated with anti-औ3 AP3, eluted with 0.57 SDS, and subjected to nonreducing or reducing SDS-7.57 PAGE and fluorography. Binding of fluorescein-labeled human fibrinogen and fibronectin were performed as previously described (5Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (935) Google Scholar). To test the effect of EDTA treatment on mAb epitope expression, transiently transfected 293T cells were preincubated in 20 mm Tris-buffered saline, pH 8.4, containing either 1 mm Ca2+, 1 mmMg2+, or 5 mm EDTA at 37 °C for 30 min, followed by washing and resuspension in 20 mm Hepes, 150 mm NaCl, 5.5 mm glucose, 17 bovine serum albumin, and 1 mm Ca2+, 1 mmMg2+ (HBS). Cells were then incubated with mAbs on ice for 30 min, followed by staining with FITC-conjugated anti-mouse IgG and flow cytometry. To test the effect of RGD peptide on mAb epitope expression, cells in HBS were incubated with 100 ॖmGRGDSP peptide at room temperature for 30 min before adding mAbs and staining as above. An integrin headpiece fragment comprising αIIb residues 1–621 and औ3 residues 1–472 was produced in Chinese hamster ovary Lec 3.2.8.1 cells stably transfected with plasmids coding for each fragment. Acid-base α-helical coiled-coil peptides were fused to the C termini of the α- and औ-subunits to increase the stability of the heterodimer. Methods were as described previously (8Takagi J. Erickson H.P. Springer T.A. Nat. Struct. Biol. 2001; 8: 412-416Crossref PubMed Scopus (226) Google Scholar). A hexahistidine tag was also attached to the C terminus of the औ-subunit to facilitate purification by nickel chelate chromatography (8Takagi J. Erickson H.P. Springer T.A. Nat. Struct. Biol. 2001; 8: 412-416Crossref PubMed Scopus (226) Google Scholar). The purified headpiece fragment was treated with 10 ॖg/ml chymotrypsin for 16 h at 25 °C to remove the C-terminal clasp, and incubated with the high affinity RGD-mimetic compound L738,167 (gift from Dr. G. D. Hartman, Merck) (30Prueksaritanont T. Gorham L.M. Naue J.A. Hamill T.G. Askew B.C. Vyas K.P. Drug Metab. Dispos. 1997; 25: 355-361PubMed Google Scholar) at 10 ॖm for 30 min at 37 °C. The mixture was cooled to room temperature; an equal volume of sample buffer containing 0.17 SDS was added, and samples were immediately subjected to nonreducing SDS-PAGE on a 4–207 gradient gel.