Critical residues for ligand binding in blade 2 of the propeller domain of the integrin αIIb subunit

Tatsushiro Tamura; Takaaki Hato; Jun Yamanouchi; Shigeru Fujita

doi:10.1160/TH03-06-0392

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2004; 91(01): 111-118
DOI: 10.1160/TH03-06-0392

Platelets and Blood Cells

Schattauer GmbH

Critical residues for ligand binding in blade 2 of the propeller domain of the integrin αIIb subunit

Tatsushiro Tamura

¹Department of Internal Medicine 1

,

Takaaki Hato

²Division of Blood Transfusion, Ehime University School of Medicine, Shigenobu, Ehime, Japan

,

Jun Yamanouchi

¹Department of Internal Medicine 1

,

Shigeru Fujita

¹Department of Internal Medicine 1

²Division of Blood Transfusion, Ehime University School of Medicine, Shigenobu, Ehime, Japan

› Institutsangaben

Abstract

Summary

Ligand binding to integrin αIIbβ3 is a key event of thrombus formation. The propeller domain of the αIIb subunit has been implicated in ligand binding. Recently, the ligand binding site of the αV propeller was determined by crystal structure analysis. However, the structural basis of ligand recognition by the αIIb propeller remains to be determined. In this study, we conducted site-directed mutagenesis of all residues located in the loops extending above blades 2 and 4 of the αIIb propeller, which are spatially close to, but distinct from, the loops that contain the binding site for an RGD ligand in the crystal structure of the αV propeller. Replacement by alanine of Q111, H112 or N114 in the loop within the blade 2 (the W2:2-3 loop in the propeller model) abolished binding of a ligand-mimetic antibody and fibrinogen to αIIbβ3 induced by different types of integrin activation including activation of αIIbβ3 by β3 cytoplasmic mutation. CHO cells stably expressing recombinant αIIbβ3 bearing Q111A, H112A or N114A mutation did not exhibit αIIbβ3mediated adhesion to fibrinogen. According to the crystal structure of αVβ3, the αV residue corresponding to αIIbN114 is exposed on the integrin surface and close to the RGD binding site. These results suggest that the Q111, H112 and N114 residues in the loop within blade 2 of the αIIb propeller are critical for ligand binding, possibly because of direct interaction with ligands or modulation of the RGD binding pocket.

Keywords

Adhesion receptors/integrins - platelet physiology - protein structure/folding - cell-matrix interactions

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Referenzen

References
1 Giancotti FG, Ruoslahti E. Transduction integrin signaling. Science 1999; 285: 1028-32.
2 Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell 2002; 110: 673-87.
3 Gottschalk KE, Kessler H. The structures of integrins and integrin-ligand complexes: implications for drug design and signal transduction. Angew Chem Int Ed Engl 2002; 41: 3767-74.
4 Coller BS. Anti-GPIIb/IIIa drugs: Current strategies and future directions. Thromb. Haemost 2001; 86: 427-43.
5 Savage B, Almus-Jacobs F. et al. Specific synergy of multiple substrate-receptor interactions in platelet thrombus formation under flow. Cell 1998; 94: 657-66.
6 Ruggeri ZM, Dent JA, Saldivar E. Contribution of distinct adhesive interactions to platelet aggregation in flowing blood. Blood 1999; 94: 172-8.
7 Tsuji S, Sugimoto M, Miyata S. et al. Realtime analysis of mural thrombus formation in various platelet aggregation disorders: Distinct shear-dependent roles of platelet receptors and adhesive proteins under flow. Blood 1999; 94: 968-75.
8 Springer TA. Folding of the N-terminal, ligand-binding region of integrin α-subunits into a β-propeller domain. Proc Natl Acad Sci USA 1997; 94: 65-72.
9 Humphries MJ. Integrin structure. Biochem. Soc. Trans 2000; 28: 311-40.
10 Xiong JP, Stehle T, Diefenbach B. et al. Crystal structure of the extracellular segment of integrin alpha Vbeta3. Science 2001; 294: 339-45.
11 Xiong JP, Stehle T, Zhang R. et al. Crystal structure of the extracellular segment of integrin alpha Vbeta3 in complex with an Arg- Gly-Asp ligand. Science 2002; 296: 151-5.
12 Felding-Habermann B, Cheresh DA. Vitronectin and its receptors. Curr Opin Cell Biol 1993; 05: 864-8.
13 Wickham TJ, Mathias P, Cheresh DA. et al. Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment. Cell 1993; 73: 309-19.
14 Hu DD, Hoyer JR, Smith JW. Ca²⁺ suppresses cell adhesion to osteopontin by attenuating binding affinity for integrin α_vβ₃ . J Biol Chem 1995; 270: 9917-25.
15 Brooks PC, Stromblad S, Sanders LC, von Schalscha TL, Aimes RT, Stetler-Stevenson WG, Quigley JP, Cheresh DA. Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin alpha v beta 3. Cell 1996; 85: 683-93.
16 Scarborough RM, Naughton MA, Teng W. et al. Design of potent and specific integrin antagonists. Peptide antagonists with high specificity for glycoprotein IIb-IIIa. J Biol Chem 1993; 268: 1066-73.
17 Honda S, Tomiyama Y, Shiraga M. et al. A two-amino acid insertion in the Cys146- Cys167 loop of the α_IIb subunit is associated with a variant of Glanzmann thrombasthenia Critical role of Asp163 in ligand binding. J Clin Invest 1998; 102: 1183-92.
18 Grimaldi CM, Chen FP, Wu CH. et al. Glycoprotein IIb Leu214Pro mutation produces glanzmann thrombasthenia with both quantitative and qualitative abnormalities in GPIIb/IIIa. Blood 1998; 91: 1562-71.
19 Basani RB, French DL, Vilaire G. et al. A naturally occurring mutation near the amino terminus of αIIb defines a new region involved in ligand binding to αIIbβ3. Blood 2000; 95: 180-8.
20 Kamata T, Irie A, Tokuhira M, Takada Y. Critical residues of integrin α_IIb subunit for binding of α_IIbβ₃(glycoprotein IIb-IIIa) to fibrinogen and ligand-mimetic antibodies (PAC-1, OP-G2, and LJ-CP3). J. Biol Chem 1996; 271: 18610-5.
21 Tozer EC, Baker EK, Ginsberg MH. et al. A mutation in the α subunit of the platelet integrin α_IIbβ₃ identifies a novel region important for ligand binding. Blood 1999; 93: 918-24.
22 Kamata T, Tieu KK, Springer TA. et al. Amino acid residues in the αIIb subunit that are critical for ligand binding to integrin αIIbβ3 are clustered in the beta- propeller model. J Biol Chem 2001; 276: 44275-83.
23 Shattil SJ, Hoxie JA, Cunningham M. et al. Changes in the platelet membrane glycoprotein IIb-IIIa complex during platelet activation. J Biol Chem 1985; 260: 11107-14.
24 Tokuhira M, Handa M, Kamata T. et al. A novel regulatory epitope defined by a murine monoclonal antibody to the platelet GPIIb-IIIa complex (αIIbβ3 integrin). Thromb Haemost 1996; 76: 1038-46.
25 Yamanouchi J, Hato T, Tamura T. et al. Identification of critical residues for ligand binding in the integrin beta3 I-domain by sitedirected mutagenesis. Thromb Haemost 2002; 87: 756-62.
26 Higuchi R, Krummel B, Saiki RK. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res 1988; 16: 7351-67.
27 Hato T, Pampori N, Shattil SJ. Complementary roles for receptor clustering and conformational change in the adhesive and signaling functions of integrin α_IIbβ₃ . J Cell Biol 1998; 141: 1685-95.
28 O’Toole TE, Katagiri Y, Faull RJ. et al. Integrin cytoplasmic domains mediate inside- out signal transduction. J Cell Biol 1994; 124: 1047-59.
29 Puzon-McLaughlin W, Kamata T. et al. Multiple discontinuous ligand-mimetic antibody binding sites define a ligand binding pocket in integrin α_IIbβ₃ . J Biol Chem 2000; 275: 7795-802.
30 Hughes PE, Diaz-Gonzalez F, Leong L. et al. Breaking the integrin hinge - A defined structural constraint regulates integrin signaling. J Biol Chem 1996; 271: 6571-4.
31 Brooks PC, Montgomery AMP, Rosenfeld M. et al. Integrin α_vβ₃ antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 1994; 79: 1157-64.
32 Kiyoi T, Tomiyama Y, Honda S. et al. A naturally occurring Tyr143Hisalpha IIb mutation abolishes alpha IIbbeta 3 function for soluble ligands but retains its ability for mediating cell adhesion and clot retraction: comparison with other mutations causing ligand-binding defects. Blood 2003; 101: 3485-91.
33 Morel-Kopp MC, Melchior C, Chen P. et al. A naturally occurring point mutation in the beta3 integrin MIDAS-like domain affects differently alphavbeta3 and alphaIIIbbeta3 receptor function. Thromb Haemost 2001; 86: 1425-34.
34 Arnaout MA, Goodman SL, Xiong JP. Coming to grips with integrin binding to ligands. Curr Opin Cell Biol 2002; 14: 641-51.
35 Yokoyama K, Zhang XP, Medved L. et al. Specific binding of integrin αvβ3 to the fibrinogen gamma and α_E chain C-terminal domains. Biochemistry 1999; 38: 5872-7.
36 Emsley J, Knight CG, Farndale RW. et al. Structural basis of collagen recognition by integrin α₂β₁ . Cell 2000; 101: 47-56.
37 Takagi J, Petre BM, Walz T. et al. Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell 2002; 110: 599-11.
38 Koivunen E, Wang B, Ruoslahti E. Isolation of a highly specific ligand for the alpha 5 beta 1 integrin from a phage display library. J Cell Biol 1994; 124: 373-80.
39 Mould AP, Burrows L, Humphries MJ. Identification of amino acid residues that form part of the ligand-binding pocket of integrin alpha5 beta1. J Biol Chem 1998; 273: 25664-72.
40 Burrows L, Clark K, Mould AP. et al. Fine mapping of inhibitory anti-alpha5 monoclonal antibody epitopes that differentially affect integrin-ligand binding. Biochem J 1999; 344 pt 2: 527-33.
41 Krukonis ES, Dersch P, Eble JA, Isberg RR. Differential effects of integrin alpha chain mutations on invasin and natural ligand interaction. J Biol Chem 1998; 273: 31837-43.