A mutation in the integrin αIIb subunit that selectively inhibits αIIbβ3 receptor function

Kim B. Perkins; Joseph C. Loftus

doi:10.1160/TH03-03-0157

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2003; 90(05): 853-862
DOI: 10.1160/TH03-03-0157

Platelets and Blood Cells

Schattauer GmbH

A mutation in the integrin α_IIb subunit that selectively inhibits α_IIbβ₃ receptor function

Authors

Kim B. Perkins

¹Mayo Clinic Scottsdale, Scottsdale, Arizona, USA
Joseph C. Loftus

¹Mayo Clinic Scottsdale, Scottsdale, Arizona, USA

Abstract

Summary

The α_IIband α_vintegrins have been shown to play a significant role in a variety of disease processes. α_IIbβ₃is a platelet-specific fibrinogen receptor that is critical for thrombosis and hemo-stasis. Determination of the basis of ligand recognition by α_IIbβ₃is essential for modulation of platelet function. To identify α_IIbresidues involved in α_IIbβ₃ligand binding function, cells expressing a constitutively active variant of α_IIbβ₃were randomly mutagenized and selected for loss of α_IIbβ₃ligand binding function. One mutant isolated in this manner contained a single amino acid substitution at position 96 in α_IIb(Ser96→Leu). Cells expressing this α_IIbmutant did not bind the ligand mimetic antibody PAC1 or adhere to fibrinogen. In addition, the mutant receptor did not bind to an RGD affinity matrix. Substitution of conserved serine residues at position 1 in β strand A of all seven repeats of α_IIbsimilarly inhibited ligand binding to α_IIbβ₃. α_IIbS96 maps to the central cavity of the β–propeller fold of the α_IIbsubunit immediately adjacent to a structurally important sequence at the center of the α and β subunit interface. In contrast, substitution of the analogous residues in α_vor α₄did not disrupt the ligand binding function of α_vβ₃or α₄β₁. These data support a potential unique structural or mechanistic role for this residue in α_IIbβ₃receptor function.

Keywords

α_IIbβ₃ - platelets - mutagenesis - flow cytometry

Full Text

References

References
1 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.
2 Xiong JP, Stehle T, Diefenbach B. et al Crystal structure of the extracellular segment of integrin α_vβ₃ . Science 2001; 294: 339-45.
3 Xiong JP, Stehle T, Zhang R. et al Crystal structure of the extracellular segment of inte-grin α_vβ₃ in complex with an Arg-Gly-Asp ligand. Science 2002; 296: 151-5.
4 Farrell DH, Thiagarajan P, Chung DW. et al Role of fibrinogen α and γ chain sites in platelet aggregation. Proc Natl Acad Sci USA 1992; 89: 10729-32.
5 Holmback K, Danton MJ, Suh TT. et al Impaired platelet aggregation and sustained bleeding in mice lacking the fibrinogen motif bound by integrin α_IIbβ₃ . EMBO J 1996; 15: 5760-71.
6 Lam SC, Plow EF, Smith MA. et al Evidence that arginyl-glycyl-aspartate peptides and fibrinogen γ chain peptides share a common binding site on platelets. J Biol Chem 1987; 262: 947-50.
7 Smith JW, Ruggeri ZM, Kunicki TJ. et al Interaction of integrins α_vβ₃ and glycoprotein IIb-IIIa with fibrinogen. Differential peptide recognition accounts for distinct binding sites. J Biol Chem 1990; 265: 12267-71.
8 Scarborough RM, Rose JW, Hsu MA. et al GPIIb-IIIa-specific integrin antagonist from the venom of Sistrurus m. barbouri. J Biol Chem 1991; 266: 9359-62.
9 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.
10 Plow EF, Cierniewski CS, Xiao Z. et al α_IIbβ₃ and its antagonism at the new millennium. Thromb Haemost 2001; 86: 34-40.
11 Baker EK, Tozer EC, Pfaff M. et al A genetic analysis of integrin function: Glanzmann thrombasthenia in vitro. Proc Natl Acad Sci USA 1997; 94: 1973-8.
12 O’Toole TE, Katagiri Y, Faull RJ. et al Integrin cytoplasmic domains mediate inside-out signal transduction. J Cell Biol 1994; 124: 1047-59.
13 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.
14 Tomiyama Y, Tsubakio T, Piotrowicz RS. et al The Arg-Gly-Asp (RGD) recognition site of platelet glycoprotein IIb-IIIa on nonactivated platelets is accessible to high-affinity macromolecules. Blood 1992; 79: 2303-12.
15 O’Toole TE, Loftus JC, Du XP. et al Affinity modulation of the α_IIbβ₃ integrin (platelet GPIIb-IIIa) is an intrinsic property of the receptor. Cell Regul 1990; 1: 883-93.
16 Frelinger AL, Cohen I, Plow EF. et al Selective inhibition of integrin function by antibodies specific for ligand-occupied receptor conformers. J Biol Chem 1990; 265: 6346-52.
17 Loftus JC, O’Toole TE, Plow EF. et al A β₃ integrin mutation abolishes ligand binding and alters divalent cation-dependent conformation. Science 1990; 249: 915-8.
18 Faull RJ, Wang J, Leavesley I D. et al A novel activating anti-β₁ integrin monoclonal antibody binds to the cysteine-rich repeats in the β₁ chain. J Biol Chem 1996; 271: 25099-106.
19 Tozer EC, Baker EK, Ginsberg MH. et al A mutation in the a subunit of the platelet inte-grin α_IIbβ₃ identifies a novel region important for ligand binding. Blood 1999; 93: 918-24.
20 Tozer EC, Liddington RC, Sutcliffe MJ. et al Ligand binding to integrin α_IIbβ₃ is dependent on a MIDAS-like domain in the β₃ subunit. J Biol Chem 1996; 271: 21978-84.
21 Ho SN, Hunt HD, Horton RM. et al Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 1989; 77: 51-9.
22 Hughes PE, O’Toole TE, Ylanne J. et al The conserved membrane-proximal region of an integrin cytoplasmic domain specifies ligand binding affinity. J Biol Chem 1995; 270: 12411-7.
23 Frelinger AL, Du XP, Plow EF. et al Monoclonal antibodies to ligand-occupied conformers of integrin α_IIbβ₃ (glycoprotein IIb-IIIa) alter receptor affinity, specificity, and function. J Biol Chem 1991; 266: 17106-1.
24 Ylanne J, Chen Y, O’Toole TE. et al Distinct functions of integrin α and β subunit cytoplasmic domains in cell spreading and formation of focal adhesions. J Cell Biol 1993; 122: 223-33.
25 French DL, Coller BS. Hematologically important mutations: Glanzmann thrombasthenia. Blood Cells Mol Dis 1997; 23 (01) 39-51.
26 Bajt ML, Loftus JC. Mutation of a ligand binding domain of β₃ integrin. Integral role of oxygenated residues in α_IIbβ₃ (GPIIb-IIIa) receptor function. J Biol Chem 1994; 269: 20913-9.
27 Kunicki TJ, Annis DS, Deng YJ. et al A molecular basis for affinity modulation of Fab ligand binding to integrin α_IIbβ₃ . J Biol Chem 1996; 271: 20315-21.
28 Plow EF, Haas TA, Zhang L. et al Ligand binding to integrins. J Biol Chem 2000; 275: 21785-8.
29 Kamata T, Puzon W, Takada Y. Identification of putative ligand-binding sites of the integrin α₄β₁ (VLA-4, CD49d/CD29). Biochem J 1995; 305: 945-51.
30 Wilcox DA, Wautier JL, Pidard D. et al A single amino acid substitution flanking the fourth calcium binding domain of α_IIb prevents maturation of the α_IIbβ₃ integrin complex. J Biol Chem 1994; 269: 4450-7.
31 Wilcox DA, Paddock CM, Lyman S. et al Glanzmann thrombasthenia resulting from a single amino acid substitution between the second and third calcium-binding domains of GPIIb. Role of the GPIIb amino terminus in integrin subunit association. J Clin Invest 1995; 95: 1553-60.
32 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.
33 Bowditch RD, Hariharan M, Tominna EF. et al Identification of a novel integrin binding site in fibronectin. Differential utilization by β₃ integrins. J Biol Chem 1994; 269: 10856-63.