A Naturally Occurring Point Mutation in the β3 Integrin MIDAS-like Domain Affects Differently αvβ3 and αIIbβ3 Receptor Function^[*]

Summary

We have investigated the effect of a new Leu¹⁹⁶Pro mutation, identified in the MIDAS-like domain of the β3 integrin subunit in a patient with type II Glanzmann thrombasthenia, on β3 integrin receptor function. Expression of the mutant β3Pro¹⁹⁶ subunit in CHO cells, either associated with recombinant human αIIb or αv, resulted in normal biosynthesis of β3 and heterodimerization with αv or IIb, but selectively interfered with αIIbβ3 maturation and transport to the cell surface. Functional analysis of the β3 mutant receptors revealed strong inhibition of αvβ3-mediated cell spreading on immobilized fibrinogen, focal contact formation, p125^FAK phosphorylation and fibrin clot retraction, as opposed to normal αIIbβ3-mediated cell interaction with immobilized fibrinogen, focal contact translocation and signaling. In contrast, antibody- or DTT-activated mutant αIIbβ3 was unable to bind soluble fibrinogen or the ligand mimetic PAC-1 monoclonal antibody, but underwent a conformational change following RGD peptide binding as demonstrated by AP5-LIBS epitope expression. These results suggest that (1) the highly conserved TL¹⁹⁶T motif in the β3 integrin subunit is located in a domain structurally important for the exposure of a functional binding site for soluble fibrinogen; and (2) that the MIDAS-like contact site in β3 is not involved in αIIbβ3-mediated cell adhesion to immobilized fibrinogen, while it is essential for αvβ3-mediated interaction with this ligand.

Keywords

Glanzmann thrombasthenia - 3 integrins - fibrinogen receptor - cell adhesion

^* This work was supported by grants from Centre de Recherche Public-Santé (CRP-Santé, Luxembourg), and CNRS (France). ⁺MC. M-K is a recipient of a European Community Marie Curie fellowship (ERBFMBICT 961531).

⁺ MC. M-K is a recipient of a European Community Marie Curie fellowship (ERBFMBICT 961531).

⁺⁺ P.C is a recipient of a fellowship from the Ministère de la Coopération, Luxembourg.

• References

• 1 Hynes RO. Integrins: Versatility, modulation and signaling in cell adhesion. Cell 1992; 69: 11-25.
• 2 Bennett JS, Vilaire G. Exposure of platelet fibrinogen receptors by ADP and epinephrine. J Clin Invest 1979; 64: 1393-401.
• 3 Peerschke EI, Zucker MB. Fibrinogen receptor exposure and aggregation of human blood platelets produced by ADP and chilling. Blood 1981; 57: 663-70.
• 4 Matsuno H, Stassen JM, Vermylen J, Deckmyn H. Inhibition of integrin function by a cyclic RGD-containing peptide prevents neointima formation. Circulation 1994; 90: 2203-6.
• 5 Brooks PC, Clark RA, Cheresh DA. Requirement of vascular integrin αvβ3 for angiogenesis. Science 1994; 264: 569-71.
• 6 Seftor REB, Seftor EA, Gehlsen KR, Stetler-Stevenson WG, Brown PD, Ruoslahti E, Hendrix MJC. Role of the αvβ3 integrin in human melanoma cell invasion. Proc Natl Acad Sci USA 1992; 89: 1557-61.
• 7 Loftus JC, Smith JW, Ginsberg MH. Integrin-mediated cell adhesion: The extracellular face. J Biol Chem 1994; 269: 25235-8.
• 8 D’Souza SE, Ginsberg MH, Burke TA, Plow EF. The ligand binding site of the platelet integrin receptor GPIIb-IIIa is proximal to the second calcium binding domain of its α subunit. J Biol Chem 1990; 265: 3440-6.
• 9 Springer T. 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.
• 10 D’Souza SE, Ginsberg MH, Burke TA, Lam SC-T, Plow EF. Localization of an Arg-Gly-Asp recognition site within an integrin adhesion receptor. Science 1988; 242: 91-3.
• 11 Smith JW, Cheresh DA. The Arg-Gly-Asp binding domain of the vitronectin receptor. J Biol Chem 1988; 263: 18726-31.
• 12 Charo IF, Nannizzi L, Phillips DR, Hsu MA, Scarborough RM. Inhibition of fibrinogen binding to GPIIbIIIa by a GPIIIa peptide. J Biol Chem 1991; 266: 1415-21.
• 13 Tozer EC, Liddington RC, Sutcliffe MJ, Smeeton AH, Loftus JC. Ligand binding to integrin αIIbβ3 is dependent on a MIDAS-like domain in the β3 subunit. J Biol Chem 1996; 271: 21978-84.
• 14 Smith JW, Cheresh D. Integrin αvβ3-ligand interaction. Identification of a heterodimeric RGD binding site on the vitronectin receptor. J Biol Chem 1990; 265: 2168-72.
• 15 Loftus JC, O’Toole TE, Plow EF, Glass A, Frelinger AL, Ginsberg MH. A β3 integrin mutation abolishes ligand binding and alters divalent cation-dependent conformation. Science 1990; 249: 915-8.
• 16 Bajt ML, Ginsberg MH, Frelinger AL, Berndt MC, Loftus JC. A spontaneous mutation of integrin αIIbβ3 (platelet glycoprotein IIb-IIIa) helps define a ligand binding site. J Biol Chem 1992; 267: 3789-94.
• 17 Lanza F, Stierle A, Fournier D, Morales M, Andre G, Nurden AT, Cazenave J-P. A new variant of Glanzmann’s thrombasthenia (Strasbourg I). Platelets with functionnally defective IIb-IIIa complexes and a glycoprotein IIIa ²¹⁴Arg – ²¹⁴Trp mutation. J Clin Invest 1992; 89: 1995-2004.
• 18 Grimaldi CM, Chen FP, Wu CH, Weiss HJ, Coller BS, French DL. Glyco-protein IIb Leu²¹⁴Pro mutation produces Glanzmann thrombasthenia with both quantitative and qualitative abnormalities in GPIIb/IIIa. Blood 1998; 91: 1562-71.
• 19 Morel-Kopp M-C, Lecompte T, Schlegel N, Hivert P, Kaplan C. Use of murine monoclonal antibodies to study thrombopathies related to GP IIb/IIIa complexes. In: Platelet Immunology: Clinical and fundamental aspects. Kaplan-Gouet C, Schlegel N, Salmon C, McGregor J. eds. Paris, France: John Libbey Paris; 1991: 161-71.
• 20 Morel-Kopp MC, Clemenceau S, Schlegel N, Lecompte T, Aurousseau MH, Kaplan C. Platelet phenotyping in carriers for Glanzmann’s thrombasthenia: a simple screening test for assessment of the molecular defect. Transfus Med 1995; 2: 123-9.
• 21 Miller SA, Dykes DD, Plesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acid Res 1987; 16: 1215-9.
• 22 Jin Y, Dietz HC, Nurden A, Bray PF. Single-strand conformation polymorphism analysis is a rapid and effective method for the identification of mutations and polymorphisms in the gene for glycoprotein IIIa. Blood 1993; 82: 2281-8.
• 23 Kieffer N, Fitzgerald LA, Wolf D, Cheresh DA, Phillips DR. Adhesive properties of the β3 integrins: Comparison of GPIIb-IIIa and the vitronectin receptor individually expressed in human melanoma cells. J Cell Biol 1991; 113: 451-61.
• 24 Schaffner-Reckinger E, Gouon V, Melchior C, Plançon S, Kieffer N. Distinct involvement of b 3 integrin cytoplasmic domain tyrosine residues 747 and 759 in integrin-mediated cytoskeletal assembly and phosphotyro-sine signaling. J Biol Chem 1998; 273: 12623-32.
• 25 Kekomaki R, Dawson B, McFarland J, Kunicki TJ. Localization of human platelet autoantigens to the cysteine-rich region of glycoprotein IIIa. J Clin Invest 1991; 88: 847-54.
• 26 Honda S, Tomiyama Y, Pelletier AJ, Annis D, Honda Y, Orchekowski R, Ruggeri R, Kunicki TJ. Topography of ligand-induced binding sites, including a novel cation-sensitive epitope (AP5) at the amino terminus of the human integrin β3 subunit. J Biol Chem 1995; 270: 11947-54.
• 27 Shattil SJ, Hoxie JA, Cunningham M, Brass LF. Changes in the platelet membrane glycoprotein IIb-IIIa complex during platelet activation. J Biol Chem 1985; 260: 11107-14.
• 28 Kouns WC, Wall CD, White MOAB, Fox CF, Jennings LK. A conformation-dependent epitope of human platelet glycoprotein IIIa. J Biol Chem 1990; 265: 20594-601.
• 29 Deckmyn H, Stanssens P, Hoet B, Declerck PJ, Lauwereys M, Gansemans Y, Tomai I, Vermylen J. An echistatin-like Arg-Gly-Asp (RGD)-containing sequence in the heavy chain CDR3 of a murine monoclonal antibody that inhibits human platelet glycoprotein IIb/IIIa function. Br J Haematol 1994; 87: 562-71.
• 30 Tomiyama Y, Tsubakio T, Piotrowicz RS, Kurata Y, Loftus JC, Kunicki TJ. 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.
• 31 Lyman S, Gilmore A, Burridge K, Gidwitz S, White GC. Integrin-mediated activation of focal adhesion kinase is independent of focal adhesion formation or integrin activation – Studies with activated and inhibitory β3 cytoplasmic domain mutants. J Biol Chem 1997; 272: 22538-47.
• 32 Tam SH, Sassoli PM, Jordan RE, Nakada MT. AbcixiMoAb (ReoPro, chimeric 7E3 Fab) demonstrates equivalent affinity and functional blockade of glycoprotein IIb/IIIa and αvβ3 integrins. Circulation 1998; 98: 1085-91.
• 33 Fitzgerald LA, Steiner B, Rall Jr SC, Lo SS, Phillips DR. Protein sequence of endothelial glycoprotein IIIa derived from a cDNA clone. Identity with platelet glycoprotein IIIa and similarity to “integrin”. J Biol Chem 1987; 262: 3936-9.
• 34 Cheresh DA, Berliner SA, Vicente V, Ruggeri ZM. Recognition of distinct adhesive sites on fibrinogen by related integrins on platelets and endothelial cells. Cell 1989; 58: 945-53.
• 35 Cherny RC, Honan MC, Thiagarajan P. Site-directed mutagenesis of the arginine-glycine-aspartic acid in vitronectin abolishes cell adhesion. J Biol Chem 1993; 268: 9725-9.
• 36 Alemany M, Concord E, Garin J, Vinçon M, Giles A, Marguerie G, Gulino D. Sequence 274-368 in the β3-subunit of the integrin αIIbbα3 provides a ligand recognition and binding domain for the γ-chain of fibrinogen that is independent of platelet activation. Blood 1996; 87: 592-601.
• 37 Farrell DH, Thiagarajan P. Binding of recombinant fibrinogen mutants to platelets. J Biol Chem 1994; 269: 226-31.
• 38 Rooney MOAB, Parise LV, Lord ST. Dissecting clot retraction and platelet aggregation. J Biol Chem 1996; 271: 8553-5.
• 39 Holmback K, Danton MJS, Suh TT, Daugherty CC, Degen JL. Impaired platelet aggregation and sustained bleeding in mice lacking the fibrinogen motif bound by integrin αIIbβ3. EMBO 1996; 15: 5760-71.
• 40 Jandrot-Perrus M, Mosesson MW, Denninger M-H, Menache D. Studies of platelet fibrinogen from a subject with a congenital plasma fibrinogen abnormality (fibrinogen Paris I). Blood 1979; 54: 1109-16.
• 41 Denninger MH, Jandrot-Perrus M, Elion O, Bertrand O, Homandberg GA, Mosesson MW, Guillin M-C. ADP-induced platelet aggregation depends on the conformational availability of the N-terminal gamma chain sequence of fibrinogen. Study of the reactivity of fibrinogen Paris I. Blood 1987; 70: 558-63.
• 42 Lee J, Rieu P, Arnaout M, Liddington R. Crystal structure of the A domain from the a subunit of integrin CR3 (CD11b/CD18). Cell 1995; 80: 631-8.
• 43 Qu A, Leahy DJ. Crystal structure of the I-domain from the CD11a/CD18 (LFA-1, αLβ2) integrin. Proc Natl Acad Sci USA 1995; 92: 10277-81.
• 44 Tuckwell DS, Humphries MJ. A structure prediction for the ligand-binding region of the integrin b subunit: evidence for the presence of a von Wille-brand factor A domain. FEBS Letters 1997; 400: 297-303.
• 45 Hu DD, White CA, Panzer-Knodle S, Page JD, Nicholson N, Smith JW. A new model of dual interacting ligand binding sites on integrin αIIbβ3. J Biol Chem 1999; 274: 4633-9.
• 46 Cierniewski CS, Byzova T, Papierak M, Haas T, Niewiarowska Zhang L, Cieslak M, Plow EF. Peptide ligands can bind to distinct sites in integrin αIIbβ3 and elicit different functional responses. J Biol Chem 1999; 274: 16923-32.