Abstract
Integrin activation leads to an increased affinity for the extracellular matrix and regulates many cellular processes such as cell adhesion and migration. To capture the process of integrin inside-out activation in a purified system, we describe here methods to isolate platelet αIIbβ3 integrins in the inactive state and to incorporate them into phospholipid nanodiscs each bearing a single lipid-embedded αIIbβ3 integrin. We delineate a simple enzyme-linked immunosorbent assay that can be used in conjunction with binding of an activation specific monoclonal antibody, PAC1, to monitor the affinity of the integrin before and after the addition of activators such as the Talin Head Domain (THD). The system has been used to show that binding of the THD to both the integrin and the phospholipid bilayer is necessary and sufficient to activate and promote molecular extension of unclustered integrins in the absence of force. This method can be used to test various integrin-binding proteins, membrane phospholipid compositions and different types of integrins. It can also serve as the nidus for synthetic assembly of adhesion plaque like complexes from purified proteins.
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References
Calderwood DA (2004) Integrin activation. J Cell Sci 117:657–666
Shattil SJ, Hoxie JA, Cunningham M et al (1985) Changes in the platelet membrane glycoprotein IIb.IIIa complex during platelet activation. J Biol Chem 260:11107–11114
Kim C, Ye F, Ginsberg MH (2011) Regulation of integrin activation. Annu Rev Cell Dev Biol 27:321–345
Anthis NJ, Wegener KL, Ye F et al (2009) The structure of an integrin/talin complex reveals the basis of inside-out signal transduction. EMBO J 28:3623–3632
Travis MA, Humphries JD, Humphries MJ (2003) An unraveling tale of how integrins are activated from within. Trends Pharmacol Sci 24:192–197
Fitzgerald LA, Leung B, Phillips DR (1985) A method for purifying the platelet membrane glycoprotein IIb–IIIa complex. Anal Biochem 151:169–177
Kouns WC, Hadvary P, Haering P et al (1992) Conformational modulation of purified glycoprotein (GP) IIb–IIIa allows proteolytic generation of active fragments from either active or inactive GPIIb–IIIa. J Biol Chem 267:18844–18851
Frelinger AL 3rd, Du XP, Plow EF et al (1991) Monoclonal antibodies to ligand-occupied conformers of integrin alpha IIb beta 3 (glycoprotein IIb–IIIa) alter receptor affinity, specificity, and function. J Biol Chem 266: 17106–17111
Denisov IG, Grinkova YV, Lazarides AA et al (2004) Directed self-assembly of monodisperse phospholipid bilayer nanodiscs with controlled size. J Am Chem Soc 126:3477–3487
Katayama H, Wang J, Tama F et al (2010) Three-dimensional structure of the anthrax toxin pore inserted into lipid nanodiscs and lipid vesicles. Proc Natl Acad Sci U S A 107: 3453–3457
Denisov IG, Sligar SG (2011) Cytochromes P450 in nanodiscs. Biochim Biophys Acta 1814:223–229
Raschle T, Hiller S, Yu TY et al (2009) Structural and functional characterization of the integral membrane protein VDAC-1 in lipid bilayer nanodiscs. J Am Chem Soc 131: 17777–17779
Ye F, Hu G, Taylor D et al (2010) Recreation of the terminal events in physiological integrin activation. J Cell Biol 188:157–173
Moser M, Legate KR, Zent R et al (2009) The tail of integrins, talin, and kindlins. Science 324:895–899
Ye F, Kim C, Ginsberg MH (2011) Molecular mechanism of inside-out integrin regulation. J Thromb Haemost 9(Suppl 1):20–25
Bouaouina M, Harburger DS, Calderwood DA (2012) Talin and signaling through integrins. Methods Mol Biol 757:325–347
Elliott PR, Goult BT, Kopp PM et al (2010) The Structure of the talin head reveals a novel extended conformation of the FERM domain. Structure 18:1289–1299
Garcia-Alvarez B, de Pereda JM, Calderwood DA et al (2003) Structural determinants of integrin recognition by talin. Mol Cell 11: 49–58
Anthis NJ, Wegener KL, Critchley DR et al (2010) Structural diversity in integrin/talin interactions. Structure 18:1654–1666
Acknowledgments
This work was supported by the American Heart Association 12SDG11610043 (A.R. Gingras) and NIH (M.H. Ginsberg).
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Gingras, A.R., Ye, F., Ginsberg, M.H. (2013). Reconstructing Integrin Activation In Vitro. In: Coutts, A. (eds) Adhesion Protein Protocols. Methods in Molecular Biology, vol 1046. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-538-5_1
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DOI: https://doi.org/10.1007/978-1-62703-538-5_1
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