Abstract
Integrin activation is a crucial event for multiple biological functions. Therefore, methods to detect integrin activation are vital. Since the main cellular function of integrins is adhesion, we and others utilize this feature to measure integrin activation. Here, we describe how to detect the activity of the fibronectin-binding integrin α5β1 using a fusion of glutathione S-transferase (GST) to the 9th, 10th, and 11th type III repeats on fibronectin (GST-FNIII9-11). Moreover, we detail how to measure αvβ3 integrin activity using the ligand-mimetic WOW-1 antibody that selectively binds unoccupied αvβ3 integrins. Finally, we describe methods of testing ligation of fibronectin-binding integrins utilizing monoclonal antibodies against ligand-induced binding sites (LIBS).
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References
Shattil SJ, Kim C, Ginsberg MH (2010) The final steps of integrin activation: the end game. Nat Rev Mol Cell Biol 11(4):288–300. https://doi.org/10.1038/nrm2871
Tzima E, del Pozo MA, Shattil SJ, Chien S, Schwartz MA (2001) Activation of integrins in endothelial cells by fluid shear stress mediates Rho-dependent cytoskeletal alignment. EMBO J 20(17):4639–4647
Hynes RO (2002) Integrins: bidirectional, allosteric signaling machines. Cell 110(6):673–687
Pampori N, Hato T, Stupack DG, Aidoudi S, Cheresh DA, Nemerow GR, Shattil SJ (1999) Mechanisms and consequences of affinity modulation of integrin alpha(V)beta(3) detected with a novel patch-engineered monovalent ligand. J Biol Chem 274(31):21609–21616
Wickham TJ, Mathias P, Cheresh DA, Nemerow GR (1993) Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment. Cell 73(2):309–319. https://doi.org/10.1016/0092-8674(93)90231-e
Frelinger AL 3rd, Lam SC, Plow EF, Smith MA, Loftus JC, Ginsberg MH (1988) Occupancy of an adhesive glycoprotein receptor modulates expression of an antigenic site involved in cell adhesion. J Biol Chem 263(25):12397–12402
Falke JJ, Koshland DE Jr (1987) Global flexibility in a sensory receptor: a site-directed cross-linking approach. Science (New York, NY) 237(4822):1596–1600. https://doi.org/10.1126/science.2820061
Parise LV, Helgerson SL, Steiner B, Nannizzi L, Phillips DR (1987) Synthetic peptides derived from fibrinogen and fibronectin change the conformation of purified platelet glycoprotein IIb-IIIa. J Biol Chem 262(26):12597–12602
Clark K, Pankov R, Travis MA, Askari JA, Mould AP, Craig SE, Newham P, Yamada KM, Humphries MJ (2005) A specific alpha5beta1-integrin conformation promotes directional integrin translocation and fibronectin matrix formation. J Cell Sci 118(Pt 2):291–300. https://doi.org/10.1242/jcs.01623. jcs.01623 [pii]
Mould AP, Garratt AN, Askari JA, Akiyama SK, Humphries MJ (1995) Identification of a novel anti-integrin monoclonal antibody that recognises a ligand-induced binding site epitope on the beta 1 subunit. FEBS Lett 363(1–2):118–122. https://doi.org/10.1016/0014-5793(95)00301-o
Frelinger AL 3rd, Du XP, Plow EF, Ginsberg MH (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(26):17106–17111
Acknowledgments
This work was supported by National Heart, Lung, and Blood Institute R01 HL098435, HL133497, HL141155, and GM121307 (to A.W.O.), by an American Heart Association Predoctoral Fellowship (19PRE34380751) and Malcolm Feist Cardiovascular Research Endowment Predoctoral Fellowship (to Z.A.Y.).
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Al-Yafeai, Z., Orr, A.W. (2021). Quantification of Integrin Activation and Ligation in Adherent Cells. In: Vicente-Manzanares, M. (eds) The Integrin Interactome. Methods in Molecular Biology, vol 2217. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0962-0_2
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DOI: https://doi.org/10.1007/978-1-0716-0962-0_2
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