Alpha5beta1 is a member of the integrin family of cell adhesion receptors which serve both the mechanical function of adhering cells and a signaling function in the control of many cell processes. It is likely that the mechanical and signaling aspects of alpha5beta1 function are linked since some alpha5beta1 signaling functions cannot be replaced by ligand binding alone. To examine the mechanical aspects of alpha5beta1 function and regulation of alpha5beta1 mediated cell adhesion, a spinning disc device has been engineered to provide a measurement of the force required to detach cells from a substrate. By applying concepts from chemical binding analysis it has been possible to convert the adhesion data to a model of monovalent receptor-ligand binding and demonstrate that there is a linear relationship between the number of integrin-extracellular matrix bonds and the force required to detach the cell. The proposal considers models to explain activation of alpha5beta1 binding to fibronectin: agonist activation, induced-fit activation, clustering activation, and tension activation. Each of these models makes different predictions for ligand, cytoskeletal, and energy requirements. These requirements would be tested using biochemical predictions. Inhibitors, and genetic analysis of fibronectin mutants, beta1 cytoplasmic domain splice variants, and beta1 cytoplasmic domain mutants. The analysis views the adhesion connection as one from fibronectin to alpha5beta1 to actin cytoskeleton and these connections, either ligand binding or cytoskeletal binding, can control the adhesion strength. Procedures have been developed to assay these connections separately. Their approach would be extended to the examination of long term (or steady-state) adhesion, including analysis of the increase in the number of alpha5beta1 receptors bound, and the role of clustering n the adhesion strengthening-cell spreading process.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Pathobiochemistry Study Section (PBC)
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Flicker, Paula F
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University of Pennsylvania
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Engler, Adam J; Chan, May; Boettiger, David et al. (2009) A novel mode of cell detachment from fibrillar fibronectin matrix under shear. J Cell Sci 122:1647-53
Friedland, Julie C; Lee, Mark H; Boettiger, David (2009) Mechanically activated integrin switch controls alpha5beta1 function. Science 323:642-4
Lee, Mark H; Boettiger, David; Composto, Russell J (2008) Biomimetic carbohydrate substrates of tunable properties using immobilized dextran hydrogels. Biomacromolecules 9:2315-21
Friedland, Julie C; Lakins, Johnathon N; Kazanietz, Marcelo G et al. (2007) alpha6beta4 integrin activates Rac-dependent p21-activated kinase 1 to drive NF-kappaB-dependent resistance to apoptosis in 3D mammary acini. J Cell Sci 120:3700-12
Boettiger, David (2007) Quantitative measurements of integrin-mediated adhesion to extracellular matrix. Methods Enzymol 426:1-25
Lee, Mark H; Ducheyne, Paul; Lynch, Laura et al. (2006) Effect of biomaterial surface properties on fibronectin-alpha5beta1 integrin interaction and cellular attachment. Biomaterials 27:1907-16
Lynch, Laura; Vodyanik, Pavel I; Boettiger, David et al. (2005) Insulin-like growth factor I controls adhesion strength mediated by alpha5beta1 integrins in motile carcinoma cells. Mol Biol Cell 16:51-63
Paszek, Matthew J; Zahir, Nastaran; Johnson, Kandice R et al. (2005) Tensional homeostasis and the malignant phenotype. Cancer Cell 8:241-54
Datta, Anirban; Huber, Francois; Boettiger, David (2002) Phosphorylation of beta3 integrin controls ligand binding strength. J Biol Chem 277:3943-9
Garcia, Andres J; Schwarzbauer, Jean E; Boettiger, David (2002) Distinct activation states of alpha5beta1 integrin show differential binding to RGD and synergy domains of fibronectin. Biochemistry 41:9063-9

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