title: Reconstructing integrin activation in vitro.
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.
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla, CA, USA.
authors.forename: Alexandre R
authors.initials: AR
authors.lastname: Gingras
authors.forename: Feng
authors.initials: F
authors.lastname: Ye
authors.forename: Mark H
authors.initials: MH
authors.lastname: Ginsberg
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism; D016466:CHO Cells; D002448:Cell Adhesion / Q000235:genetics* / Q000276:immunology; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D066298:In Vitro Techniques; D008967:Molecular Biology / Q000379:methods*; D010743:Phospholipids / Q000378:metabolism; D015539:Platelet Activation / Q000235:genetics / Q000276:immunology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D015398:Signal Transduction / Q000276:immunology
chemical_list: D010743:Phospholipids; D019039:Platelet Glycoprotein GPIIb-IIIa Complex