The Regulation of Conformation and Binding Kinetics of Integrin alphaLbeta2
The interaction mediated by integrin alphaLbeta2 and its ligand plays major role in many immune responses by regulating leukocyte adhesion. This study investigated the conformational regulation of alphaLbeta2 and the effects of conformational change on the ligand binding of alphaLbeta2. Micropipette adhesion frequency assay was used to measure the two-dimensional binding affinity and kinetics of alphaLbeta2 on K562 cells and neutrophils. The conformations of alphaLbeta2 were regulated by mutations, antibodies, small molecule antagonists, as well as divalent cations. Our results indicated that the change in binding affinity and off-rate was mostly due to the alphaL I domain conformational change. Without affecting the I domain conformation, the extension of alphaLbeta2 only increases the on-rate for several fold by providing a better orientation and accessibility of the molecule on cell surface. The binding characteristics of divalent cations to I domain MIDAS and other metal ion binding sites in alphaLbeta2 are determined by the nature of divalent cations, Mn2+ has higher binding affinity to the metal ion binding sites than Mg2+. The conformation of I domain also affected the binding of divalent cations. Open and intermediate I domains have higher binding affinity for Mn2+ and Mg2+ than WT and closed I domains. Divalent cations dissociate from I domain MIDAS very slowly but from those metal ion binding sites that important for conformational change of alphaLbeta2 rapidly. One of the most important biological processes mediated by alphaLbeta2 and other beta2 integrins is the recruitment and migration of neutrophils during inflammation. The activation of beta2 integrins by E-selectin binding to neutrophils in this process was also investigated. The binding of E-selectin, but not P- or L-selectin, activates beta2 integrins in a timescale of ~ 5 seconds and the activation may require the crosslink of E-selectin ligands. These results provide insights into the relationship between the conformational change and the function of alphaLbeta2 and most importantly would contribute to the understanding of integrin regulation mechanisms.
Advisor:McIntire, Larry; Springer, Timothy; Selvaraj, Periasamy; Garcia, Andres; Zhu, Cheng; Babensee , Julia
School:Georgia Institute of Technology
School Location:USA - Georgia
Source Type:Master's Thesis
Date of Publication:07/09/2007