Ann Kimble Hill - Native Ligands Change Integrin Sequestering but Not Oligomerization in Raft-Mimicking Lipid Mixtures

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      Publication Details (including relevant citation   information): Siegel, Amanda P., Kimble-Hill, Ann,   Garg, Sumit, Jordan, Rainer, Naumann, Christoph A.,   Biophysical Journal, 2011, 101  (7), pp 1642-1650

      Abstract: Distinct lipid environments, including   lipid rafts, are increasingly recognized as a crucial factor   affecting membrane protein function in plasma membranes.   Unfortunately, an understanding of their role in membrane protein   activation and oligomerization has remained elusive due to the   challenge of characterizing these often small and transient   plasma membrane heterogeneities in live cells. To address this   difficulty, we present an experimental model membrane platform   based on polymer-supported lipid bilayers containing stable   raft-mimicking domains (type I) and homogeneous cholesterol-lipid   mixtures (type II) into which transmembrane proteins are   incorporated (αvβ3 and α5β1 integrins). These flexible lipid   platforms enable the use of confocal fluorescence spectroscopy,   including the photon counting histogram method, in tandem with   epifluorescence microscopy to quantitatively probe the effect of   the binding of native ligands from the extracellular matrix   ligands (vitronectin and fibronectin for αvβ3 and α5β1,   respectively) on domain-specific protein sequestration and on   protein oligomerization state. We found that both αvβ3 and α5β1   sequester preferentially to nonraft domains in the absence of   extracellular matrix ligands, but upon ligand addition, αvβ3   sequesters strongly into raft-like domains and α5β1 loses   preference for either raft-like or nonraft-like domains.   A corresponding photon counting histogram analysis showed   that integrins exist predominantly in a monomeric state. No   change was detected in oligomerization state upon ligand binding   in either type I or type II bilayers, but a moderate increase in   oligomerization state was observed for increasing concentrations   of cholesterol. The combined findings suggest a mechanism in   which changes in integrin sequestering are caused by   ligand-induced changes in integrin conformation and/or dynamics   that affect integrin-lipid interactions without altering the   integrin oligomerization state.

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