Bilayer Asymmetry, Cholesterol Content, and Ligand Binding Influence Membrane Protein Sequestering in Raft-Mimicking Lipid Mixtures

Abstract

Lipid heterogeneities and membrane proteins are widely considered to show an important functional relationship in plasma membranes. However, the analysis of this intriguing relationship is challenging, due to the small size and transient nature of lipid heterogeneities in the plasma membrane environment. Recently, we presented a powerful model membrane platform that allows the thorough analysis of membrane protein sequestering and oligomerization in well-defined heterogeneous lipid environments using confocal fluorescence intensity analysis paired with a photon counting histogram (PCH) method (1). By applying this experimental approach, here we show that bilayer asymmetry has a significant influence on the sequestering of αvβ3 and α5β1 integrins. Our experiments demonstrate a higher affinity of integrins for the liquid-disordered (ld) phase in the presence of bilayer-spanning liquid-ordered (lo)-ld phase separations. In contrast, a preference for the lo phase is observed in an asymmetric bilayer with lo-ld phase separations, which are exclusively located in the top leaflet of the bilayer. Importantly, PCH analysis shows that the observed changes in integrin sequestering are not caused by altered receptor oligomerization states. In another set of experiments, we also demonstrate that changes in cholesterol may have a profound impact on integrin sequestering without altering receptor oligomerization state. The obtained results are discussed in terms of potential changes in lipid packing density and hydrophobic thickness within the model membrane. The experimental model membrane approach is also applied to explore the functionally important relationship between the sequestering, level of dimerization, and ligand binding of the GPI-anchored urokinase receptor.(1) Siegel, A. P. et al. (2011) Biophys J101(7): 1642-1650.