Integrin Sequestering in Raft-Mimicking Lipid Mixtures: The Role of Bilayer Asymmetry and Cholesterol Content

Abstract

It is widely accepted that lipid microdomains play an important functional role in plasma membranes. However, small size and transient nature of lipid/membrane heterogeneities in the plasma membrane make a characterization of microdomains and microdomain-related membrane processes in cellular systems quite challenging. To address this important problem, we recently introduced a powerful model membrane system that allows the investigation of membrane protein sequestering and oligomerization in raft-mimicking lipid mixtures using combined confocal fluorescence spectroscopy, photon counting histogram (PCH), and epifluorescence microscopy (1). Our experiments on bilayer-spanning domains showed that αvβ3 and α5β1 integrins predominantly exist as monomers and sequester preferentially to the liquid-disordered (ld) phase in the absence of ligands. Notably, addition of vitronectin (αvβ3) and fibronectin (α5β1) caused substantial translocations of integrins into the liquid-ordered (lo) phase without altering receptor oligomerization state. Here we expand our previous studies and report on the sequestering and oligomerization state of αvβ3 and α5β1 in asymmetric bilayer compositions containing coexisting lo and ld phases located exclusively in the top leaflet of the bilayer (bottom leaflet shows only ld phase). Remarkably, in such a membrane environment, both integrins show a higher affinity for the top leaflet-restricted lo domains in the absence of their respective ligands. This sequestering behavior of integrins was only slightly modified after addition of their respective native ligands. Our findings show that cholesterol content has a substantial influence on integrin sequestering and oligomerization in raft-mimicking lipid mixtures. The described experimental results highlight the potential importance of membrane asymmetry and lipid composition in the sequestering of membrane proteins in biological membranes.(1) Siegel, A. P. et al. (2011) Biophys J 101(7): 1642-1650.