Unraveling the mechanism of cholesterol-mediated regulation of receptor dimerization in plasma membranes in vivo

Abstract

Membrane cholesterol can alter the signaling pathways of living cells. However, the process that modulates the interaction of receptor proteins is still unclear. We performed single-molecule optical tracking of ligand-induced dimerization of epidermal growth factor receptors (EGFRs) in two cancerous cell lines (HeLa and A431) and one normal endothelial cell line (MCF12A). We discovered that unliganded EGFRs typically reside in non-raft regions of the plasma membrane and can move into raft domains upon ligand binding. This ligand-induced motion could be a common behavior in live cells. We found that the amount of membrane cholesterol significantly affects the stability of EGFR dimers by manipulating the total amount of membrane cholesterol with methyl-β-cyclodextrin and the local concentration of cholesterol with nystatin. The EGFR dimers in the plasma membrane of normal cells are more sensitive to changes in the local concentration of cholesterol compared with the cancer cells. Our methodology can yield useful information for understanding cholesterol-mediated protein-protein interactions in live cells.