The LDL receptor is regulated by membrane cholesterol as revealed by fluorescence fluctuation analysis

Membrane cholesterol-rich domains have been shown to be important for regulating a range of membrane protein activities. Low-density lipoprotein receptor (LDLR)-mediated internalization of cholesterol-rich LDL particles is tightly regulated by feedback mechanisms involving intracellular sterol sensors. Since LDLR plays a role in maintaining cellular cholesterol homeostasis, we explore the role that membrane domains may have in regulating LDLR activity. We expressed a fluorescent LDLR-mEGFP construct in HEK293T cells and imaged the unligated receptor or bound to an LDL/DiI fluorescent ligand using total internal reflection fluorescence microscopy. We studied the receptor's spatiotemporal dynamics using fluorescence fluctuation analysis methods. Image cross correlation spectroscopy reveals a lower LDL-to-LDLR binding fraction when membrane cholesterol concentrations are augmented using cholesterol esterase, and a higher binding fraction when the cells are treated with methyl-β-cyclodextrin) to lower membrane cholesterol. This suggests that LDLR's ability to metabolize LDL particles is negatively correlated to membrane cholesterol concentrations. We then tested if a change in activity is accompanied by a change in membrane localization. Image mean-square displacement analysis reveals that unligated LDLR-mEGFP and ligated LDLR-mEGFP/LDL-DiI constructs are transiently confined on the cell membrane, and the size of their confinement domains increases with augmented cholesterol concentrations. Receptor diffusion within the domains and their domain-escape probabilities decrease upon treatment with methyl-β-cyclodextrin, consistent with a change in receptor populations to more confined domains, likely clathrin-coated pits. We propose a feedback model to account for regulation of LDLR within the cell membrane: when membrane cholesterol concentrations are high, LDLR is sequestered in cholesterol-rich domains. These LDLR populations are attenuated in their efficacy to bind and internalize LDL. However, when membrane cholesterol levels drop, LDL has a higher binding affinity to its receptor and the LDLR transits to nascent clathrin-coated domains, where it diffuses at a slower rate while awaiting internalization.