Membrane cholesterol regulates endocytosis and trafficking of the serotonin receptor: Insights from acute cholesterol depletion.

Endocytosis and intracellular trafficking constitute important regulatory features associated with G protein-coupled receptor (GPCR) function. GPCR endocytosis involves several remodeling events at the plasma membrane orchestrated by a concerted interplay of a large number of proteins and membrane lipids. Although considerable literature exists on the protein framework underlying GPCR endocytosis, the role of membrane lipids in this process remains largely unexplored. In order to explore the role of membrane cholesterol (an essential and important lipid in higher eukaryotes) in GPCR endocytosis, we monitored the effect of acute cholesterol depletion using methyl-β-cyclodextrin (MβCD) on endocytosis and intracellular trafficking of the serotonin receptor, an important neurotransmitter GPCR. Our results show that the serotonin receptor exhibits agonist-induced clathrin-mediated endocytosis with a concentration-dependent inhibition in internalization with increasing concentrations of MβCD, which was restored upon cholesterol replenishment. Interestingly, subsequent to internalization under these conditions, serotonin receptors were re-routed toward lysosomal degradation, instead of endosomal recycling observed under normal conditions, thereby implicating membrane cholesterol in modulation of intracellular trafficking of the receptor. This raises the possibility of a novel cholesterol-dependent role of intracellular sorting proteins in GPCR trafficking. These results differ from our previous observations on the endocytosis of the serotonin receptor upon statin-induced chronic cholesterol depletion, in terms of endocytic pathway. We conclude that analysis of complex cellular trafficking events such as GPCR endocytosis under acute and chronic cholesterol depletion conditions should be carried out with caution due to fundamental differences underlying these processes.