Abstract 21: Sortilin Is a Novel Mediator of Cellular Low-Density Lipoprotein Uptake

Abstract

Using genome-wide association studies and follow-up functional studies, we recently identified the SORT1 gene as a novel regulator of blood low-density-lipoprotein cholesterol (LDL-C) levels. SORT1 encodes sortilin, also known as neurotensin receptor 3, a protein that functions as a multiligand sorting receptor. Sortilin localizes to various intracellular compartments including the Golgi apparatus and the plasma membrane, and it has roles in both endocytosis and intracellular trafficking of other proteins. To test whether sortilin regulates blood LDL-C levels by influencing cellular uptake of LDL particles, we used a lentiviral delivery system to stably overexpress the wild-type protein or either of two trafficking mutants_the first, designated LAYA, traffics to the plasma membrane but cannot undergo endocytosis, and the second, designated STOP, remains trapped in the Golgi apparatus. When overexpressed in cultured human hepatocellular carcinoma cells, we found that wild-type sortilin dramatically increases LDL uptake into the endolysosomal compartment for degradation, and the LAYA mutant robustly traps LDL particles in the plasma membrane, whereas the STOP mutant does not affect LDL uptake. We observed similar effects in skin fibroblasts taken from a patient with familial hypercholesterolemia_and thus are deficient in LDL receptor function_suggesting that sortilin can facilitate LDL uptake independently of the LDL receptor. As a complementary approach, we have generated human SORT1 knockout cells, using the novel TAL effector nuclease (TALEN) technology, to introduce frameshift mutations into the coding sequence and thereby disrupt the SORT1 alleles. Besides providing a proof-of-principle that we can for the first time rapidly and efficiently generate gene knockouts in human cells, the SORT1 knockout cells are allowing us to assess the effects of sortilin deficiency on LDL uptake.