Abstract
The low density lipoprotein (LDL) receptor plays a major role in maintaining human plasma cholesterol levels and mutations in the gene cause familial hypercholesterolemia. The LDL receptor (LDLR) pathway has been well characterized, but little is known of proteins involved in its complex intracellular sorting and trafficking. Sorting nexin 17 (SNX17) has recently been implicated in LDLR intracellular trafficking. We show here that endogenous SNX17 is highly expressed in several cell types and is localized partially in early endosomes. We found that the PX domain of SNX17 is required for its endosomal localization but does not interact directly with the LDL receptor. A novel domain containing a FERM-like domain of SNX17 is needed for its interaction with the LDL receptor. Mutations in the NPXY motif of the LDL-receptor cytoplasmic tail that disrupt internalization also disrupt its interaction with SNX17, whereas mutations elsewhere had little effect. When transiently overexpressed in Chinese hamster ovary cells, SNX17 localized to large vesicular structures and disrupted normal trafficking of the LDL receptor in a PX domain-dependent manner. These results suggest that SNX17 plays a role in the cellular trafficking of the LDL receptor through interaction with the NPVY motif in its cytoplasmic domain and interaction of the PX domain with subcellular membrane compartments.
Highlights
The low density lipoprotein (LDL)1 receptor mediates cellular uptake and degradation of extracellular LDL by high affinity binding of LDL to receptors on the cell surface followed by internalization of the receptor-ligand complex in clathrincoated vesicles
Screen of a Liver Yeast Two-hybrid Library for Proteins Interacting with the Cytoplasmic Tail of the LDL Receptor—When ϳ1.8 ϫ 106 clones of a human liver cDNA library were screened with amino acid residues 789 – 839 of the LDL-receptor cytoplasmic tail, 31 clones reproducibly showed strong to moderate interaction
The NPVY Motif in the LDL Receptor Is Required for Its Interaction with sorting nexin-17 (SNX17)—Because it has been suggested that SNX17 is involved in LDL receptor trafficking, we investigated the effect of mutations in the LDL receptor that have been shown to influence different aspects of its trafficking, for example internalization [10, 16] or basolateral sorting [4]
Summary
The low density lipoprotein (LDL) receptor mediates cellular uptake and degradation of extracellular LDL by high affinity binding of LDL to receptors on the cell surface followed by internalization of the receptor-ligand complex in clathrincoated vesicles. A basolateral sorting signal comprising a proximal motif that overlaps with the NPXY internalization motif and a second distal motif has been shown to be important both for sorting of newly synthesized receptor to the basolateral plasma membrane and for sorting of the receptor after internalization [4] Mutations in this region of the LDL receptor that interfere with its basolateral sorting have been identified in patients with familial hypercholesterolemia [5], but the one or more proteins that contribute to the sorting machinery have yet to be identified conclusively. SNX17-LDL Receptor Interaction homologue of SNX17 as a protein that bound to the cytoplasmic tail of murine LR-8, another member of the LDL-receptor gene family These authors reported that stable expression of SNX17 in mouse embryonic fibroblasts increased the internalization index of LDL receptor-mediated uptake of LDL and concluded that SNX17 must be involved in trafficking of the LDL receptor. To investigate how SNX17 functions in intracellular trafficking, we have mapped the domains or residues in the two proteins that are important for their interaction and have investigated which domains of SNX17 determine its subcellular localization
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