Quantitative fluorescence imaging reveals point of release for lipoproteins during LDLR-dependent uptake

Abstract

This article is available online at http://www.jlr.org by internalization of LDLR-lipoprotein complexes through clathrin-coated pits into endocytic vesicles. Nascent endocytic vesicles recruit the early endosome antigen 1 (EEA1) protein ( 1, 2 ), which is part of the molecular machinery that drives fusion of endocytic vesicles with each other and with early endosomes. Endocytic vesicles and endosomes acidify via the V-pump and lose calcium via the TRPV2 calcium channel ( 3-6 ). Both acidic pH and loss of calcium accelerate lipoprotein dissociation from the LDLR ( 7,8 ). After release, the LDLR recycles back to the surface for additional rounds of uptake, while released lipoproteins progress through the endosomal system, eventually reaching lysosomes, where hydrolysis occurs.The molecular details of how acidic pH and loss of calcium drive release are generally well understood; however, the importance of each process for release during endocytosis is not clear. The LDLR consists of seven LDLR type A (LA) repeats; two EGF-like repeats (EGF-A and EGF-B); six YWTD repeats that form a ␤ -propeller; a third EGF-like repeat (EGF-C); a single transmembrane domain; and a short cytoplasmic tail. LDL binds to the LDLR through interaction of apolipoprotein B100 (apoB100) of LDL with LA3-7 and the EGF-A modules of the LDLR ( 9, 10 ). VLDL remnants bind to the LDLR through interaction of apolipoprotein E (apoE) of remnants with LA4 and LA5 of the LDLR ( 10, 11 ). LA repeats are calcium-binding modules, and loss of calcium results in a conformational change that accelerates release ( 8,(12)(13)(14). LA4, which is required for both apoB100 and apoE binding, has particularly weak calcium-binding affi nity ( 15 ), suggesting that loss of calcium from LA4 initiates lipoprotein dissociation by the calcium release process. Acidic pH promotes loss of calcium by protonating the glutamates and aspartates that coordinate calcium in LA repeats. Acidic pH also accelerates lipoprotein release through a calcium-independent Abstract The LDL receptor (LDLR) supports effi cient uptake of both LDL and VLDL remnants by binding lipoprotein at the cell surface, internalizing lipoprotein through coated pits, and releasing lipoprotein in endocytic compartments before returning to the surface for further rounds of uptake. While many aspects of lipoprotein binding and receptor entry are well understood, it is less clear where, when, and how the LDLR releases lipoprotein. To address these questions, the current study employed quantitative fl uorescence imaging to visualize the uptake and endosomal processing of LDL and the VLDL remnant ␤ -VLDL. We fi nd that lipoprotein release is rapid, with most release occurring prior to entry of lipoprotein into early endosomes. Published biochemical studies have identifi ed two mechanisms of lipoprotein release: one that involves the ␤ -propeller module of the LDLR and a second that is independent of this module. Quantitative imaging comparing uptake supported by the normal LDLR or by an LDLR variant incapable of ␤...

Identification of roles for H264, H306, H439, and H635 in acid-dependent lipoprotein release by the LDL receptor

“…Retro-endocytosis occurs when LDLRs commit to the recycling pathway prior to releasing lipoprotein, thereby allowing internalized lipoprotein to return to the cell surface. In LDLR-BC cells, whose LDLR cannot utilize the acid-dependent release mechanism, 70% of the internalized LDL is lost to retroendocytosis (30). By labeling LDL or -VLDL with a pHinsensitive dye, the net accumulation (internalization minus retro-endocytosis) of these lipoproteins can easily be measured by flow cytometry.…”

“…Prior work has shown that endosomal lipoprotein release normally occurs rapidly in fibroblasts (30). In these cells, the calcium release mechanism is sufficient for -VLDL release, though the acid-dependent mechanism accelerates the rate of release (30).…”

“…In these cells, the calcium release mechanism is sufficient for -VLDL release, though the acid-dependent mechanism accelerates the rate of release (30). The calcium release mechanism also drives LDL release in fibroblasts; however, the acceleration imparted by the acid-dependent mechanism is necessary for efficient delivery of LDL to lysosomes because inhibition of acid-dependent release increases the fraction of internalized LDL that is lost through the process of retro-endocytosis (30,55,56).…”

“…These observations suggest that binding of the -propeller induces an allosteric change in LA5 that accelerates lipoprotein dissociation. Both the calcium release and acid-dependent release processes contribute to lipoprotein dissociation in endosomes (21,30).…”

“…These observations suggest that binding of the -propeller induces an allosteric change in LA5 that accelerates lipoprotein dissociation. Both the calcium release and acid-dependent release processes contribute to lipoprotein dissociation in endosomes (21,30).Crystal structures of the LDLR ectodomain have been solved at both neutral and acidic pH (28, 31). These structures have shown that the EGF-B module pivots about its linker to the -propeller, allowing the LA repeats to either wrap around the -propeller at acidic pH or extend away from the -propeller at neutral pH (Fig.…”