The Low Density Lipoprotein Receptor-related Protein (LRP) Is a Novel β-Secretase (BACE1) Substrate

Abstract

BACE is a transmembrane protease with ␤-secretase activity that cleaves the amyloid precursor protein (APP). After BACE cleavage, APP becomes a substrate for ␥-secretase, leading to release of amyloid-␤ peptide (A␤), which accumulates in senile plaques in Alzheimer disease. APP and BACE are co-internalized from the cell surface to early endosomes. APP is also known to interact at the cell surface and be internalized by the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic and signaling receptor. Using a new fluorescence resonance energy transfer (FRET)-based assay of protein proximity, fluorescence lifetime imaging (FLIM), and co-immunoprecipitation we demonstrate that the light chain of LRP interacts with BACE on the cell surface in association with lipid rafts. Surprisingly, the BACE-LRP interaction leads to an increase in LRP C-terminal fragment, release of secreted LRP in the media and subsequent release of the LRP intracellular domain from the membrane. Taken together, these data suggest that there is a close interaction between BACE and LRP on the cell surface, and that LRP is a novel BACE substrate. BACE1 (␤ site of APP-cleaving enzyme) is a type I membrane-associated aspartyl protease that cleaves APP (1-4). Besides APP, the few BACE substrates that have been identified include the APP homologues APLP1 and -2 (5), P-selectin glycoprotein ligand-1 (PSGL-1), and a membrane-bound sialyltransferase (6). Post-translational processing of BACE involves N-glycosylation, removal of its prodomain by a furin-like protease, and further complex glycosylation (7-9). After glycosylation, BACE co-traffics with APP and is rapidly transported to the Golgi apparatus and distal secretory pathway (9). Measurable amounts of APP and BACE are present on the plasma membrane (10 -12) and in lipid rafts (12)(13)(14). BACE and APP are internalized from the cell surface to early endosomes and cycle between the cell membrane and endosomes (10,11,15).The low density lipoprotein receptor-related protein, LRP, is a type I integral membrane protein with a 515-kDa extracellular ␣-chain non-convalently bound to the 85 kDa membranespanning ␤-chain. It is also found on the cell surface and cycles between the cell membrane and endosomes. Multiple intracellular adaptor and scaffolding proteins bind the LRP 100 amino acid cytoplasmic tail (16, 17); its four extracellular binding domains mediate endocytosis of a wide array of ligands, including several of potential importance for Alzheimer disease pathophysiology: APP, apolipoprotein E and ␣ 2 -macroglobulin (16 -18). The LRP ligand binding domains interact with KPIcontaining forms of APP. In addition, an interaction between the C-terminal domain of APP and LRP, mediated by the cytoplasmic adaptor protein Fe65, impacts APP internalization (18 -23). In addition to its role in endocytosis, LRP has an interesting pattern of proteolysis that parallels APP in some ways. Ectodomain shedding of LRP has been described (24) and proteolysis of LRP by matrix metalloproteas...

Differential Regulation of Extracellular Tissue Inhibitor of Metalloproteinases-3 Levels by Cell Membrane-bound and Shed Low Density Lipoprotein Receptor-related Protein 1

“…The rate of TIMP-3 endocytosis was not constant over 24 h. After addition of [ 35 S]TIMP-3 to cells, endocytosis was initially rapid, but then slowed, so that only minimal endocytosis was observed after 10 h. We demonstrated that this reduction in endocytosis is due to interaction of TIMP-3 with a soluble form of LRP-1 shed from the cell surface into the medium. Different LRP-1 "sheddases" have been described, including ADAM10, ADAM12, ADAM17, MT1-MMP, and ␤-secretase (23,(35)(36)(37)(38). Although the specific sites at which these enzymes cleave LRP-1 have not been characterized, shedding is known to involve cleavage of the ␤-chain of LRP-1, releasing a fragment of the ␤-chain in complex with the entire ligand binding ␣-chain from the cell membrane (39).…”

A shed form of LDL receptor–related protein–1 regulates peripheral nerve injury and neuropathic pain in rodents

“…This reaction occurs near the center of the LRP1 α-chain, inactivating the ligand-binding function of the receptor. The LRP1 β-chain is a target for BACE-like proteases (22,23), which are known to be present in the PNS (24). Cleavage of the β-chain releases a shed form of the receptor with an apparently intact α-chain.…”

Visualizing interaction of proteins relevant to Alzheimer’s disease in intact cells

“…In summary, we have shown that FRET measured by FLIM is a versatile method to detect protein-protein interactions [6,8,9,16,19,21,23], protein dimerisation [24,25], changes in protein phosphorylation [17,18] or conformational changes of proteins [6,21] in intact cells (Fig. 2) with high relevance in AD research, therefore leading to a better comprehension of the molecular and cellular mechanisms in memory decline.…”

“…A FITC/Cy3-FRET-pair with FITC as donor and Cy3 as acceptor was used to immunolabel the proteins of interest. The FLIM assay was performed in general as previously described [16][17][18][19] and is explained in detail in the following protocol.…”