γ-Secretase-mediated Release of the Low Density Lipoprotein Receptor-related Protein 1B Intracellular Domain Suppresses Anchorage-independent Growth of Neuroglioma Cells

Abstract

The low density lipoprotein receptor related protein 1B (LRP1B) is a large endocytic receptor that was first identified as a candidate tumor suppressor gene. In the current investigation we demonstrate that LRP1B undergoes regulated intramembrane proteolysis in a ␥-secretase-dependent process. The released intracellular domain (ICD) then translocates to the nucleus via a nuclear localization signal that is present within this domain. ICD release first requires shedding of the LRP1B ectodomain, which appears to be catalyzed by a member of the metalloproteinase family. Employing site-directed mutagenesis studies, we identified lysine residues 4432 and 4435 and arginine 4442 as key amino acids important for ectodomain shedding of LRP1B. We also demonstrate that an LRP1B minireceptor as well as the ICD domain alone suppresses anchorage-independent growth of LRP1B-deficient neuroglioma cells (H4 cells). Interestingly, abrogating ectodomain shedding resulted in a loss of the ability of LRP1B minireceptors to suppress anchorage-independent growth. Together, these studies reveal that LRP1B has tumor suppression function that is mediated by proteolytic processing of the receptor resulting in ICD release. The low density lipoprotein (LDL)2 receptor-related protein 1B (LRP1B) is a member of the LDL receptor gene family and was initially identified as a candidate tumor suppressor gene by positional cloning (1, 2). The LRP1B gene is frequently inactivated by homozygous genomic deletions or by the expression of aberrant mRNA transcripts (1). In human esophagus cancer cell lines and primary tumors the LRP1B gene was shown to be inactivated by transcriptional silencing through hypermethylation of its promoter CpG island (3). Homozygous deletions of LRP1B have also been reported in human liver cancers (4) and urothelial cancers (5). By conventional and array-based comparative genomic hybridization analysis, loss of the LRP1B allele was noted in endocervical adenocarcinomas of uterus (6). Thus, collective data indicate that this gene is inactivated during cellular transformation, suggesting that LRP1B is a candidate tumor suppressor gene.Structurally, LRP1B is closely related to LRP1, a receptor that recognizes numerous ligands and is essential for normal development (7). Like LRP1, LRP1B contains a furin cleavage site (REKR) within a ␤-propeller domain that is cleaved by furin during receptor trafficking (8 -10). The major structural differences between LRP1 and LRP1B occur within their intracellular domains (ICD). First, the LRP1B ICD contains an alternatively spliced exon (exon 90) that is not present in LRP1 (1). Second, the LRP1B ICD contains a cluster of basic residues (KRKRRTK) that is similar to basic clusters in BRCA2 tumor suppressor which have been shown to function as a nuclear localization sequence (NLS) (11).Currently, the physiological function of LRP1B remains unknown. In mice, LRP1B expression is primarily restricted to the brain, adrenal glands, salivary gland, and testis (12). However, in humans, LRP1B is widel...

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Chromosomal, epigenetic and microRNA-mediated inactivation of LRP1B, a modulator of the extracellular environment of thyroid cancer cells

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Regulated Intramembrane Proteolysis: Signaling Pathways and Biological Functions

“…For other ␥-secretase substrates, such as the closely related SorLA/LR11 (5), low-density lipoprotein-related protein (LRP) members (33,42,44), and megalin (73), the study of RIP nuclear signaling is still in its infancy. Although ␥-secretasedependent cleavage of these substrates occurs in what appears to be a manner very similar to that described for Notch and APP, the triggers and biologically relevant signaling-dependent effects remain largely unknown.…”

“…With respect to SorLA, the ICD could be detected in the nucleus of cells heterologously overexpressing SorLA-ICD, but the transcriptional potential appears to be weak (5). LRP1B has recently been shown to undergo intramembrane proteolysis in a manner that give rises to an LRP1B-ICD that accumulates in the nucleus and that alone possesses the capacity to suppress tumor cell anchorage-independent growth (33). Megalin, a scavenger receptor with a central role in renal protein absorption, colocalizes with and is processed by presenlin-1/␥-secretase in the brush border of the kidney proximal tubules (73).…”