Abstract

The LOW-density lipoprotein related protein 6 (LRP6) receptor is an important effector of canonical Wnt signaling, a developmental pathway, whose dysregulation has been implicated in various diseases including cancer. The membrane proximal low-density lipoprotein (LDL) receptor repeats in LRP6 exhibit homology to ligand binding repeats in the LDL receptor (LDLR), but lack known function. We generated single amino acid substitutions of LRP6-LDLR repeat residues, which are highly conserved in the human LDLR and mutated in patients with Familial Hypercholesteremia (FH). These substitutions negatively impacted LRP6 internalization and activation of Wnt signaling. By mass spectrometry, we observed that the Itch E3 ubiquitin ligase associated with and ubiquitinated wild type LRP6 but not the LDLR repeat mutants. These findings establish the involvement of LRP6-LDLR repeats in the regulation of canonical Wnt signaling.

Highlights

  • Wnt/β-catenin signaling is critical in embryonic development, and aberrations of this pathway play important roles in a variety of human diseases including cancer [1, 2]. β-catenin serves as a major Wnt transcriptional effector, whose functions are regulated by casein kinase 1α (CK1α) and glycogen synthase kinase 3β (GSK3β) phosphorylation, which target it for proteosomal degradation by beta-transducing repeat-containing protein (β-TrCP) in the absence of Wnt ligand stimulation [3,4,5]

  • We focused on amino acid sequences from 139 to 270 encoded by exons four and five of the LDL receptor (LDLR) that showed highest homology to the lipoprotein related protein 6 (LRP6)-LDLR repeats (LDLRR) sequence (Figure 1 A)

  • Within this 122 amino acid span in the LRP6-LDLRR (1239-1360), we identified previously reported point mutations in the LDLR that are causative of familial hypercholesterolemia (FH) [23] (Table 1)

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Summary

Introduction

Wnt/β-catenin signaling is critical in embryonic development, and aberrations of this pathway play important roles in a variety of human diseases including cancer [1, 2]. β-catenin serves as a major Wnt transcriptional effector, whose functions are regulated by casein kinase 1α (CK1α) and glycogen synthase kinase 3β (GSK3β) phosphorylation, which target it for proteosomal degradation by beta-transducing repeat-containing protein (β-TrCP) in the absence of Wnt ligand stimulation [3,4,5]. Several independent studies have shown that the caveolin endocytic pathway plays a critical role in Wnt mediated LRP6 internalization [10, 13]. A tetrameric sequence, Asn-Pro-Val-Tyr (NPxY), in the cytoplasmic region is implicated in the internalization of several cell surface receptors including the LDLR [14]. This sequence is absent in LRP6, and it is not known whether there is any specific LRP6 motif or sequence whose modification in response to Wnt, induces assembly of the endocytic machinery to internalize the LRP6 receptor complex and activate the Wnt/β-catenin pathway

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