The canonical Wnt pathway plays a crucial role in embryonic development, and its deregulation is involved in human diseases. The LRP6 single-span transmembrane coreceptor is essential for transmission of canonical Wnt signaling. However, due to the lack of immunological reagents, our understanding of LRP6 structure and function has relied on studies involving its overexpression, and regulation of the endogenous receptor by the Wnt ligand has remained unexplored. Using a highly sensitive and specific antibody to LRP6, we demonstrate that the endogenous receptor is modified by N-glycosylation and is phosphorylated in response to Wnt stimulation in a sustained yet ligand-dependent manner. Moreover, following triggering by Wnt, endogenous LRP6 is internalized and recycled back to the cellular membrane within hours of the initial stimulus. Finally, we have identified a novel feedback mechanism by which Wnt, acting through ␤-catenin, negatively regulates LRP6 at the mRNA level. Together, these findings contribute significantly to our understanding of LRP6 function and uncover a new level of regulation of Wnt signaling. In light of the direct role that the Wnt pathway plays in human bone diseases and malignancies, our findings may support the development of novel therapeutic approaches that target Wnt signaling through LRP6.The highly conserved canonical Wnt pathway plays a critical role in cell fate determination and tissue development (7, 23). Moreover, aberrant activation of Wnt signaling is causally involved in human cancers (9, 28). Members of this family of secreted glycoproteins interact with two coreceptors, the Frizzled seven-pass transmembrane receptor and the low-density lipoprotein (LDL) receptor-related protein LRP5/6. Wnt-receptor interactions lead to inhibition of ␤-catenin phosphorylation by casein kinase 1-␣ (CK1-␣) and glycogen synthase kinase-␤, which occurs within a protein complex containing axin and the tumor suppressor adenomatous polyposis coli. Inhibition of ␤-catenin phosphorylation impairs its degradation and results in accumulation of the uncomplexed cytosolic molecule, which translocates to the nucleus and interacts with TCF/LEF factors to activate transcription (9,13,24).Frizzled receptors are known to mediate signaling through both the Wnt-␤-catenin "canonical" pathway and other, "noncanonical" ones, such as the planar cell polarity and Wnt/Ca 2ϩ pathways. In contrast, the LRP6 receptor and the family member LRP5 specifically function in the Wnt-␤-catenin pathway (5, 13, 17). In fact, inactivation of the LRP5/6 homologue arrow in Drosophila melanogaster results in a phenotype similar to that of the wingless mutant, and injection of LRP6 mRNA into Xenopus laevis embryos enhances Wnt-induced developmental defects (33, 35). Moreover, mice deficient for LRP6 exhibit defects resembling those caused by the loss of various Wnt proteins (27). There is evidence supporting a dual-receptor model in which independent binding of Wnt to Frizzled and LRP6 recruits these two proteins into a receptor comp...

Protein ectodomain shedding is a specialized type of regulated proteolysis that releases the extracellular domain of transmembrane proteins. The metalloprotease disintegrin tumor necrosis factor-␣-converting enzyme (TACE) has been convincingly shown to play a central role in ectodomain shedding, but despite its broad interest, very little is known about the mechanisms that regulate its activity. An analysis of the biosynthesis of TACE in mutant cell lines that have a gross defect in ectodomain shedding (M1 and M2) shows a defective removal of the prodomain that keeps TACE in an inactive form. Using LoVo, a cell line that lacks of active furin, and ␣ 1 -Antitrypsin Portland, a protein inhibitor of proprotein convertases, we show that TACE is normally processed by furin and other proprotein convertases. The defect in M1 and M2 cells is due to a blockade of the exit of TACE from the endoplasmic reticulum. The processing of other zincdependent metalloproteases, previously suggested to participate in activated ectodomain shedding is normal in the mutant cells, indicating that the component mutated is highly specific for TACE. In summary, the characterization of shedding-defective somatic cell mutants unveils the existence of a specific mechanism that directs the proteolytic activation of TACE through the control of its exit from the ER.Protein ectodomain shedding is a specialized type of proteolysis that releases the extracellular domain of cell surface proteins, leaving the transmembrane-cytoplasmic region bound to the plasma membrane. Ectodomain shedding can regulate the function of transmembrane growth factors, turn growth factor receptors into agonist or antagonist of the ligands they bind, or modulate cell-cell and cell-extracellular matrix interactions (1). Furthermore, the shedding of the ␤-amyloid precursor protein (also known as ␣-secretase cleavage) prevents the formation of the ␤-amyloid peptide, the main component of the senile plaques found in brains of patients with Alzheimer's disease (2).Experiments with specific inhibitors indicate that practically all of the shedding events analyzed to date are mediated by zinc-dependent metalloproteases that belong the metzincin family (3). In addition, overexpression of certain metzincins or their dominant-negative forms point to the participation of these metalloproteases in ectodomain shedding (for example see Refs. 4 and 5). A definitive confirmation of the role of a member of the metalloprotease disintegrins (also known as ADAMs or MDCs), a subfamily of the metzincins, in ectodomain shedding came with the development of mice genetically deficient in the tumor necrosis factor-␣-converting enzyme (TACE 1 /ADAM17) (6). The characterization of cell lines derived from TACE knock-out mice (TACEϪ/Ϫ cells) confirmed the involvement of this metalloprotease disintegrin in the shedding of a surprisingly large group of cell surface proteins (reviewed in Ref. 1).Metzincins are synthesized as zymogens containing a prodomain with an odd cysteine, which coordinates the zinc p...

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