Cooperative Folding and Ligand-binding Properties of LRP6 β-Propeller Domains

Abstract

Wnt/␤-catenin signaling controls cell growth during development, and its misregulation in adults can cause human diseases. LRP6, the essential co-receptor for the Wnt pathway, consists of four ␤-propeller domains flanked by epidermal growth factor repeats in its extracellular region. To understand the maturation and ligand-binding properties of individual BP domains, we generated soluble receptor consisting of individual BPs, as well as combinations of these domains. We show that BP1, BP2, and BP4 each can be folded and secreted, and their secretion was enhanced by co-expression of Mesd, a molecular chaperone essential for LRP6 folding and maturation. BP3 is not secreted when expressed on its own or in combination with BP2 or BP1 and 2 (BP12); however, folding and secretion of BP3 is vastly enhanced when expressed together with BP4. Similar cooperative folding and maturation was observed between BP1 and BP2. These results suggest that BP1 forms a functional folding unit with BP2, whereas BP3 folds together with BP4. Using these BP constructs, we also found that BP12 and BP34 constitute independent ligand-binding domains capable of binding Wnt3a, Dkk1, and Mesd. The ability of Mesd to block the binding of both Wnt3a and Dkk1 to LRP6 enables this specialized chaperone to function as a Wnt signaling modulator. Together, our studies reveal unique properties of the LRP6 BP domains and provide novel tools to understand LRP6 function in ligand binding and Wnt signaling. Our results also support the development of soluble LRP6 receptors and Mesd as potential therapeutic molecules that target Wnt signaling.

Reconstitution of a Frizzled8·Wnt3a·LRP6 Signaling Complex Reveals Multiple Wnt and Dkk1 Binding Sites on LRP6

“…Although Wnt3a has been shown to bind to E1E2 in an enzyme-linked immunosorbent assay (35), this experiment was performed with conditioned media containing E1E2, raising questions about whether other proteins are involved in mediating this binding event. The differences seen between the direct binding studies reported here and interaction analyses based either on enzyme-linked immunosorbent assay (35) or indirect transcriptional readouts (9) stress the importance of well defined biochemical assays using purified proteins.…”

Retinoic Acid Isomers Facilitate Apolipoprotein E Production and Lipidation in Astrocytes through the Retinoid X Receptor/Retinoic Acid Receptor Pathway

“…To rule out the possibility that all-trans-RA, 9-cis-RA, 13-cis-RA, and bexarotene increase apoE levels in the conditioned media by suppressing its cellular clearance, we examined the levels of two major apoE metabolic receptors LDLR and LRP1 (32). We found that receptor expression at both protein and mRNA levels was not changed by tested compounds (Fig.…”

International Union of Basic and Clinical Pharmacology. LXXX. The Class Frizzled Receptors

“…LRP5/6 collaborate with FZDs through formation of a ternary WNT/FZD/LRP5/6 complex (Tamai et al, 2000;Wehrli et al, 2000). In contrast to FZDs, LRP5/6 do not contain any CRD-WNT binding domains; rather, their N-terminal ␤ propeller epidermal growth factor repeats are essential for the interaction with the WNT/FZD complex (Hey et al, 1998;Liu et al, 2009;Bourhis et al, 2010). In fact, a recent mapping study identified unique binding patterns of different WNTs to the extracellular domains of LRP6, showing that several WNTs, such as WNT-3A and WNT-9B, can bind simultaneously (Bourhis et al, 2010).…”