Wnt/␤-catenin signaling is initiated at the cell surface by association of secreted Wnt with its receptors Frizzled (Fz) and low density lipoprotein receptor-related protein 5/6 (LRP5/6). The study of these molecular interactions has been a significant technical challenge because the proteins have been inaccessible in sufficient purity and quantity. In this report we describe insect cell expression and purification of soluble mouse Fz8 cysteine-rich domain and human LRP6 extracellular domain and show that they inhibit Wnt/␤-catenin signaling in cellular assays. We determine the binding affinities of Wnts and Dickkopf 1 (Dkk1) to the relevant co-receptors and reconstitute in vitro the Fz8 CRD⅐Wnt3a⅐LRP6 signaling complex. Using purified fragments of LRP6, we further show that Wnt3a binds to a region including only the third and fourth ␤-propeller domains of LRP6 (E3E4). Surprisingly, we find that Wnt9b binds to a different part of the LRP6 extracellular domain, E1E2, and we demonstrate that Wnt3a and Wnt9b can bind to LRP6 simultaneously. Dkk1 binds to both E1E2 and E3E4 fragments and competes with both Wnt3a and Wnt9b for binding to LRP6. The existence of multiple, independent Wnt binding sites on the LRP6 co-receptor suggests new possibilities for the architecture of Wnt signaling complexes and a model for broad-spectrum inhibition of Wnt/␤-catenin signaling by Dkk1.Since its discovery more than 30 years ago, the Wnt signaling pathway has captured the interest of researchers due to its role in development, progression of cancer, and self-renewal and differentiation of stem cells (1, 2). Wnt signaling is initiated at the cell surface where secreted Wnt glycoprotein forms a complex with the receptors Frizzled (Fz) 2 (3) and low density lipoprotein receptor-related proteins 5 or 6 (LRP5/6) (4, 5). This results in stabilization of intracellular ␤-catenin ("Wnt/␤-catenin pathway") and its translocation to the nucleus. Nuclear ␤-catenin then initiates T-cell factor-dependent transcription of downstream Wnt target genes (6, 7). The interactions of LRP5/6 and Fz with Wnt are critical for mediating Wnt/␤-catenin signaling (5,8,9). Underscoring the central role that these receptors play in Wnt signaling, suppression of either Fz or LRPs has been shown to generate phenotypes associated with Wnt mutations (10). Furthermore, mechanisms have evolved to regulate the Wnt⅐Fz⅐LRP complex through secreted proteins that interfere with these extracellular interactions. One such example is Dickkopf-1 (Dkk1), which inhibits Wnt signaling by binding to LRP6 (9,11,12). Dkk1 has been shown to play a crucial role in bone formation, vertebrate embryogenesis, and suppression of cancer cell proliferation (13-15). Therefore, Wnt ligands, receptors Fz and LRP, and their natural inhibitors are all of great therapeutic interest. Indeed, recent findings show that the soluble extracellular domain of Fz8 interferes with Wnt-driven tumor growth in vivo (16).The detailed molecular arrangement of the Wnt⅐Fz⅐LRP ternary complex at the cell surface is ...

The Wnt pathway inhibitors DKK1 and sclerostin (SOST) are important therapeutic targets in diseases involving bone loss or damage. It has been appreciated that Wnt coreceptors LRP5/6 are also important, as human missense mutations that result in bone overgrowth (bone mineral density, or BMD, mutations) cluster to the E1 propeller domain of LRP5. Here, we report a crystal structure of LRP6 E1 bound to an antibody, revealing that the E1 domain is a peptide recognition module. Remarkably, the consensus E1 binding sequence is a close match to a conserved tripeptide motif present in all Wnt inhibitors that bind LRP5/6. We show that this motif is important for DKK1 and SOST binding to LRP6 and for inhibitory function, providing a detailed structural explanation for the effect of the BMD mutations.

In rod phototransduction, cyclic GMP synthesis by membrane bound guanylate cyclase ROS-GC1 is under Ca 2+ -dependent negative feedback control mediated by guanylate cyclase-activating proteins, GCAP-1 and GCAP-2. The cellular concentration of GCAP-1 and GCAP-2 approximately sums to the cellular concentration of a functional ROS-GC1 dimer. Both GCAPs increase the catalytic efficiency (k cat /K m ) of ROS-GC1. However, the presence of a myristoyl group in GCAP-1 has a strong impact on the regulation of ROS-GC1, this is in contrast to GCAP-2. Catalytic efficiency of ROS-GC1 increases 25-fold when it is reconstituted with myristoylated GCAP-1, but only by a factor of 3.4 with nonmyristoylated GCAP-1. In contrast to GCAP1, myristoylation of GCAP-2 has only a minor effect on k cat /K m . The increase with both myristoylated and nonmyristoylated GCAP-2 is 10 to 13-fold. GCAPs also confer different Ca 2+ -sensitivities to ROS-GC1. Activation of the cyclase by GCAP-1 is half-maximal at 707 nM free [Ca 2+ ], while that by GCAP-2 is at 100 nM. The findings show that differences in catalytic efficiency and Ca 2+ -sensitivity of ROS-GC1 are conferred by GCAP-1 and GCAP-2. The results further indicate the concerted operation of two ÔGCAP modesÕ that would extend the dynamic range of cyclase regulation within the physiological range of free cytoplasmic Ca 2+ in photoreceptor cells.

DNA double-strand breaks (DSB) can arise during DNA replication, or after exposure to DNA-damaging agents, and their correct repair is fundamental for cell survival and genomic stability. Here, we show that the Smc5-Smc6 complex is recruited to DSBs de novo to support their repair by homologous recombination between sister chromatids. In addition, we demonstrate that Smc5-Smc6 is necessary to suppress gross chromosomal rearrangements. Our findings show that the Smc5-Smc6 complex is essential for genome stability as it promotes repair of DSBs by error-free sister-chromatid recombination (SCR), thereby suppressing inappropriate non-sister recombination events.