Reconstitution of a frizzled8.Wnt3a.LRP6 signaling complex reveals multiple Wnt and Dkk1 binding sites on LRP6.
Wnt/beta-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/beta-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 CRD.Wnt3a.LRP6 signaling complex. Using purified fragments of LRP6, we further show that Wnt3a binds to a region including only the third and fourth beta-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/beta-catenin signaling by Dkk1.
Bourhis
Eric
E
Department of Protein Engineering, Genentech, Inc, South San Francisco, California 94080, USA.
Tam
Christine
C
Franke
Yvonne
Y
Bazan
J Fernando
JF
Ernst
James
J
Hwang
Jiyoung
J
Costa
Mike
M
Cochran
Andrea G
AG
Hannoush
Rami N
RN
D000818:Animals; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D006801:Humans; D020128:Inhibitory Concentration 50; D007313:Insecta; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D007700:Kinetics; D026502:LDL-Receptor Related Proteins / Q000378:metabolism*; D060488:Low Density Lipoprotein Receptor-Related Protein-6; D051379:Mice; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D051153:Wnt Proteins / Q000378:metabolism*; D060508:Wnt3 Protein; D060509:Wnt3A Protein; D051176:beta Catenin / Q000378:metabolism
D036341:Intercellular Signaling Peptides and Proteins; D026502:LDL-Receptor Related Proteins; D060488:Low Density Lipoprotein Receptor-Related Protein-6; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D051153:Wnt Proteins; D060508:Wnt3 Protein; D060509:Wnt3A Protein; D051176:beta Catenin; C501126:DKK1 protein, human; C501127:Dkk1 protein, mouse; C529739:Fzd8 protein, human; C555475:LRP6 protein, human; C555476:Lrp6 protein, mouse; C555510:WNT3A protein, human; C555511:Wnt3a protein, mouse; C511610:Fzd8 protein, mouse