In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 109, No. 14 ( 2012-04-03)
Abstract:
Taken together, these findings significantly change the current view of how Fz relays its signals to Dvl, giving the Fz third loop region and the Dvl DEP-C region a central role. These results provide a mechanistic explanation for a number of functionally defective Drosophila fz gene types, in which mutated residues map to the newly identified third loop Dvl binding motifs ( 3 , 4 ). The previously undescribed Fz-Dvl binding mode implicates a conformation-sensitive interface, which offers possibilities for regulation of the interaction by allosteric means. Understanding how Fz and Dvl interact will help guide the ongoing design of therapeutics that interfere with Wnt pathway activation in cancer. What part of Dvl binds the newly identified motifs in the third intracellular loop of Fz? We confirmed that the Dvl PDZ domain binds the classic KTxxxW motif in vitro, but no interactions were detected with the Fz third loop. Screening of multiple Dvl fragments against the Fz peptide library revealed that, unexpectedly, the Dvl DEP domain plus C terminus (DEP-C) fully recapitulated binding to all three motifs in the Fz receptor. Indeed, the isolated DEP-C domain, but not the PDZ domain, is efficiently recruited to form stable complexes with Fz at the plasma membrane in cells. These findings strongly suggest that the DEP-C domain comprises the Fz binding module in the Dvl protein. Our results further show that the DEP domain mainly contacts the classic motif in the Fz C-tail, whereas the Dvl C-terminal domain binds the third intracellular loop of Fz. Mutational analysis suggests that the very 13 C-terminal amino acids of the Dvl protein comprise a previously unrecognized small functional unit, which helps to stabilize binding of Dvl to the Fz receptor. In this work, we shed light on this issue by using a combinatorial peptide library, which mimics the cytoplasmic interface of the Fz receptor and allows for the identification of complex molecular-recognition modes. We show that Dvl binds a three-segmented, discontinuous site in Fz composed of two previously undescribed motifs in the third intracellular loop and the classic KTxxxW motif in the C-terminal tail ( Fig. P1 ). Furthermore, we demonstrate the functional importance of the highly conserved third loop motifs in Dvl binding and signaling in the context of full-length Fz proteins in human cells and Xenopus embryos. Mutations of single amino acids within these motifs but not in flanking residues disrupt Dvl binding and signaling, suggesting that each of the individual motifs is required but not sufficient for Fz–Dvl complex formation and signal relay. Currently, no Fz receptor crystal structure is available; however, based on homology with related proteins, we show that the three identified motifs are likely to compose a single binding surface on a favorable conformation of the receptor in the membrane ( Fig. P1 ). Wnt binds to receptors on the cell surface called Frizzled (Fz) proteins, which then recruit another protein, Dishevelled (Dvl), usually located in the cell's cytoplasm, to the plasma membrane. Fz–Dvl complexes can then activate signaling cascades that control cellular developmental processes by affecting gene activity. Fz proteins traverse the lipid bilayer seven times, exposing three loops and an extended C-terminal tail in the cytoplasm ( Fig. P1 ). Over a decade ago, a conserved linear motif was identified in the Fz C-terminal tail (KTxxxW) that mediates membrane relocalization of Dvl and activation of Wnt signaling through the β-catenin pathway ( 1 ). Subsequently, a protein interaction module in Dvl, the PDZ domain, was shown to bind the KTxxxW sequence in vitro, and microinjection of Fz peptides comprising this motif were found to interfere with Wnt/β-catenin signaling, confirming the functional importance of the Fz C-terminal tail ( 2 ). Despite this progress, insight into how the interaction between Fz and Dvl drives pathway activation has been scarce. Wnt proteins control embryonic development and are implicated in tumorigenesis. The complex molecular mechanisms mediating Wnt-induced cell responses are of wide interest, and the underlying protein interactions provide an attractive drug target, particularly in cancer treatment. However, the initiating step by which Wnts steer cellular signaling cascades and, ultimately, drive gene activation remains a major unresolved question. Here, we provide key mechanistic insight into how Wnt forms stable protein signaling complexes at the plasma membrane to induce downstream gene activation.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1114802109
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2012
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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