Functional dissection of the N-terminal extracellular domains of Frizzled 6 reveals their roles for receptor localization and Dishevelled recruitment

Abstract

The Frizzled (FZD) proteins belong to class F of G proteincoupled receptors (GPCRs) and are essential for various pathways involving the secreted lipoglycoproteins of the wingless/ int-1 (WNT) family. A WNT-binding cysteine-rich domain (CRD) in FZDs is N-terminally located and connected to the seven transmembrane domain-spanning receptor core by a linker domain that has a variable length in different FZD homologs. However, the function and importance of this linker domain are poorly understood. Here we used systematic mutagenesis of FZD 6 to define the minimal N-terminal domain sufficient for receptor surface expression and recruitment of the intracellular scaffold protein Dishevelled (DVL). Further, we identified a triad of evolutionarily conserved cysteines in the FZD linker domain that is crucial for receptor membrane expression and recruitment of DVL. Our results are in agreement with the concept that the conserved cysteines in the linker domain of FZDs assist with the formation of a common secondary structure in this region. We propose that this structure could be involved in agonist binding and receptor activation mechanisms that are similar to the binding and activation mechanisms known for other GPCRs.

Familial Exudative Vitreoretinopathy-Related Disease-Causing Genes and Norrin/β-Catenin Signal Pathway: Structure, Function, and Mutation Spectrums

“…Several amino residuals in intracellular loops 1, 2, and 3 and the flanking region near to intracellular loop 3 were also important for the intracellular location of Dishevelled while the mutant impaired the binding of Dishevelled [73–77]. Research based on FZD6 also showed that the linker domain, especially some conserved cystines, between the CRD domain and seven transmembrane core was imperative for Dishevelled recruitment [78]. One potential mechanism for FZD4 activation would be a Wnt/Norrin-induced movement of the seventh transmembrane domain to expose the key FZD4-Dishevelled interaction site [79].…”

Structural insight into small molecule action on Frizzleds

“…DVL is a central mediator of the β-catenin-dependent and PCP-like WNT signaling pathways and its recruitment to FZD is an initial step in DVL-dependent signaling 12,47,48 . Simultaneous overexpression of DVL and FZD leads to FZD-dependent membrane recruitment of DVL even in the absence of a ligand [49][50][51] colocalization and recruitment of cytosolic DVL present in punctate aggregates to membrane-expressed FZDs 47,[49][50][51] . However, quantification of recruitment and measurement of ligandinduced dynamics were not possible.…”

“…In order to assess ligand-induced effects on DVL-FZD BRET, we chose an acceptor:donor ratio corresponding to the plateau part of the saturation curves for both setups. In addition, we did not treat the cells with the porcupine inhibitor C59 to block secretion of endogenous WNTs as their presence had no significant effect on the basal recruitment of the overexpressed DVL2 to the overexpressed FZD 6 as recently reported 51 and presented in Supplementary Fig. 15a, b.…”

“…FLAG-FZD 6 -Nluc and ΔCRD FLAG-FZD 6 -Nluc were subcloned from FLAG-FZD 6 -His and ΔCRD FLAG-FZD 6 -His, respectively, into pNluc-N1 with BgIII and AgeI. SNAP-FZD 6 -Rluc8, SNAP-FZD 7 -Rluc8, SMO-Rluc8, Nluc-DVL2, FLAG-FZD 6 -His, ΔCRD FLAG-FZD 6 -His, and FZD 4 -Nluc were generated and validated in our previous studies 15,51 .…”

Structural insight into Class F receptors – What have we learnt regarding agonist‐induced activation?

“…For example, it is still a mystery how the phosphoprotein dishevelled (DVL), which acts as a central signalling relay and FZD‐interacting scaffold in several β‐catenin‐dependent and β‐catenin‐independent WNT signalling pathways, biochemically mediates WNT‐induced and FZD‐mediated signal initiation and transmission – despite its polymerization in agonist‐induced signalosomes. Further, it remains unclear, if DVL interaction with FZDs is weakened or strengthened upon agonist stimulation in the light of conflicting findings . In this context, it remains obscure if DVL could act as a FZD binding partner to form a ternary complex in a pharmacological sense as previously hypothesized .…”