Biophysical and functional characterization of Norrin signaling through Frizzled4

Abstract

Wnt signaling is initiated by Wnt ligand binding to the extracellular ligand binding domain, called the cysteine-rich domain (CRD), of a Frizzled (Fzd) receptor. Norrin, an atypical Fzd ligand, specifically interacts with Fzd4 to activate β-catenin-dependent canonical Wnt signaling. Much of the molecular basis that confers Norrin selectivity in binding to Fzd4 was revealed through the structural study of the Fzd4-Norrin complex. However, how the ligand interaction, seemingly localized at the CRD, is transmitted across full-length Fzd4 to the cytoplasm remains largely unknown. Here, we show that a flexible linker domain, which connects the CRD to the transmembrane domain, plays an important role in Norrin signaling. The linker domain directly contributes to the high-affinity interaction between Fzd4 and Norrin as shown by ∼10-fold higher binding affinity of Fzd4 to Norrin in the presence of the linker. Swapping the Fzd4 linker with the Fzd5 linker resulted in the loss of Norrin signaling, suggesting the importance of the linker in ligand-specific cellular response. In addition, structural dynamics of Fzd4 associated with Norrin binding investigated by hydrogen/deuterium exchange MS revealed Norrin-induced conformational changes on the linker domain and the intracellular loop 3 (ICL3) region of Fzd4. Cell-based functional assays showed that linker deletion, L430A and L433A mutations at ICL3, and C-terminal tail truncation displayed reduced β-catenin-dependent signaling activity, indicating the functional significance of these sites. Together, our results provide functional and biochemical dissection of Fzd4 in Norrin signaling.

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

“…stimulation actually promotes FZD-DVL recruitment (44). From this study, we conclude that no agonist input is required for the basal interaction between overexpressed FZD and DVL because the ⌬CRD-FZD 6 , a construct devoid of the orthosteric binding site for WNTs, is capable of recruiting DVL, even in the absence of WNTs.…”

“…It is attractive to speculate that the linker domain does not only maintain receptor surface expression but that it also has a role in the agonist-induced dynamics and a potential two-step agonist binding mode, as described for class B receptors (53,54). Very recently it was shown for FZD 4 that the linker domain indeed has a role in defining functional high-affinity binding of the WNT-unrelated, FZD 4 -selective ligand Norrin (44). The study supports our speculation of a CRD-and linker-mediated two-step binding mode also in the case of FZD-WNT interaction.…”

A glimpse into the molecular mechanism of integral membrane proteins through hydrogen–deuterium exchange mass spectrometry

“…Norrin is a cysteine‐knot growth factor, distinct from WnT ligands, that activates the canonical Wnt pathway by interacting with receptor Frizzled4 cysteine‐rich domain (Fz4 CRD ) . To understand the rules of selectivity toward Norrin and the pathway for signaling, Bang et al combined biochemical data with biophysical and computational studies . They found out that the flexible linker domain that connects the cysteine‐rich domain (CRD) to the TM domain plays an important role in signal transmission.…”

Structural basis of recognition and destabilization of histone H2B ubiquitinated nucleosome by DOT1L histone H3 Lys79 methyltransferase

“…For non-deuterated samples (ND), 5µL of protein samples was mixed with 25 µL of H2O buffer (20 mM HEPES, pH 7.5, 30 mM NaCl, 1mM EDTA and 1mM DTT in H2O) and quenched with 30 µL of ice-cold quench buffer. The quenched samples were digested by passing through an immobilized pepsin column, and the peptide masses were analyzed as described previously (Bang et al 2018). Peptides from non-deuterated samples were identified with ProteinLynx Global Server (PLGS) 2.4 (Waters, Milford, MA, USA).…”