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Wnt/β-catenin signaling directs animal development and tissue renewal in a tightly controlled, cell- and tissue-specific manner. In the mammalian central nervous system, the atypical ligand Norrin controls angiogenesis and maintenance of the blood-brain barrier and blood-retina barrier through the Wnt/β-catenin pathway. Like Wnt, Norrin activates signaling by binding and heterodimerizing the receptors Frizzled (Fzd) and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), leading to membrane recruitment of the intracellular transducer Dishevelled (Dvl) and ultimately stabilizing the transcriptional coactivator β-catenin. Unlike Wnt, the cystine knot ligand Norrin only signals through Fzd4 and additionally requires the co-receptor Tetraspanin12 (Tspan12); however, the mechanism underlying Tspan12-mediated signal enhancement is unclear. It has been proposed that Tspan12 integrates into the Norrin-Fzd4 complex to enhance Norrin-Fzd4 affinity or otherwise allosterically modulate Fzd4 signaling. Here, we measure direct, high-affinity binding between purified Norrin and Tspan12 in a lipid environment and use AlphaFold models to interrogate this interaction interface. We find that Tspan12 and Fzd4 can simultaneously bind Norrin and that a pre-formed Tspan12/Fzd4 heterodimer, as well as cells co-expressing Tspan12 and Fzd4, more efficiently capture low concentrations of Norrin than Fzd4 alone. We also show that Tspan12 competes with both heparan sulfate proteoglycans and LRP6 for Norrin binding and that Tspan12 does not impact Fzd4-Dvl affinity in the presence or absence of Norrin. Our findings suggest that Tspan12 does not allosterically enhance Fzd4 binding to Norrin or Dvl, but instead functions to directly capture Norrin upstream of signaling., Competing Interests: EB, JM, WW No competing interests declared, (© 2024, Bruguera et al.)

Wnt/β-catenin signaling directs animal development and tissue renewal in a tightly controlled, cell- and tissue-specific manner. In the mammalian central nervous system, the atypical ligand Norrin controls angiogenesis and maintenance of the blood-brain barrier and blood-retina barrier through the Wnt/β-catenin pathway. Like Wnt, Norrin activates signaling by binding and heterodimerizing the receptors Frizzled (Fzd) and Low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6), leading to membrane recruitment of the intracellular transducer Dishevelled (Dvl) and ultimately stabilizing the transcriptional coactivator β-catenin. Unlike Wnt, the cystine-knot ligand Norrin only signals through Fzd4 and additionally requires the co-receptor Tetraspanin12 (Tspan12); however, the mechanism underlying Tspan12-mediated signal enhancement is unclear. It has been proposed that Tspan12 integrates into the Norrin-Fzd4 complex to enhance Norrin-Fzd4 affinity or otherwise allosterically modulate Fzd4 signaling. Here, we measure direct, high-affinity binding between purified Norrin and Tspan12 in a lipid environment and use AlphaFold models to interrogate this interaction interface. We find that Tspan12 and Fzd4 can simultaneously bind Norrin and that a pre-formed Tspan12/Fzd4 heterodimer, as well as cells co-expressing Tspan12 and Fzd4, more efficiently capture low concentrations of Norrin than Fzd4 alone. We also show that Tspan12 competes with both heparan sulfate proteoglycans and LRP6 for Norrin binding and that Tspan12 does not impact Fzd4-Dvl affinity in the presence or absence of Norrin. Our findings suggest that Tspan12 does not allosterically enhance Fzd4 binding to Norrin or Dvl, but instead functions to directly capture Norrin upstream of signaling.

In the Wnt-β-catenin pathway, Wnt binding to Frizzled (Fzd) and LRP5 or LRP6 (LRP5/6) co-receptors inhibits the degradation of the transcriptional coactivator β-catenin by recruiting the cytosolic effector Dishevelled (Dvl). Polymerization of Dvl at the plasma membrane recruits the β-catenin destruction complex, enabling the phosphorylation of LRP5/6, a key step in inhibiting β-catenin degradation. Using purified Fzd proteins reconstituted in lipid nanodiscs, we investigated the factors that promote the recruitment of Dvl to the plasma membrane. We found that the affinity of Fzd for Dvl was not affected by Wnt ligands, in contrast to other members of the GPCR superfamily for which the binding of extracellular ligands affects the affinity for downstream transducers. Instead, Fzd-Dvl binding was enhanced by increased concentration of the lipid PI(4,5)P 2 , which is generated by Dvl-associated lipid kinases in response to Wnt and which is required for LRP5/6 phosphorylation. Moreover, binding to Fzd did not promote Dvl DEP domain dimerization, which has been proposed to be required for signaling downstream of Fzd. Our findings suggest a positive feedback loop in which Wnt-stimulated local PI(4,5)P 2 production enhances Dvl recruitment and further PI(4,5)P 2 production to support Dvl polymerization, LRP5/6 phosphorylation, and β-catenin stabilization.

Many membrane proteins function as dimers or larger oligomers, including transporters, channels, certain signaling receptors, and adhesion molecules. In some cases, the interactions between individual proteins may be weak and/or dependent on specific lipids, such that detergent solubilization used for biochemical and structural studies disrupts functional oligomerization. Solubilized membrane protein oligomers can be captured in lipid nanodiscs, but this is an inefficient process that can produce stoichiometrically and topologically heterogeneous preparations. Here, we describe a technique to obtain purified homogeneous membrane protein dimers in nanodiscs using a split GFP (sGFP) tether. Complementary sGFP tags associate to tether the coexpressed dimers and control both stoichiometry and orientation within the nanodiscs, as assessed by quantitative Western blotting and negative-stain EM. The sGFP tether confers several advantages over other methods: it is highly stable in solution and in SDS-PAGE, which facilitates screening of dimer expression and purification by fluorescence, and also provides a dimer-specific purification handle for use with GFP nanobody-conjugated resin. We used this method to purify a Frizzled-4 homodimer and a Frizzled-4/low-density lipoprotein receptor-related protein 6 heterodimer in nanodiscs. These examples demonstrate the utility and flexibility of this method, which enables subsequent mechanistic molecular and structural studies of membrane protein pairs., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Mammalian odorant receptors are a diverse and rapidly evolving set of G protein-coupled receptors expressed in olfactory cilia membranes. Most odorant receptors show little to no cell surface expression in nonolfactory cells due to endoplasmic reticulum retention, which has slowed down biochemical studies. Here we provide evidence that structural instability and divergence from conserved residues of individual odorant receptors underlie intracellular retention using a combination of large-scale screening of odorant receptors cell surface expression in heterologous cells, point mutations, structural modeling, and machine learning techniques. We demonstrate the importance of conserved residues by synthesizing consensus odorant receptors that show high levels of cell surface expression similar to conventional G protein-coupled receptors. Furthermore, we associate in silico structural instability with poor cell surface expression using molecular dynamics simulations. We propose an enhanced evolutionary capacitance of olfactory sensory neurons that enable the functional expression of odorant receptors with cryptic mutations., Competing Interests: Competing interest statement: K.I., C.A.d.M., M.H.N., and H.M. filed a provisional patent application relevant to this work. H.M. receives royalties from Chemcom. The remaining authors declare no competing interests.