Molecular Mechanisms of Wnt Transport

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ABSTRACT Wnts are a family of evolutionary conserved, secreted ligands that act at short range to coordinate morphogenetic movements and cell fate decisions. Secretion of Wnt and subsequent recognition by its cognate receptor require its O-palmitoleation in the endoplasmic reticulum. This modification is carried out by PORCN, that then transfers Wnt to WLS, its dedicated integral membrane transporter. How WLS associates with Wnt in the ER, and subsequently delivers it to the receptor Frizzled on the receiving cell remains largely unknown, and is the focus of this Application. Currently, there is a limited understanding of how WLS binds Wnts and transport them to the cell membrane. Here we present as recently published data the structure of human WLS, in a complex with WNT8A, determined using single-particle cryo-electron microscopy to 3.2 Å resolution. The WLS membrane domain resembles a G protein-coupled receptor (GPCR), albeit with one additional transmembrane helix. The O-palmitoleated Wnt hairpin loop 2 inserts into a conserved central cavity of the GPCR-related domain, with the attached palmitoleate protruding out into the lipid bilayer. Highly conserved patches on the outward-facing surfaces of the transmembrane domain of WLS, adjacent to a hydrophobic gateway, suggest potential mechanisms for Wnt transfer from PORCN and to Frizzled. We propose to further study the interaction between WLS and Wnt, using the available structural information as starting point. First, we will investigate how Wnt associate with WLS. We propose to determine the structure of WLS in its apo – absent Wnt – state, as well as probe the putative site of entrance of Wnt into WLS by structure- guided mutagenesis and biochemical assays. Our preliminary results suggest a direct interaction between WLS and PORCN. Second, we will test how changes in sequence and glycosylation pattern can affect Wnt secretion and destination, as well as the effect of pH on the interaction between the two proteins, using structure-guided mutagenesis as well as Wnt transport and delivery assays in primary intestinal cells. Third, we propose to study the release of Wnt to subsequent receptors. Our preliminary results suggest a direct interaction between WLS and the Frizzled receptor, and we will test this hypothesis using detailed structure function analyses.