Molecular Mechanisms of Wnt Transport

摘要 Wnt是一个进化上保守的分泌配体家族，其在短距离内协调形态发生 运动和细胞命运的决定。Wnt的分泌和随后被其同源受体识别需要 内质网中的O-棕榈油酸化。此修改由PORCN执行，然后将 Wnt到WLS，其专用的整体膜转运蛋白。WLS如何在ER中与Wnt关联，以及 随后将其传递到接收细胞上的受体Frizzled在很大程度上仍然未知， 本应用程序。 目前，对WLS如何结合Wnt并将其转运到细胞膜的理解有限。 在这里，我们提出了最近发表的数据，在与WNT 8A的复合物中，确定了人WLS的结构， 使用单粒子低温电子显微镜至3.2 μ m分辨率。WLS膜结构域类似于G 蛋白偶联受体（GPCR），尽管有一个额外的跨膜螺旋。O-棕榈油酸化Wnt 发夹环2插入GPCR相关结构域的保守中心空腔，并连接棕榈油酸酯 突出到脂质双层中。高度保守的补丁上的外向表面的 WLS的跨膜结构域，邻近疏水通道，提示Wnt的潜在机制 从PORCN转移到Frizzled。 我们建议进一步研究WLS和Wnt之间的相互作用，使用现有的结构信息， 起点首先，我们将研究Wnt如何与WLS关联。我们建议确定 WLS在其apo -缺失的Wnt -状态下，以及通过结构- 定向诱变和生物化学测定。我们的初步结果表明，WLS之间的直接相互作用 关于PORCN其次，我们将测试序列和糖基化模式的变化如何影响Wnt分泌 和目的地，以及pH值对两种蛋白质之间的相互作用的影响，使用结构指导的 诱变以及原代肠细胞中的Wnt转运和递送测定。第三，我们建议研究 Wnt向后续受体的释放。我们的初步结果表明，WLS之间的直接相互作用 和卷曲受体，我们将测试这一假设使用详细的结构功能分析。

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.