The role of "lipid raft" microdomains in the PDZ-adaptor protein-mediated NHE3 Regulation in the intestine

Na+/H+交换异构体3是最重要的肠道Na+吸收途径。NHE3有一个很长的细胞质尾巴，它含有多个调节蛋白的结合位点以及蛋白激酶的磷酸化共识序列。NHERF(Na+/H+调节因子)家族的PDZ受体蛋白在转运蛋白、受体、锚蛋白、蛋白激酶和细胞骨架之间NHE3多蛋白复合体的形成中起着重要的作用。虽然在异源表达系统中已经证明了简单的NHERF-NHE3-锚定蛋白-蛋白激酶复合体的形成，但关于NHERF蛋白在天然组织中的调节作用的信息很少。对于更好地理解NHE3调节的分子生理学以及靶向NHERF相互作用的治疗潜力，最重要的是更好地理解如何在给定的受体激活的信号通路中建立NHERF、NHE3和信号分子之间相互作用的选择性和保密性(将信号事件包含在靶点位置，避免靶外效应)。信号特异性的一个潜在机制可能是NHE3和相互作用的蛋白质在膜微域内的协同分离，也称为脂筏。这些富含鞘磷脂的结构域是质膜内蛋白质聚集的区域，因此可以作为相互作用的平台。我们最近证实NHERF与肠道刷状缘膜内的脂筏和非脂筏结构域有不同的联系。通过使用基因敲除小鼠的各自的NHERF，我们还发现，不同的NHERF分布决定了NHE3在木筏上的分布。在本项目中，我们旨在研究NHE3、NHERF、cGMP信号通路的信号分子和小鼠肠道刷状缘膜上的脂筏之间的动态相互作用。在人肠道器官(HIO)培养建立和鉴定后，我们将研究人肠道刷状缘膜的脂筏平台是否也起到将NHE3区分为RAFT相关和非RAFT NHERF介导的蛋白质复合体的作用，以及STA诱导的NHE3调控的分子机制是否在小鼠和人的肠刷状缘相似。

The Na+/H+ exchanger isoform 3 is the most important intestinal Na+ absorptive pathway. NHE3 has a long cytoplasmic tail which harbors multiple binding sites for regulatory proteins as well as phosphorylation consensus sequences for protein kinases. The NHERF (Na+/H+ regulatory factor) family of PDZ-adaptor proteins has a prominent role in mediating the es-tablishment of NHE3 multiprotein complex formation between the transporter, receptors, an-chor proteins, protein kinases and the cytoskeleton. While the formation of simple NHERF-NHE3-anchor protein-protein kinase complexes has been documented in heterologous expres-sion systems, information about the regulatory role of the NHERF proteins in native tissues is scarce. Of prime importance for both a better understanding of the molecular physiology of NHE3 regulation as well as the therapeutic potential of targeting NHERF interactions is a better understanding of how selectivity and privacy (containment of the signaling event at the site of target localization, avoidance of off-target effects) of the interaction between one of the NHERFs, NHE3 and the signaling molecules within a given receptor-activated signaling pathway is established. One potential mechanism for signal specificity could be the cosegregation of NHE3 and inter-acting proteins within membrane microdomains, also called lipid rafts. These sphingolipid-rich domains are regions of Protein clustering within the plasma membrane and can thus act as interaction platforms. We recently demonstrated that the NHERFs differentially associate with the lipid raft and the nonraft domains within the intestinal brush border membrane. By using knockout mice for the respective NHERFs, we also found that the differential NHERF distribution determines the distribution of NHE3 to the rafts. In the proposed project, we aim to study the dynamic interaction between NHE3, the NHERFs, the signaling molecules of the cGMP signaling pathway and the lipid rafts in the murine intestinal brush border membrane in vivo. After the establishment and characterization of human intestinal organoid (HIO) culture, we will investigate whether the lipid raft platforms of human intestinal brush border membrane also serves to differentially segregate NHE3 to raft-associated and nonraft NHERF-mediated protein complexes, and whether the molecular mechanisms i.e. in STa-induced NHE3 regulation are similar in the mouse and human intesti-nal brush border.