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

Na+/H+交换器亚型3是小肠Na+吸收的最重要途径。NHE 3具有长的胞质尾，其具有调节蛋白的多个结合位点以及蛋白激酶的磷酸化共有序列。PDZ-接头蛋白NHERF（Na+/H+调节因子）家族在介导NHE 3多蛋白复合物的形成中具有重要作用，该复合物是转运蛋白、受体、锚蛋白、蛋白激酶和细胞骨架之间的复合物。虽然在异源表达系统中已经记录了简单的NHERF-NHE 3-锚蛋白-蛋白激酶复合物的形成，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NHE 3调节的分子生理学以及靶向NHERF相互作用的治疗潜力来说，最重要的是更好地理解选择性和隐私性如何影响NHE 3调节。（在靶定位位点抑制信号传导事件，避免脱靶效应），NHE 3和信号分子在给定的受体激活的信号传导途径内建立。信号特异性的一个潜在机制可能是NHE 3和膜微区（也称为脂筏）内相互作用蛋白的共分离。这些富含鞘脂的结构域是质膜内蛋白质聚集的区域，因此可以充当相互作用平台。我们最近证明，NHERFs差异与脂筏和非筏结构域内肠刷状缘膜。通过使用敲除小鼠的各个NHERF，我们还发现，差异NHERF分布决定了NHE 3的筏的分布。本研究的目的是研究NHE 3、NHERFs、cGMP信号通路中的信号分子和小鼠小肠刷状缘膜脂筏之间的动态相互作用。在人肠类器官（HIO）培养的建立和表征之后，我们将研究人肠刷状缘膜的脂筏平台是否也用于将NHE 3差异地分离为筏相关和非筏NHERF介导的蛋白复合物，以及在小鼠和人肠刷状缘中STa诱导的NHE 3调节的分子机制是否相似。

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.