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

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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.