Abstract
Background: Sorting Nexin 27 (SNX27) is a 62-kDa protein localized to early endosomes and known to regulate the intracellular trafficking of ion channels and receptors. In addition to a PX domain common among all of the sorting nexin family, SNX27 is the only sorting family member that contains a PDZ domain. To identify novel SNX27–PDZ binding partners, we performed a proteomic screen in mouse principal kidney cortical collecting duct cells (mpkCCD) using a GST-SNX27 fusion construct as bait. We found that the C-terminal type I PDZ binding motif (DTDL) of β-catenin, an adherens junction scaffolding protein and transcriptional co-activator, interacts directly with SNX27. Using biochemical and immunofluorescent techniques, β-catenin was identified in endosomal compartments where co-localization with SNX27 was observed. Furthermore, E-cadherin, but not Axin, GSK3 or Lef-1 was located in SNX27 protein complexes. While overexpression of wild-type β-catenin protein increased TCF-LEF dependent transcriptional activity, an enhanced transcriptional activity was not observed in cells expressing β-Catenin ΔFDTDL or diminished SNX27 expression.These results imply importance of the C-terminal PDZ binding motif for the transcriptional activity of β-catenin and propose that SNX27 might be involved in the assembly of β-catenin complexes in the endosome.
Highlights
Many signaling pathways are regulated by endocytosis
The endosomal network is composed of several intracellular compartments that begin at the plasma membrane where cargo such as a ligand-bound receptor or ion channel is internalized into the early sorting endosome [1,2,3]
Lysates from mouse principal kidney cortical collecting duct cells (mpkCCD) cells were incubated with GST alone, GST-PDZ or a mutant PDZ protein where a conserved G G (GYGF in Sorting Nexin 27 (SNX27)) is mutated to GGGG as previously described [11]
Summary
Many signaling pathways are regulated by endocytosis. This process plays a critical role in signaling component activation, transduction of downstream signals and signal termination. Each sorting nexin (SNX) contains a phox homology (PX) domain that binds phosphoinositide-enriched membranes [4,5,7]. Sorting Nexin 27 (SNX27) is a 62-kDa protein localized to early endosomes and known to regulate the intracellular trafficking of ion channels and receptors. While overexpression of wild-type β-catenin protein increased TCF-LEF dependent transcriptional activity, an enhanced transcriptional activity was not observed in cells expressing β-Catenin FDTDL or diminished SNX27 expression. These results imply importance of the C-terminal PDZ binding motif for the transcriptional activity of β-catenin and propose that SNX27 might be involved in the assembly of β-catenin complexes in the endosome
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