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]

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Summary

Introduction

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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Conclusion

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