Sorting Nexin 27 Protein Regulates Trafficking of a p21-activated Kinase (PAK) Interacting Exchange Factor ( -Pix)-G Protein-coupled Receptor Kinase Interacting Protein (GIT) Complex via a PDZ Domain Interaction

Cell Biology and Physiology Center, NHLBI, National Institutes of Health, Bethesda, Maryland 20982, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2011; 286(45):39403-16. DOI: 10.1074/jbc.M111.260802
Source: PubMed


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, 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 using a GST-SNX27 fusion construct as bait. We found that β-Pix (p21-activated kinase-interactive exchange factor),
a guanine nucleotide exchange factor for the Rho family of small GTPases known to regulate cell motility directly interacted
with SNX27. The association of β-Pix and SNX27 is specific for β-Pix isoforms terminating in the type-1 PDZ binding motif
(ETNL). In the same screen we also identified Git1/2 as a potential SNX27 interacting protein. The interaction between SNX27
and Git1/2 is indirect and mediated by β-Pix. Furthermore, we show recruitment of the β-Pix·Git complex to endosomal sites
in a SNX27-dependent manner. Finally, migration assays revealed that depletion of SNX27 from HeLa and mouse principal kidney
cortical collecting duct cells significantly decreases cell motility. We propose a model by which SNX27 regulates trafficking
of β-Pix to focal adhesions and thereby influences cell motility.

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    • "SNX27 has also been annotated to possess a Ras-association domain and an extended C-terminal region (Seet and Hong 2006; Cullen 2008). Relevant to NHE3, SNX27 has been implicated in mediating PDZdirected sorting of proteins from endosomes to the plasma membrane (Joubert et al., 2004; Lunn et al., 2007; Lauffer et al., 2010; Cai et al., 2011; Valdes et al., 2011). "
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    ABSTRACT: Sorting Nexin 27 (SNX27) contains a PDZ domain which is phylogenetically related to the PDZ domain of the NHERF proteins. Studies on non-epithelial cells have shown that this protein is located in endosomes where it regulates trafficking of cargo proteins in a PDZ domain dependent manner. However the role of SNX27 in trafficking of cargo proteins in epithelial cells has not been adequately explored. Here we show that SNX27 directly interacts with NHE3 (C-terminus) which is primarily through SNX27 PDZ domain. A combination of knock down and reconstitution experiments with wild type or a PDZ domain mutant (GYGF→GAGA) of SNX27 demonstrated that the PDZ domain of SNX27 is required to maintain basal NHE3 activity and surface expression of NHE3 in polarized epithelial cells. Biotinylation based recycling and degradation studies in intestinal epithelial cells showed that SNX27 is required for the exocytosis (not endocytosis) of NHE3 from early endosome to plasma membrane. SNX27 is also required to regulate the retention of NHE3 on the plasma membrane. The findings of the present study extend our understanding of PDZ mediated recycling of cargo proteins from endosome to plasma membrane in epithelial cells. © 2015 by The American Society for Cell Biology.
    Full-text · Article · Apr 2015 · Molecular biology of the cell
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    • "Functionally, SNX17 promotes recycling and suppresses the degradation of its membrane cargo proteins [60], [61]. In the case of SNX27, it contains an additional PDZ domain that targets proteins containing a PDZ-binding motif, including beta-Pix (p21-activated kinase-interactive exchange factor) [62] and multidrug resistance-associated protein 4 (MRP4/ABCC4) [63]. Functionally, SNX27 in general enhances the recycling of plasma membrane (cf. "
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    ABSTRACT: Uroplakins (UP), a group of integral membrane proteins, are major urothelial differentiation products that form 2D crystals of 16-nm particles (urothelial plaques) covering the apical surface of mammalian bladder urothelium. They contribute to the urothelial barrier function and, one of them, UPIa, serves as the receptor for uropathogenic Escherichia coli. It is therefore important to understand the mechanism by which these surface-associated uroplakins are degraded. While it is known that endocytosed uroplakin plaques are targeted to and line the multivesicular bodies (MVBs), it is unclear how these rigid-looking plaques can go to the highly curved membranes of intraluminal vesicles (ILVs). From a cDNA subtraction library, we identified a highly urothelium-specific sorting nexin, SNX31. SNX31 is expressed, like uroplakins, in terminally differentiated urothelial umbrella cells where it is predominantly associated with MVBs. Apical membrane proteins including uroplakins that are surface biotin-tagged are endocytosed and targeted to the SNX31-positive MVBs. EM localization demonstrated that SNX31 and uroplakins are both associated not only with the limiting membranes of MVBs containing uroplakin plaques, but also with ILVs. SNX31 can bind, on one hand, the PtdIns3P-enriched lipids via its N-terminal PX-domain, and, on the other hand, it binds uroplakins as demonstrated by co-immunoprecipitation and proximity ligation assay, and by its reduced membrane association in uroplakin II-deficient urothelium. The fact that in urothelial umbrella cells MVBs are the only major intracellular organelles enriched in both PtdIns3P and uroplakins may explain SNX31's MVB-specificity in these cells. However, in MDCK and other cultured cells transfected SNX31 can bind to early endosomes possibly via lipids. These data support a model in which SNX31 mediates the endocytic degradation of uroplakins by disassembling/collapsing the MVB-associated uroplakin plaques, thus enabling the uroplakin-containing (but 'softened') membranes to bud and form the ILVs for lysosomal degradation and/or exosome formation.
    Full-text · Article · Jun 2014 · PLoS ONE
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    ABSTRACT: Multidrug resistance-associated protein 4 (MRP4/ABCC4) makes a vital contribution to the bodily distribution of drugs and endogenous compounds because of its cellular efflux abilities. However, little is known about the mechanism regulating its cell surface expression. MRP4 has a PDZ-binding motif, which is a potential sequence that modulates the membrane expression of MRP4 via interaction with PDZ adaptor proteins. To investigate this possible relationship, we performed GST pull-down assays and subsequent analysis with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. This method identified sorting nexin 27 (SNX27) as the interacting PDZ adaptor protein with a PDZ-binding motif of MRP4. Its interaction was confirmed by a coimmunoprecipitation study using HEK293 cells. Knockdown of SNX27 by siRNA in HEK293 cells raised MRP4 expression on the plasma membrane, increased the extrusion of 6-[(14)C]mercaptopurine, an MRP4 substrate, and conferred resistance against 6-[(14)C]mercaptopurine. Cell surface biotinylation studies indicated that the inhibition of MRP4 internalization was responsible for these results. Immunocytochemistry and cell surface biotinylation studies using COS-1 cells showed that SNX27 localized to both the early endosome and the plasma membrane. These data suggest that SNX27 interacts with MRP4 near the plasma membrane and promotes endocytosis of MRP4 and thereby negatively regulates its cell surface expression and transport function.
    No preview · Article · Mar 2012 · Journal of Biological Chemistry
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