scholarly journals Characterization of the interaction between β-catenin and sorting nexin 27: contribution of the type I PDZ-binding motif to Wnt signaling

2019 ◽  
Vol 39 (11) ◽  
Author(s):  
Brian J. DuChez ◽  
Christina L. Hueschen ◽  
Seth P. Zimmerman ◽  
Yvonne Baumer ◽  
Stephen Wincovitch ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Collecting Duct ◽  
Pdz Domain ◽  
Scaffolding Protein ◽  
Binding Motif ◽  
Type I ◽  
Cortical Collecting Duct ◽  
Fusion Construct ◽  
Sorting Nexin ◽  
Early Endosomes

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.

scholarly journals Sorting nexin 27 (SNX27) associates with zonula occludens-2 (ZO-2) and modulates the epithelial tight junction

2013 ◽  
Vol 455 (1) ◽  
pp. 95-106 ◽  
Author(s):  
Seth P. Zimmerman ◽  
Christina L. Hueschen ◽  
Daniela Malide ◽  
Sharon L. Milgram ◽  
Martin P. Playford
Keyword(s):  
Tight Junction ◽  
Collecting Duct ◽  
Pdz Domain ◽  
Binding Motif ◽  
Sorting Nexin ◽  
Zonula Occludens ◽  
Proteomic Approach ◽  
Early Endosomes ◽  
Junction Protein ◽  
Epithelial Tight Junction

Proteins of the SNX (sorting nexin) superfamily are characterized by the presence of a PX (Phox homology) domain and associate with PtdIns3P (phosphatidylinositol-3-monophosphate)-rich regions of the endosomal system. SNX27 is the only sorting nexin that contains a PDZ domain. In the present study, we used a proteomic approach to identify a novel interaction between SNX27 and ZO-2 [zonula occludens-2; also known as TJP2 (tight junction protein 2)], a component of the epithelial tight junction. The SNX27–ZO-2 interaction requires the PDZ domain of SNX27 and the C-terminal PDZ-binding motif of ZO-2. When tight junctions were perturbed by chelation of extracellular Ca2+, ZO-2 transiently localized to SNX27-positive early endosomes. Depletion of SNX27 in mpkCCD (mouse primary kidney cortical collecting duct) cell monolayers resulted in a decrease in the rate of ZO-2, but not ZO-1, mobility at cell–cell contact regions after photobleaching and an increase in junctional permeability to large solutes. The findings of the present study identify an important new SNX27-binding partner and suggest a role for endocytic pathways in the intracellular trafficking of ZO-2 and possibly other tight junction proteins. Our results also indicate a role for SNX27–ZO-2 interactions in tight junction maintenance and function.

Evidence of corticosteroid action along the nephron

1984 ◽  
Vol 246 (2) ◽  
pp. F111-F123 ◽  
Author(s):  
D. Marver
Keyword(s):  
Collecting Duct ◽  
Type I ◽  
Type Ii ◽  
Cortical Collecting Duct ◽  
Distal Nephron ◽  
Type Iii ◽  
Collecting Ducts ◽  
Collecting Tubule ◽  
Cortical Collecting Tubule

The kidney contains three classes of corticosteroid-binding proteins receptors. They include a mineralocorticoid-specific (Type I), a glucocorticoid-specific (Type II), and a corticosterone-specific (Type III) site. The Type I and Type III sites roughly parallel each other along the nephron, with maximal binding occurring in the late distal convoluted or connecting segment and the cortical and medullary collecting ducts. Type II sites occur throughout the nephron, with maximal concentrations appearing in the proximal tubule and the late distal convoluted-cortical collecting duct region. The function of the Type I sites in the connecting segment is unclear since chronic mineralocorticoid therapy does not influence the potential difference in this segment as it does in the cortical collecting tubule. Furthermore, the specific role of Type II versus Type III sites in the distal nephron is unknown. Finally, the possible influence of sodium on both latent and steroid-induced renal cortical and medullary Na-K-ATPase is discussed.

scholarly journals Regulation of the endosomal SNX27-retromer by OTULIN

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Aurelia Stangl ◽  
Paul R. Elliott ◽  
Adan Pinto-Fernandez ◽  
Sarah Bonham ◽  
Luke Harrison ◽  
...  
Keyword(s):  
Cell Surface ◽  
Membrane Trafficking ◽  
Pdz Domain ◽  
Binding Motif ◽  
Ubiquitin Chain ◽  
Sorting Nexin ◽  
Catalytic Function ◽  
Retromer Complex ◽  
Mass Spectrometric Identification ◽  
Affinity Interaction

Abstract OTULIN (OTU Deubiquitinase With Linear Linkage Specificity) specifically hydrolyzes methionine1 (Met1)-linked ubiquitin chains conjugated by LUBAC (linear ubiquitin chain assembly complex). Here we report on the mass spectrometric identification of the OTULIN interactor SNX27 (sorting nexin 27), an adaptor of the endosomal retromer complex responsible for protein recycling to the cell surface. The C-terminal PDZ-binding motif (PDZbm) in OTULIN associates with the cargo-binding site in the PDZ domain of SNX27. By solving the structure of the OTU domain in complex with the PDZ domain, we demonstrate that a second interface contributes to the selective, high affinity interaction of OTULIN and SNX27. SNX27 does not affect OTULIN catalytic activity, OTULIN-LUBAC binding or Met1-linked ubiquitin chain homeostasis. However, via association, OTULIN antagonizes SNX27-dependent cargo loading, binding of SNX27 to the VPS26A-retromer subunit and endosome-to-plasma membrane trafficking. Thus, we define an additional, non-catalytic function of OTULIN in the regulation of SNX27-retromer assembly and recycling to the cell surface.

scholarly journals Cx50 requires an intact PDZ-binding motif and ZO-1 for the formation of functional intercellular channels

2011 ◽  
Vol 22 (23) ◽  
pp. 4503-4512 ◽  
Author(s):  
Zhifang Chai ◽  
Daniel A. Goodenough ◽  
David L. Paul
Keyword(s):  
Gap Junction ◽  
Hela Cells ◽  
Pdz Domain ◽  
Cell Communication ◽  
Normal Function ◽  
Scaffolding Protein ◽  
Binding Motif ◽  
Interacting Protein ◽  
And Function

The three connexins expressed in the ocular lens each contain PDZ domain–binding motifs directing a physical association with the scaffolding protein ZO-1, but the significance of the interaction is unknown. We found that Cx50 with PDZ-binding motif mutations did not form gap junction plaques or induce cell–cell communication in HeLa cells, whereas the addition of a seven–amino acid PDZ-binding motif restored normal function to Cx50 lacking its entire C-terminal cytoplasmic domain. C-Terminal deletion had a similar although weaker effect on Cx46 but little if any effect on targeting and function of Cx43. Furthermore, small interfering RNA knockdown of ZO-1 completely inhibited the formation of gap junctions by wild-type Cx50 in HeLa cells. Thus both a PDZ-binding motif and ZO-1 are necessary for Cx50 intercellular channel formation in HeLa cells. Knock-in mice expressing Cx50 with a PDZ-binding motif mutation phenocopied Cx50 knockouts. Furthermore, differentiating lens fibers in the knock-in displayed extensive intracellular Cx50, whereas plaques in mature fibers contained only Cx46. Thus normal Cx50 function in vivo also requires an intact PDZ domain–binding motif. This is the first demonstration of a connexin-specific requirement for a connexin-interacting protein in gap junction assembly.

scholarly journals SNX27 suppresses SARS-CoV-2 infection by inhibiting viral lysosome/late endosome entry

2022 ◽  
Vol 119 (4) ◽  
pp. e2117576119
Author(s):  
Bo Yang ◽  
Yuanyuan Jia ◽  
Yumin Meng ◽  
Ying Xue ◽  
Kefang Liu ◽  
...  
Keyword(s):  
Cell Surface ◽  
Host Cell ◽  
Viral Entry ◽  
Complex Structure ◽  
Pdz Domain ◽  
Late Endosome ◽  
Endocytic Pathway ◽  
Cell Surface Receptor ◽  
Binding Motif ◽  
Type I

After binding to its cell surface receptor angiotensin converting enzyme 2 (ACE2), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the host cell through directly fusing with plasma membrane (cell surface pathway) or undergoing endocytosis traveling to lysosome/late endosome for membrane fusion (endocytic pathway). However, the endocytic entry regulation by host cell remains elusive. Recent studies show ACE2 possesses a type I PDZ binding motif (PBM) through which it could interact with a PDZ domain-containing protein such as sorting nexin 27 (SNX27). In this study, we determined the ACE2-PBM/SNX27-PDZ complex structure, and, through a series of functional analyses, we found SNX27 plays an important role in regulating the homeostasis of ACE2 receptor. More importantly, we demonstrated SNX27, together with retromer complex (the core component of the endosomal protein sorting machinery), prevents ACE2/virus complex from entering lysosome/late endosome, resulting in decreased viral entry in cells where the endocytic pathway dominates. The ACE2/virus retrieval mediated by SNX27–retromer could be considered as a countermeasure against invasion of ACE2 receptor-using SARS coronaviruses.

Cellular origin and hormonal regulation of K+-ATPase activities sensitive to Sch-28080 in rat collecting duct

2000 ◽  
Vol 279 (6) ◽  
pp. F1053-F1059 ◽  
Author(s):  
Nicolas Laroche-Joubert ◽  
Sophie Marsy ◽  
Alain Doucet
Keyword(s):  
Hormonal Regulation ◽  
Collecting Duct ◽  
Type I ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Type Iii ◽  
Principal Cells ◽  
Cellular Origin ◽  
Hormone Effects ◽  
A Cell

Rat collecting ducts exhibit type I or type III K+-ATPase activities when animals are fed a normal (NK) or a K+-depleted diet (LK). This study aimed at determining functionally the cell origin of these two K+-ATPases. For this purpose, we searched for an effect on K+-ATPases of hormones that trigger cAMP production in a cell-specific fashion. The effects of 1-deamino-8-d-arginine vasopressin (dD-AVP), calcitonin, and isoproterenol in principal cells, α-intercalated cells, and β-intercalated cells of cortical collecting duct (CCD), respectively, and of dD-AVP and glucagon in principal and α-intercalated cells of outer medullary collecting duct (OMCD), respectively, were examined. In CCDs, K+-ATPase was stimulated by calcitonin and isoproterenol in NK rats (type I K+-ATPase) and by dD-AVP in LK rats (type III K+-ATPase). In OMCDs, dD-AVP and glucagon stimulated type III but not type I K+-ATPase. These hormone effects were mimicked by the cAMP-permeant analog dibutyryl-cAMP. In conclusion, in NK rats, cAMP stimulates type I K+-ATPase activity in α- and β-intercalated CCD cells, whereas in LK rats it stimulates type III K+-ATPase in principal cells of both CCD and OMCD and in OMCD intercalated cells.

scholarly journals Insights into acidosis-induced regulation of SLC26A4 (pendrin) and SLC4A9 (AE4) transporters using three-dimensional morphometric analysis of β-intercalated cells

2014 ◽  
Vol 307 (5) ◽  
pp. F601-F611 ◽  
Author(s):  
Jeffrey M. Purkerson ◽  
Eric V. Heintz ◽  
Aya Nakamori ◽  
George J. Schwartz
Keyword(s):  
Collecting Duct ◽  
Three Dimensional ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Acid Base ◽  
Recycling Endosomes ◽  
Coordinate Regulation ◽  
Anion Transporters ◽  
Early Endosomes ◽  
Zona Occludens

The purpose of this study was to examine the three-dimensional (3-D) expression and distribution of anion transporters pendrin (SLC26A4) and anion exchanger (AE)4 (SLC4A9) in β-intercalated cells (β-ICs) of the rabbit cortical collecting duct (CCD) to better characterize the adaptation to acid-base disturbances. Confocal analysis and 3-D reconstruction of β-ICs, using identifiers of the nucleus and zona occludens, permitted the specific orientation of cells from normal, acidotic, and recovering rabbits, so that adaptive changes could be quantified and compared. The pendrin cap likely mediates apical Cl−/HCO3− exchange, but it was also found beneath the zona occludens and in early endosomes, some of which may recycle back to the apical membrane via Rab11a+ vesicles. Acidosis reduced the size of the pendrin cap, observed as a large decrease in cap volume above and below the zona occludens, and the volume of the Rab11a+ apical recycling compartment. Correction of the acidosis over 12–18 h reversed these changes. Consistent with its proposed function in the basolateral exit of Na+ via Na+-HCO3− cotransport, AE4 was expressed as a barrel-like structure in the lateral membrane of β-ICs. Acidosis reduced AE4 expression in β-ICs, but this was rapidly reversed during the recovery from acidosis. The coordinate regulation of pendrin and AE4 during acidosis and recovery is likely to affect the magnitude of acid-base and possibly Na+ transport across the CCD. In conclusion, acidosis induces a downregulation of AE expression in β-ICs and a diminished presence of pendrin in apical recycling endosomes.

scholarly journals Chronic hypoxia-induced acid-sensitive ion channel expression in chemoafferent neurons contributes to chemoreceptor hypersensitivity

2011 ◽  
Vol 301 (6) ◽  
pp. L985-L992 ◽  
Author(s):  
X. Liu ◽  
L. He ◽  
B. Dinger ◽  
S. J. Fidone
Keyword(s):  
Inflammatory Cytokines ◽  
Carotid Body ◽  
Chronic Hypoxia ◽  
Immune Cell ◽  
Pdz Domain ◽  
Scaffolding Protein ◽  
Electrophysiological Recording ◽  
Type I ◽  
Type I Cells ◽  
I Cells

Previously we demonstrated that chronic hypoxia (CH) induces an inflammatory condition characterized by immune cell invasion and increased expression of inflammatory cytokines in rat carotid body. It is well established that chronic inflammatory pain induces the expression of acid-sensitive ion channels (ASIC) in primary sensory neurons, where they contribute to hyperalgesia and allodynia. The present study examines the effect of CH on ASIC expression in petrosal ganglion (PG), which contains chemoafferent neurons that innervate oxygen-sensitive type I cells in the carotid body. Five isoforms of ASIC transcript were increased ∼1.5–2.5-fold in PG following exposure of rats to 1, 3, or 7 days of hypobaric hypoxia (380 Torr). ASIC transcript was not increased in the sympathetic superior cervical ganglion (SCG). In the PG, CH also increased the expression of channel-interacting PDZ domain protein, a scaffolding protein known to enhance the surface expression and the low pH-induced current density mediated by ASIC3. Western immunoblot analysis showed that CH elevated ASIC3 protein in PG, but not in SCG or the (sensory) nodose ganglion. ASIC3 transcript was likewise elevated in PG neurons cultured in the presence of inflammatory cytokines. Increased ASIC expression was blocked in CH rats concurrently treated with the nonsteroidal anti-inflammatory drug ibuprofen (4 mg·kg−1·day−1). Electrophysiological recording of carotid sinus nerve (CSN) activity in vitro showed that the specific ASIC antagonist A-317567 (100 μM) did not significantly alter hypoxia-evoked activity in normal preparations but blocked ∼50% of the hypoxic response following CH. Likewise, a high concentration of ibuprofen, which is known to block ASIC1a, reduced hypoxia-evoked CSN activity by ∼50% in CH preparations. Our findings indicate that CH induces inflammation-dependent phenotypic adjustments in chemoafferent neurons. Following CH, ASIC are important participants in chemotransmission between type I cells and chemoafferent nerve terminals, and these proton-gated channels appear to enhance chemoreceptor sensitivity.

scholarly journals Sorting Nexin 27 (SNX27): A Novel Regulator of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Trafficking

2018 ◽  
Author(s):  
Mark I. McDermott ◽  
William R. Thelin ◽  
Yun Chen ◽  
Patrick T. Lyons ◽  
Gabrielle Reilly ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Pdz Domain ◽  
Type I ◽  
Apical Surface ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Sorting Nexin ◽  
Cystic Fibrosis Transmembrane

AbstractThe underlying defect in cystic fibrosis is mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel expressed at the apical surface of lung epithelia. In addition to its export and maintenance at the cell surface, CFTR regulation involves repeated cycles of transport through the endosomal trafficking system, including endocytosis and recycling. Many of the known disease mutations cause CFTR intracellular trafficking defects that result in failure of ion channel delivery to the apical plasma membrane. Corrective maneuvers directed at improving transport to the plasma membrane are thwarted by rapid internalization and degradation of the mutant CFTR proteins. The molecular mechanisms involved in these processes are not completely understood but may involve protein-protein interactions with the C-terminal type I PDZ-binding motif of CFTR. Using a proteomic approach, we identify sorting nexin 27 (SNX27) as a novel CFTR binding partner in human airway epithelial Calu-3 cells. SNX27 and CFTR interact directly, with the SNX27 PDZ domain being both necessary and sufficient for this interaction. SNX27 co-localizes with internalized CFTR at sub-apical endosomal sites in polarized Calu-3 cells, and either knockdown of the endogenous SNX27, or over-expression of a dominant-negative SNX27 mutant, resulted in significant decreases in cell surface CFTR levels. CFTR internalization was not affected by SNX27 knockdown, but defects were observed in the recycling arm of CFTR trafficking through the endosomal system. Furthermore, knockdown of SNX27 in Calu-3 cells resulted in significant decreases in CFTR protein levels, consistent with degradation of the internalized pool. These data identify SNX27 as a physiologically significant regulator of CFTR trafficking and homeostasis in epithelial cells.

Abstract 134: Activation of the Mineralocorticoid Receptor by O-glycosylation Through Hyperglycemia

Hypertension ◽  
2015 ◽  
Vol 66 (suppl_1) ◽  
Author(s):  
Maniselvan Kuppusamy ◽  
Elise Gomez-Sanchez ◽  
Celso Gomez-Sanchez
Keyword(s):  
High Glucose ◽  
Mineralocorticoid Receptor ◽  
Transcriptional Activity ◽  
Collecting Duct ◽  
Cardiovascular Complications ◽  
Luciferase Reporter ◽  
Cortical Collecting Duct ◽  
Luciferase Reporter Gene ◽  
Reporter Activity ◽  
The Metabolic Syndrome

Introduction: Hypertension and diabetes are independent risk factors for cardiovascular disease, however why they frequently occur together is not clear. Inappropriate mineralocorticoid receptor (MR) activation is associated with diabetes and the metabolic syndrome, as well as hypertension and abnormal cardiovascular remodeling. MR antagonists are effective in reducing hypertension and delaying the onset of renal and cardiovascular complications in diabetes despite circulating aldosterone levels that are within normal limits. Glucose concentrations increase O-glycosylation of many proteins, thus alter their function. Hence, we hypothesized that increased O-GlcNac modification of the MR by high glucose enhances MR activation. Methods: MR transcriptional activity was studied in a mouse cortical collecting duct (M1) cell line stably transfected with a cDNA construct including the MR and one with a hormone-response element driving a Gaussia luciferase reporter gene. The cells were incubated for 48 h with low (5mM) or high (25mM) glucose media with and without Thiamet-G (TMG), an O-GlcNAcase inhibitor to inhibit deglycosylation, and 6-diazo-5-oxonorleucine (DON), a glucosamine-fructose-6-phosphate amidotransferase inhibitor (GFAT) to reduce O-GlcNAc levels. Additionally, MR and GR antagonists were used to identify receptor specificity under low and high glucose conditions. O-GlcNac-modified MR was co-immunoprecipitated with an MR antibody and detected with an O-GlcNAc antibody. Results: 1. Co-immunoprecipitation assays showed that high glucose and TMG increased O-GlcNac-MR by 3-fold. 2. Compared to low glucose, treatment with high glucose and with TMG increased the transcriptional activity of MR by 300%. 3. DON decreased MR-reporter activity by 75%. 4. High glucose alone had no significant basal effect but significantly increased MR activation by aldosterone. 5. MR reporter activity was increased similarly by aldosterone and corticosterone. Conclusion: High glucose increased glycosylation of the MR, augmenting its transcriptional activity. Enhancement of MR activation by hyperglycemia may explain how MRs play a significant role in the cardiorenal pathology in Diabetes.

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