[Show abstract][Hide abstract] ABSTRACT: Shank2 is a PDZ-based adaptor that has been suggested to regulate membrane transporting proteins in the brain and epithelial tissues. Here, we report that Shank2-mutant (Shank2-/-) mice exhibit aberrant fluid and ion transport in the intestine. Molecular characterization using epithelial tissues from Shank2+/+ and Shank2-/- mice revealed that a long spliceoform of Shank2 (Shank2E) is predominantly expressed in the pancreatic, renal, and intestinal epithelia. In functional assays, deletion of Shank2 increased the cystic fibrosis transmembrane conductance regulator (CFTR)-dependent short-circuit currents by 84% (P < 0.05) and 101% (P < 0.05) in the mouse colon and rectum, respectively. Disruption of the CFTR-Shank2-phosphodiesterase 4D protein complex appeared to be mostly responsible for the changes in the CFTR activities. Notably, Shank2 deletion profoundly increased cholera toxin-induced fluid accumulation in the mouse intestine (~90%, P < 0.01). Analyses with chemical inhibitors confirmed that the hyperactivation of CFTR channel function is responsible for the increased response to cholera toxin. These results suggest that Shank2 is a key molecule that participates in epithelial homeostasis, in particular to prevent overt secretory responses caused by epithelial pathogens.
No preview · Article · Jan 2014 · The Journal of Physiology
[Show abstract][Hide abstract] ABSTRACT: Na(+)/H(+) exchanger regulatory factor 3 (NHERF3) is a PSD-95/discs large/ZO-1 (PDZ)-based adaptor protein that regulates several membrane-transporting proteins in epithelia. However, the in vivo physiologic role of NHERF3 in transepithelial transport remains poorly understood. Multidrug resistance protein 4 (MRP4) is an ATP binding cassette transporter that mediates the efflux of organic molecules, such as nucleoside analogs, in the gastrointestinal and renal epithelia. Here, we report that Nherf3 knockout (Nherf3(-/-)) mice exhibit profound reductions in Mrp4 expression and Mrp4-mediated drug transport in the kidney. A search for the binding partners of the COOH-terminal PDZ binding motif of MRP4 among several epithelial PDZ proteins indicated that MRP4 associated most strongly with NHERF3. When expressed in HEK293 cells, NHERF3 increased membrane expression of MRP4 by reducing internalization of cell surface MRP4 and consequently, augmented MRP4-mediated efflux of adefovir, a nucleoside-based antiviral agent and well known substrate of MRP4. Examination of wild-type and Nherf3(-/-) mice revealed that Nherf3 is most abundantly expressed in the kidney and has a prominent role in modulating Mrp4 levels. Deletion of Nherf3 in mice caused a profound reduction in Mrp4 expression at the apical membrane of renal proximal tubules and evoked a significant increase in the plasma and kidney concentrations of adefovir, with a corresponding decrease in the systemic clearance of this drug. These results suggest that NHERF3 is a key regulator of organic transport in the kidney, particularly MRP4-mediated clearance of drug molecules.
No preview · Article · Jan 2014 · Journal of the American Society of Nephrology
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: The atopic diseases, which are the most common chronic diseases of childhood, are complex genetic diseases that involve the contribution of multiple genetic factors to disease pathophysiology. Chitotriosidase is involved in innate immunity, but the association of chitotriosidase with allergic diseases remains unclear. OBJECTIVE: To examine the contribution of genetic variation of the chitotriosidase-encoding gene CHIT1 to atopic phenotypes in a Korean cohort of children. METHODS: We identified CHIT1 variations in a Korean population and conducted association analyses using 295 atopic and 242 nonatopic children. An independent replication study was performed using DNA samples from 148 atopic and 243 nonatopic children. All children were unrelated. We performed Western blot analysis in each genotype in vitro to see whether the CHIT1 A442G variation affects the final protein expression levels. RESULTS: In the case-control association analysis, atopy was significantly associated with a single A442G (rs1065761) polymorphism in CHIT1 (odds ratio = 1.32, P = .01). Children with the c.442G risk allele had significantly higher blood eosinophils (P = .001), total serum IgE (P = .007), and eosinophil cationic protein (P = .02) levels. The results of the replication stage analysis confirmed a significant association between the A442G polymorphism and childhood atopy. The joint analysis of the exploratory and replication studies displayed a stronger significant association. The relative protein expression levels of chitotriosidase were significantly higher in both cell lysate and media with the G transfection compared with the wild type. CONCLUSION: These results indicate that the nonsynonymous A442G polymorphism in CHIT1 is associated with risk of atopy.
No preview · Article · Jun 2013 · Annals of allergy, asthma & immunology: official publication of the American College of Allergy, Asthma, & Immunology
[Show abstract][Hide abstract] ABSTRACT: SLC26A3 functions as a chloride/bicarbonate anion exchanger expressed in the secretory epithelial cells in the intestine, pancreas, and salivary glands. SLC26A3 has a C-terminal class I PDZ binding motif that assembles regulatory factors or other transporters by anchoring to various PDZ scaffold proteins. NHERF4 is an epithelial-enriched PDZ domain scaffold protein that has attracted attention because of its enriched tissue expression in the intestine and kidney. In this study, we identified SLC26A3 as a novel binding transporter of NHERF4. We investigated the functional role of NHERF4 in the regulation of SLC26A3 by using integrated biochemical and physiological approaches. A direct protein-protein interaction was identified between the PDZ-binding motif of SLC26A3 and the third PDZ domain of NHERF4. Interaction with NHERF4 decreased the level of SLC26A3 expression on the plasma membrane, which led to reduced SLC26A3 anion exchange activity. Notably, interaction with NHERF4 induced rapid internalisation of SLC26A3 from the plasma membrane. The SLC26A3-NHERF4 interaction was modulated by phosphorylation; serine 329 of NHERF4-PDZ3 played a critical role in modulating binding selectivity. Our findings suggest that NHERF4 is a novel modulator of luminal fluidity in the intestine by adjusting SLC26A3 expression and activity through a phosphorylation-dependent mechanism.
No preview · Article · May 2012 · Cellular Signalling