Activity and PI3-kinase dependent trafficking of the intestinal anion exchanger downregulated in adenoma depend on its PDZ interaction and on lipid rafts.

Medical Dept., Univ. of Tübingen, Germany.
AJP Gastrointestinal and Liver Physiology (Impact Factor: 3.65). 10/2010; 299(4):G907-20. DOI: 10.1152/ajpgi.00191.2010
Source: PubMed

ABSTRACT The Cl/HCO(3) exchanger downregulated in adenoma (DRA) mediates electroneutral NaCl absorption in the intestine together with the apical Na/H exchanger NHE3. Lipid rafts (LR) modulate transport activity and are involved in phosphatidylinositol 3-kinase (PI3-kinase)-dependent trafficking of NHE3. Although DRA and NHE3 interact via PDZ adaptor proteins of the NHERF family, the role of LR and PDZ proteins in the regulation of DRA is unknown. We examined the association of DRA with LR using the nonionic detergent Triton X-100. DRA cofractionated with LR independently of its PDZ binding motif. Furthermore, DRA interacts with PDZK1, E3KARP, and IKEPP in LR, although their localization within lipid rafts is independent of DRA. Disruption of LR integrity resulted in the disappearance of DRA from LR, in a decrease of its surface expression and in a reduction of its activity. In HEK cells the inhibition of DRA by LR disruption was entirely dependent on the presence of the PDZ interaction motif. In addition, in Caco-2/BBE cells the inhibition by LR disruption was more pronounced in wild-type DRA than in mutated DRA (DRA-ETKFminus; lacking the PDZ binding motif)-expressing cells. Inhibition of PI3-kinase decreased the activity and the cell surface expression of wild-type DRA but not of DRA-ETKFminus; the partitioning into LR was unaffected. Furthermore, simultaneous inhibition of PI3-kinase and disruption of LR did not further decrease DRA activity and cell surface expression compared with LR disruption only. These results suggest that the activity of DRA depends on its LR association, on its PDZ interaction, and on PI3-kinase activity.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Background/Aims: Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na(+)/H(+) exchanger NHE3 is regulated by the Na(+)/H(+) Exchanger Regulatory Factor (NHERF) family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of these NHERFs. Thus, we studied the association of NHE3, NHERF1 (EBP50), NHERF2 (E3KARP), and NHERF3 (PDZK1) with lipid rafts in murine small intestinal BBM. Methods: Detergent resistant membranes ("lipid rafts") were isolated by floatation of Triton X-incubated small intestinal BBM from a variety of knockout mouse strains in an Optiprep step gradient. Acid-activated NHE3 activity was measured fluorometrically in BCECF-loaded microdissected villi, or by assessment of CO2/HCO3(-) mediated increase in fluid absorption in perfused jejunal loops of anethetized mice. Results: NHE3 was found to partially associate with lipid rafts in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity and regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. NHERF2 expression enhanced the localization of NHE3 to membrane rafts. The use of acid sphingomyelinase-deficient mice, which have altered membrane lipid as well as lipid raft composition, allowed us to test the validity of the lipid raft concept in vivo. Conclusions: The differential association of the NHERFs with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is one component of the differential and signal-specific NHE3 regulation by the different NHERFs. © 2013 S. Karger AG, Basel.
    Cellular Physiology and Biochemistry 11/2013; 32(5):1386-1402. · 3.42 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The duodenal villus brush border membrane expresses several ion transporters and/or channels, including the solute carrier 26 anion transporters Slc26a3 (DRA) and Slc26a6 (PAT-1), the Na(+)/H(+) exchanger isoform 3 (NHE3), as well as the anion channels CFTR and Slc26a9. Using genetically engineered mouse models lacking Scl26a3, Slc26a6, Slc26a9, or Slc9a3 (Na(+)/H(+) exchanger isoform 3, NHE3), the study was carried out to assess the role of these transporters in mediating the protective duodenal HCO3(-) secretory response (DBS-R) to luminal acid; and to compare it to their role in DBS-R elicited by the adenylyl cyclase agonist forskolin (FSK). While basal DBS was reduced in the absence of any of the three Slc26 isoforms, the DBS-R to FSK was not altered. In contrast, the DBS-R to a 5 minute exposure to luminal acid, pH 2.5, was strongly reduced in the absence of Slc26a3 or Slc26a9, but not Slc26a6. CFTR inhibitor [CFTR(Inh)-172] reduced the first phase of the acid-induced DBS-R, while NHE3 inhibition (or knockout) abolished the sustained phase of the DBS-R. Luminal acid exposure resulted in the activation of multiple intracellular signalling pathways, including SPAK, AKT and p38 phosphorylation. It induced a biphasic trafficking of NHE3, first rapidly into the brush border membrane, followed by endocytosis later. We conclude that the long-lasting DBS-R to luminal acid exposure activates multiple duodenocyte signalling pathways and involves changes in trafficking and/or activity of CFTR, Slc26 isoforms Slc26a3 and Slc26a9, and NHE3.
    The Journal of Physiology 09/2013; · 4.38 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: NHERF (Na+/H+ exchanger regulatory factor) proteins are a family of PDZ scaffolding proteins, three of which (NHERFs 1-3) are localized to the brush border in kidney and intestinal epithelial cells. All NHERF proteins are involved in anchoring membrane proteins which contain PDZ recognition motifs to form multiprotein signaling complexes. In contrast to their predicted immobility, NHERF1, NHERF2 and NHERF3 were all shown by FRAP/confocal microscopy to be surprisingly mobile in the microvilli of the renal proximal tubule OK cell line. Their diffusion coefficients, while different among the three, were all of the same magnitude as that of transmembrane proteins, suggesting they are all anchored in the microvilli but to different extents. NHERF3 moves faster than NHERF1, while NHERF2 moves the slowest. Several chimeras and mutants of NHERF1 and NHERF2 were made to determine which part of NHERF2 confers the slower mobility rate. Surprisingly, the slower mobility rate of NHERF2 was determined by a unique C-terminal domain, which includes a non-conserved region along with the ERM binding domain. Also, this C-terminal domain of NHERF2 determined its greater detergent insolubility and was necessary for the formation of larger multiprotein NHERF2 complexes. In addition, this NHERF2 domain was functionally significant in NHE3 regulation, being necessary for stimulation by LPA of activity and increased mobility of NHE3, as well as necessary for inhibition of NHE3 activity by calcium ionophore 4-Br-A23187. Thus, multiple functions of NHERF2 require involvement of an additional domain in this protein.
    Journal of Biological Chemistry 04/2013; · 4.65 Impact Factor