Mo1748 Absorptive Enterocytes Are Poorly Differentiated in the CFTR Knockout Mouse Small Intestine

Abstract

Background: BB NHE3 (Na+/H+ exchanger 3) plus at least 7 other proteins are assembled into 600 kDa -1000 kDa macro-complexes, which serve as the signaling platforms required for the regulation of NHE3. NHERF proteins are a family of multi-PDZ-scaffolding proteins. NHERF1, NHERF2 and NHERF3 are localized to the areas of and below the BB and are involved in NHE3 regulation. Of these, both NHERF2 and NHERF3 but not NHERF1 were required for the carbachol inhibition of NHE3 activity, suggesting possible roles of NHERF dimerization in NHE3 regulation. The purpose of this study was 1) to compare the dimerization strength of all possible NHERF-NHERF interactions and 2) to test the hypothesis that NHERF2-NHERF3 hetero-dimerization contributes to NHE3 macro-complexes and is required for the acute carbachol inhibition of NHE3. Methods: FLAG-tagged NHERF proteins were used for Co-Immunoprecipitation (CO-IP) with anti-FLAG magnetic beads. Bacterially produced recombinant GST-fusion and MBP-fusion NHERF proteins were purified for pulldown (PD) assays to study direct protein-protein interactions in vitro. NHERF2 PDZ (GYGF to GAGA) mutants and NHERF3-4Ala (last 4 aa were Ala) mutant were used to define their interaction domains. Inhibition of NHE3 activity by carbachol was determined with BCECF/ fluorometry in Caco-2 cells expressing HA-NHE3. shRNAwas used to knock-down NHERF3. Adenovirus was used to express the FLAG-tagged rat wild type and mutant NHERF3. Results: NHERF1-NHERF3 and NHERF2-NHERF3 hetero-dimers were detected by both CO-IP and PD assays. NHERF1 and NHERF2 homo-dimers and NHERF1-NHERF2 hetero-dimers were detected minimally by PD and not by CO-IP. There was no significant NHERF3 homodimerization. Hetero-dimerization of NHERF2-NHERF3 was stronger than that of NHERF1NHERF3. The single-PDZ domain defective mutants of NHERF2 had reduced binding to NHERF3, whereas the double-PDZ domain mutant did not bind NHERF3 at all. NHERF34A did not bind NHERF2. This lack of interactions was confirmed by FRET in live OK (opossum kidney proximal tubule) cells. Macro-complexes could be assembled in vitro from NHERF2, NHERF3 and NHE3. NHE3 activity was inhibited by carbachol in wild-type Caco2 cells, but not in NHERF3 KD cells. This could be rescued by overexpression of shRNAresistant rat NHERF3 but not the rat NHERF3-4Amutant. Conclusions: 1) NHERF2-NHERF3 hetero-dimerization is strongest among all possible NHERF dimerizations. 2) NHERF2 and NHERF3 interact via the PDZ domains of NHERF2 and C-terminal PDZ recognition motif of NHERF3. 3) NHERF3 prefers binding to PDZ1 domain of NHERF2 while NHE3 prefers PDZ2 domain. 4) The NHERF2-NHERF3 hetero-dimerization mediated NHE3 macro-complex is necessary for the inhibition of NHE3 activity by carbachol. 5) Hetero-dimerization of NHER2-NHERF3 explains why both NHERF2 and NHERF3 are required for carbachol inhibition of NHE3.