Differential Association of the Na⁺/H⁺Exchanger Regulatory Factor (NHERF) Family of Adaptor Proteins with the Raft- and the Non-Raft Brush Border Membrane Fractions of NHE3

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.

Evidence for a causal link between adaptor protein PDZK1 downregulation and Na+/H+ exchanger NHE3 dysfunction in human and murine colitis

“…NHERF2 mRNA was not investigated because its absence does not result in a decrease in acid-activated NHE3 activity in murine colon [ 8 ], and because of its low colonic expression [ 19 ]. In addition, the cross-reactivity of the NHERF2 antibody with the much more strongly expressed NHERF1 [ 35 ] would make interpretation of the results difficult. We also studied three inflammatory mouse models, each of which had an immunologically mediated intestinal inflammation, but with different segment predilection, acuity, and severity of inflammation.…”

Role of epithelial ion transports in inflammatory bowel disease

“…Moreover, NHE3 activity and membrane expression were reduced, although mRNA levels were not affected (21). One study found NHERF2 Ϫ/Ϫ mice to have lower ileal NHE3 activity due to impaired trafficking or retention of NHE3 in the apical membrane (99), which is possibly related to NHERF2 association with lipid-raft NHE3 (136). In fact, lysophosphatidic acid (LPA, released in inflammatory settings to repair wounded tissue) stimulates NHE3 activity and expression through NHERF2-dependent mechanisms in ileum (99) and kidney brush border (27, 84).…”

The Role of Lipid Rafts in the Early Stage of Enterovirus 71 Infection

“…They perform crucial roles in the establishment of cell polarity [9,10] and act as platforms mediating signal transduction and protein trafficking [11,12,13]. A number of raft-associated proteins have been identified, including tyrosine kinase cascades, G protein-coupled receptors, Src family kinases, and cytoskeletal and intermediate filament proteins [14].…”