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

• Authors: Ayesha Sultan, Hannover Medical School; Min Luo, Hannover Medical School; Qin Yu, Hannover Medical School; Brigitte Riederer, Hannover Medical School; Weiliang Xia, Hannover Medical School; Mingmin Chen, Hannover Medical School; Simone Lissner, University of Tübingen; Johannes E. Gessner, Hannover Medical School; Mark Donowitz, John Hopkins School of Medicine; Chris Yun, Emory University; Hugo deJonge, Erasmus MC; Georg Lamprecht, University of Rostock; Ursula Seidler, Hannover Medical School
• Journal Title: Cellular Physiology and Biochemistry
• Volume: Volume 32, Number 5
• Publisher: Karger Publishers Open Access | 2013-01-01, Pages 1386-1402
• Type of Work: Article | Final Publisher PDF
• Publisher DOI: http://doi.org/10.1159/000356577
• Subjects: Health Sciences, General
• Keywords: Science & Technology; Life Sciences & Biomedicine; Cell Biology; Physiology; PDZ-domain adaptor proteins; Membrane rafts; Cholesterol; Intestine; Salt absorption; Ezrin; SPHINGOMYELINASE DEFICIENT MICE; INTESTINAL ION-TRANSPORT; NIEMANN-PICK-DISEASE; T-CELL-ACTIVATION; LIPID RAFTS; ACID SPHINGOMYELINASE; TARGETED DISRUPTION; ACTIN CYTOSKELETON; ABSORPTIVE DEFECTS; FLUID ABSORPTION

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 CO 2 /HCO 3 - 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.