title: 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: OBJECTIVE
  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.
authors.forename: Ayesha
authors.initials: A
authors.lastname: Sultan
authors.forename: Min
authors.initials: M
authors.lastname: Luo
authors.forename: Qin
authors.initials: Q
authors.lastname: Yu
authors.forename: Brigitte
authors.initials: B
authors.lastname: Riederer
authors.forename: Weiliang
authors.initials: W
authors.lastname: Xia
authors.forename: Mingmin
authors.initials: M
authors.lastname: Chen
authors.forename: Simone
authors.initials: S
authors.lastname: Lissner
authors.forename: Johannes E
authors.initials: JE
authors.lastname: Gessner
authors.forename: Mark
authors.initials: M
authors.lastname: Donowitz
authors.forename: C Chris
authors.initials: CC
authors.lastname: Yun
authors.forename: Hugo
authors.initials: H
authors.lastname: deJonge
authors.forename: Georg
authors.initials: G
authors.lastname: Lamprecht
authors.forename: Ursula
authors.initials: U
authors.lastname: Seidler
mesh_terms: D000818:Animals; D007421:Intestine, Small / Q000166:cytology* / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D007583:Jejunum / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism; D008565:Membrane Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008871:Microvilli / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D013108:Sphingomyelin Phosphodiesterase / Q000235:genetics
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C514896:PDZK1 protein, mouse; D010750:Phosphoproteins; C000613386:Slc9a3 protein, mouse; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D013108:Sphingomyelin Phosphodiesterase