title: The Extracellular C-loop Domain Plays an Important Role in the Cell Adhesion Function of Aquaporin 0.
abstract: Although aquaporin 0 (AQP0) is a member of the AQP family, it has limited water permeability compared with other members. AQP0 may also have cell adhesion-related functions, but the evidence is still limited. Here, we studied the relationship of AQP0 to cell adhesion and determined the region required for cell adhesion.
  
  
  
  L-cell fibroblasts stably expressing AQP0 or AQP1 (L-AQP0 or L-AQP1) were established. One group of cells was stained with CellTracker Red and cultured into a confluent monolayer, whereas the other group was loaded with CellTracker Blue and seeded over the monolayer. To study cell adhesion, the percentages of lower and upper layer cells were measured using flow cytometry. To determine the region of AQP0 required for adhesion, activity was done by pull-down assay using glutathione S-transferase fusion proteins. To study the water permeability, Xenopus laevis oocyte expressing AQP0 wild-type or AQP0 mutated in C-loop was transferred to a hypotonic solution and photographed, and the diameter was measured to calculate the volume.
  
  
  
  More cells adhered to the lower cells in the L-AQP0 homotypic pair than other pairs such as L-AQP1 homotypic or L-AQP0/L-AQP1 heterotypic pairs. Pull-down assays revealed that AQP0 could bind to itself via the C-loop extracellular domain. Furthermore, we determined that109Pro and110Pro in the C-loop were important for cell adhesion. However, mutation of the C-loop in AQP0 did not affect its water permeability.
  
  
  
  AQP0 is known to bind lipids in the opposing membrane. Our data suggest that this cell-to-cell adhesion occurs not only in the AQP0/liquids but also via AQP0/AQP0 interaction through the C-loop domain. Mutations in the C-loop amino acids did not affect the water permeability of AQP0 but did affect its cell adhesion function. These independent dual functions of AQP0 are important for lens transparency.
authors.affiliation: a Faculty of Pharmacy , Keio University , Tokyo , Japan.
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Nakazawa
authors.affiliation: b Center for Pharmaceutical Education , Yokohama University of Pharmacy , Kanagawa , Japan.
authors.forename: Mikako
authors.initials: M
authors.lastname: Oka
authors.affiliation: a Faculty of Pharmacy , Keio University , Tokyo , Japan.
authors.forename: Megumi
authors.initials: M
authors.lastname: Funakoshi-Tago
authors.affiliation: a Faculty of Pharmacy , Keio University , Tokyo , Japan.
authors.forename: Hiroomi
authors.initials: H
authors.lastname: Tamura
authors.affiliation: a Faculty of Pharmacy , Keio University , Tokyo , Japan.|b Center for Pharmaceutical Education , Yokohama University of Pharmacy , Kanagawa , Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Takehana
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020346:Aquaporins / Q000737:chemistry / Q000502:physiology*; D002448:Cell Adhesion / Q000502:physiology*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D005136:Eye Proteins / Q000737:chemistry / Q000502:physiology*; D005347:Fibroblasts; D005434:Flow Cytometry; D005822:Genetic Vectors; D007908:Lens, Crystalline / Q000378:metabolism; D051379:Mice; D010957:Plasmids; D017354:Point Mutation; D000072417:Protein Domains / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014867:Water / Q000378:metabolism; D014882:Water-Electrolyte Balance / Q000502:physiology; D014982:Xenopus laevis
chemical_list: D020346:Aquaporins; D005136:Eye Proteins; C030328:aquaporin 0; D014867:Water
keywords: Aquaporin; cataract; cell adhesion; lens; water channel