Role of NHERF1, cystic fibrosis transmembrane conductance regulator, and cAMP in the regulation of aquaporin 9.
Water and solute transport across the plasma membrane of cells is a crucial biological function that is mediated mainly by aquaporins and aquaglyceroporins. The regulation of these membrane proteins is still incompletely understood. Using the male reproductive tract as a model system in which water and glycerol transport are critical for the establishment of fertility, we now report a novel pathway for the regulation of aquaporin 9 (AQP9) permeability. AQP9 is the major aquaglyceroporin of the epididymis, liver, and peripheral leukocytes, and its COOH-terminal portion contains a putative PDZ binding motif (SVIM). Here we show that NHERF1, cystic fibrosis transmembrane conductance regulator (CFTR), and AQP9 co-localize in the apical membrane of principal cells of the epididymis and the vas deferens, and that both NHERF1 and CFTR co-immunoprecipitate with AQP9. Overlay assays revealed that AQP9 binds to both the PDZ1 and PDZ2 domains of NHERF1, with an apparently higher affinity for PDZ1 versus PDZ2. Pull-down assays showed that the AQP9 COOH-terminal SVIM motif is essential for interaction with NHERF1. Functional assays on isolated tubules perfused in vitro showed a high permeability of the apical membrane to glycerol, which is inhibited by the AQP9 inhibitor, phloretin, and is markedly activated by cAMP. The CFTR inhibitors DPC, GlyH-101 and CFTRinh-172 all significantly reduced the cAMP-activated glycerol-induced cell swelling. We propose that CFTR is an important regulator of AQP9 and that the interaction between AQP9, NHERF1, and CFTR may facilitate the activation of AQP9 by cAMP.
Pietrement
Christine
C
Center for Systems Biology, Program in Membrane Biology/Nephrology Division, Simches Research Center, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114, USA.
Da Silva
Nicolas
N
Silberstein
Claudia
C
James
Marianne
M
Marsolais
Mireille
M
Van Hoek
Alfred
A
Brown
Dennis
D
Pastor-Soler
Nuria
N
Ameen
Nadia
N
Laprade
Raynald
R
Ramesh
Vijaya
V
Breton
Sylvie
S
D000595:Amino Acid Sequence; D000818:Animals; D020346:Aquaporins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D000242:Cyclic AMP / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004822:Epididymis / Q000378:metabolism; D005298:Fertility / Q000502:physiology; D066298:In Vitro Techniques; D008297:Male; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014649:Vas Deferens / Q000378:metabolism
C491637:Aqp9 protein, rat; D020346:Aquaporins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP