Parathyroid hormone regulation of NA+,K+-ATPase requires the PDZ 1 domain of sodium hydrogen exchanger regulatory factor-1 in opossum kidney cells.
It was demonstrated that expression of murine sodium hydrogen exchanger regulatory factor (NHERF-1) lacking the ezrin-binding domain blocks parathyroid hormone (PTH) regulation of Na+,K+-ATPase in opossum kidney (OK) cells. The hypothesis that the NHERF-1 PDZ domains contribute to PTH regulation of Na+,K+-ATPase was tested by comparison of PTH regulation of Na+,K+-ATPase in wild-type OK (OK-WT) cells, NHERF-deficient OKH cells, OK-WT transfected with siRNA for NHERF (NHERF siRNA OK-WT), and OKH cells that were stably transfected with full-length NHERF-1 or constructs with mutated PDZ domains. OKH cells and NHERF siRNA OK-WT showed decreased expression of NHERF-1 but equivalent expression of ezrin and Na+,K+-ATPase alpha1 subunit when compared with OK-WT cells. PTH decreased Na+,K+-ATPase activity and stimulated phosphorylation of the Na+,K+-ATPase alpha1 in OK-WT cells but not in NHERF-deficient cells. Rubidium (86Rb) uptake was equivalent in OK-WT, OKH, and OKH cells that were transfected with all but the double PDZ domain mutants. PTH decreased 86Rb uptake significantly in OK-WT but not in OKH cells. PTH also significantly inhibited 86Rb uptake in OKH cells that were transfected with full-length NHERF-1 or NHERF-1 with mutated PDZ 2 but not in OKH cells that were transfected with mutated PDZ 1. Transfection with NHERF expressing both mutated PDZ domains resulted in diminished basal 86Rb uptake that was not inhibited further by PTH. PTH stimulated protein kinase Calpha activity and alpha1 subunit phosphorylation in OK-WT but not in NHERF-deficient cells. Transfection of OKH cells with NHERF constructs that contained an intact PDZ1 domain restored PTH-stimulated protein kinase Calpha activity and alpha1 subunit phosphorylation. These results demonstrate that NHERF-1 is necessary for PTH-mediated inhibition of Na+,K+-ATPase activity and that the inhibition is mediated through the PDZ1, not PDZ2, domain.
Department of Medicine, University of Louisville, Kidney Disease Program, Louisville, KY 40202, USA. syed.khundmiri@louisville.edu
Syed Jalal
SJ
Khundmiri
Edward J
EJ
Weinman
Deborah
D
Steplock
Judith
J
Cole
Aamir
A
Ahmad
Patrick D
PD
Baumann
Michelle
M
Barati
Madhavi J
MJ
Rane
Eleanor
E
Lederer
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects; D051379:Mice; D009893:Opossums; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010750:Phosphoproteins / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection
D010281:Parathyroid Hormone; D010750:Phosphoproteins; D021122:Protein Subunits; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D051571:Protein Kinase C-alpha; D000254:Sodium-Potassium-Exchanging ATPase