Dynamics of PTH-induced disassembly of Npt2a/NHERF-1 complexes in living OK cells.
Parathyroid hormone (PTH) inhibits the reabsorption of phosphate in the renal proximal tubule by disrupting the binding of the sodium-dependent phosphate transporter 2A (Npt2a) to the adapter protein sodium-hydrogen exchanger regulatory factor-1 (NHERF-1), a process initiated by activation of protein kinase C (PKC). To gain additional insights into the dynamic sequence of events, the time course of these responses was studied in living opossum kidney (OK) cells. Using a FRET-based biosensor, we found that PTH activated intracellular PKC within seconds to minutes. In cells expressing GFP-Npt2a and mCherry-NHERF, PTH did not affect the relative abundance of NHERF-1 but there was a significant and time-dependent decrease in the Npt2a/NHERF-1 ratio. The half-time to maximal dissociation was 15 to 20 min. By contrast, PTH had no effect on the fluorescence ratio for GFP-ezrin compared with mCherry-NHERF-1 at the apical surface. These experiments establish that PTH treatment of proximal tubule OK cells leads to rapid activation of PKC with the subsequent dissociation of Npt2a/NHERF-1 complexes. The association of NHERF-1 with Ezrin and their localization at the apical membrane, however, was unperturbed by PTH, thereby enabling the rapid recruitment and membrane reinsertion of Npt2a and other NHERF-1 targets on termination of the hormone response.
University of Maryland School of Medicine, Department of Veterans Affairs Medical Center, Baltimore, MD 21202, USA. eweinman@medicine.umaryland.edu
Edward J
EJ
Weinman
Deborah
D
Steplock
Shirish
S
Shenolikar
Thomas A
TA
Blanpied
D000818:Animals; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000378:metabolism; D005260:Female; D007668:Kidney / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000187:drug effects / Q000378:metabolism*; D009893:Opossums; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010750:Phosphoproteins / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa / Q000378:metabolism*; D000099149:Ezrin
D003598:Cytoskeletal Proteins; D046912:Multiprotein Complexes; D010281:Parathyroid Hormone; D010750:Phosphoproteins; D017923:Sodium-Hydrogen Exchangers; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D000099149:Ezrin; C093376:sodium-hydrogen exchanger regulatory factor