PTH transiently increases the percent mobile fraction of Npt2a in OK cells as determined by FRAP.
Renal sodium-dependent phosphate transporter 2a (Npt2a) binds to a number of PDZ adaptor proteins including sodium-hydrogen exchanger regulatory factor-1 (NHERF-1), which regulates its retention in the apical membrane of renal proximal tubule cells and the response to parathyroid hormone (PTH). The present experiments were designed to study the lateral mobility of enhanced green fluorescent protein (EGFP)-Npt2a in proximal tubule-like opossum kidney (OK) cells using fluorescence recovery after photobleaching (FRAP) and to determine the role of PDZ binding proteins in mediating the effects of PTH. The mobile fraction of wild-type Npt2a (EGFP-Npt2a-TRL) under basal conditions was approximately 17%. Treatment of the cells with Bis(sulfosuccinimidyl) suberate, a water-soluble cross-linker, abolished recovery nearly completely, indicating that recovery represented lateral diffusion in the plasma membrane and not the exocytosis or synthesis of unbleached transporter. Substitution of the C-terminal amino acid PDZ binding sequence TRL with AAA (EGFP-Npt2a-AAA) resulted in a nearly twofold increase in percent mobile fraction of Npt2a. Treatment of cells with PTH resulted in a rapid increase in the percent mobile fraction to >30% followed by a time-dependent decrease to baseline or below. PTH had no effect on the mobility of EGFP-Npt2a-AAA expressed in native OK cells or on wild-type EGFP-Npt2a-TRL expressed in OK-H cells deficient in NHERF-1. These findings indicate that the association of Npt2a with PDZ binding proteins limits the lateral mobility of the transporter in the apical membrane of renal proximal tubule cells. Treatment with PTH, presumably by dissociating NHERF-1/Npt2a complexes, transiently increases the mobility of Npt2a, suggesting that freeing of Npt2a from the cytoskeleton precedes PTH-mediated endocytosis.
Dept. of Medicine, Div. of Nephrology, Univ. of Maryland School of Medicine, Baltimore, MD 21202, USA. eweinman@medicine.umaryland.edu
Edward J
EJ
Weinman
Deborah
D
Steplock
Boyoung
B
Cha
Olga
O
Kovbasnjuk
Nicholas A
NA
Frost
Rochelle
R
Cunningham
Shirish
S
Shenolikar
Thomas A
TA
Blanpied
Mark
M
Donowitz
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D015536:Down-Regulation; D036681:Fluorescence Recovery After Photobleaching; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D009893:Opossums; D054731:PDZ Domains; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010750:Phosphoproteins / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa / Q000378:metabolism*; D013997:Time Factors; D015854:Up-Regulation
D048868:Adaptor Proteins, Signal Transducing; D010281:Parathyroid Hormone; D010750:Phosphoproteins; D017923:Sodium-Hydrogen Exchangers; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; C093376:sodium-hydrogen exchanger regulatory factor