Submitted on November 4, 2008
Revised on September 4, 2009
Accepted on September 28, 2009
PTH transiently increases the percent mobile fraction of Npt2a in OK cells as determined by FRAP
Edward J. Weinman1*, Deborah Steplock, Boyoung Cha2, Olga Kovbasnjuk3, Nicholas A. Frost4, Rochelle Cunningham4, Shirish Shenolikar5, Thomas A. Blanpied6, and Mark Donowitz7
1 University of Maryland School of Medicine, Department of Veterans Affairs Medical Center, Baltimore
2 The Johns Hopkins Univ. School of Med.
3 JHSOM
4 University of Maryland School of Medicine
5 Duke University Medical Center
6 Univ. Maryland
7 Johns Hopkins Un Sch of Med
* To whom correspondence should be addressed. E-mail: eweinman{at}medicine.umaryland.edu.
The renal sodium-dependent phosphate transporter 2a (Npt2a) binds to a number of PDZ adaptor proteins including the 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 EGFP-Npt2a in proximal tubule-like 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 two-fold increase in percent mobile fraction of Npt2a. Treatment of cells with PTH resulted in a rapid increase in the percent mobile fraction to over 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.
