Na+-dependent phosphate transporters in the murine osteoclast: cellular distribution
and protein interactions.
Khadeer, Mohammed A., Zhihui Tang, Harriet S. Tenenhouse, Maribeth V. Eiden, Heini
Murer, Natividad Hernando, Edward J. Weinman, Meenakshi A. Chellaiah, and
Anandarup Gupta.
1Department of Oral and Craniofacial Biological Sciences, University of Maryland,
Baltimore, Maryland 21201; 2Departments of Pediatrics and Human Genetics, Montreal
Children's Hospital Research Institute, McGill University, Montreal, Quebec, H3Z 2Z3
Canada; 3Laboratory of Cellular and Molecular Regulation, National Institute of Mental
Health, Bethesda, Maryland 20892; 4Physiologisches Institut, Universität Zürich-Irchel,
Zürich, CH-8057, Switzerland; and, 5Department of Medicine, University of Maryland,
Baltimore, Maryland 21201
APStracts 10:0079C, 2003.
We previously demonstrated that inhibition of Na-dependent phosphate (Pi) transport in
osteoclasts led to reduced ATP levels and diminished bone resorption. These findings
suggested that Na/Pi cotransporters in the osteoclast plasma membrane provide Pi for
ATP synthesis and that the osteoclast may utilize part of the Pi released from bone
resorption for this purpose. The present study was undertaken to define the cellular
localization of Na/Pi cotransporters in the mouse osteoclast and to identify the proteins
with which they interact. Using glutathione S-transferase (GST) fusion constructs, we
demonstrate that the type IIa Na/Pi cotransporter (Npt2a) in osteoclast lysates interacts
with the Na/H exchanger regulatory factor, NHERF-1, a PDZ protein that is essential for
the regulation of various membrane transporters. In addition, NHERF-1 in osteoclast
lysates interacts with Npt2a in spite of deletion of a putative PDZ-binding domain within
the carboxy terminus of Npt2a. In contrast, deletion of the carboxy terminal TRL amino
acid motif of Npt2a significantly reduced its interaction with NHERF-1 in kidney lysates.
Studies in osteoclasts transfected with green fluorescent protein-Npt2a constructs
indicated that Npt2a colocalizes with NHERF-1 and actin at or near the plasma
membrane of the osteoclast and associates with ezrin, a linker protein associated with the
actin cytoskeleton, likely via NHERF-1. Furthermore, we demonstrate by RT/PCR of
osteoclast RNA and in situ hybridization that the type III Na/Pi cotransporter, PiT-1, is
also expressed in mouse osteoclasts. To examine the cellular distribution of PiT-1, we
infected mouse osteoclasts with a retroviral vector encoding PiT-1 fused to an epitope
tag. PiT-1 colocalizes with actin and is present on the basolateral membrane of the
polarized osteoclast, similar to that previously reported for Npt2a. Taken together, our
data suggest that association of Npt2a with NHERF-1, ezrin, and actin, and of PiT-1 with
actin, may be responsible for membrane sorting and regulation of these Na/Pi
cotransporters in the osteoclast.
Received 12 December 2002; accepted in final form 21 February 2003
APS Manuscript Number C580-2.
Article publication pending Am J Physiol Cell Physiol
ISSN 1080-4757 Copyright 2003 The American Physiological Society.
Published in APStracts on 25 March 2003