We previously demonstrated that inhibition of Na-dependent
phosphate (P(i)) transport in osteoclasts led to reduced
ATP levels and diminished
bone resorption. These findings suggested that Na/P(i) cotransporters in the osteoclast plasma membrane provide P(i) for
ATP synthesis and that the osteoclast may utilize part of the P(i) released from
bone resorption for this purpose. The present study was undertaken to define the cellular localization of Na/P(i) 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/P(i) 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/P(i) 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/P(i) cotransporters in the osteoclast.