Na+-dependent phosphate transporters in the murine osteoclast: cellular distribution and protein interactions

Wepreviously demonstrated that inhibition of Na-dependent phosphate(P_i) transport in osteoclasts led to reduced ATP levels anddiminished bone resorption. These findings suggested that Na/P_i cotransporters in the osteoclast plasma membraneprovide P_i for ATP synthesis and that the osteoclast mayutilize part of the P_i released from bone resorption forthis purpose. The present study was undertaken to define the cellularlocalization of Na/P_i cotransporters in the mouseosteoclast and to identify the proteins with which they interact. Usingglutathione S-transferase (GST) fusion constructs, wedemonstrate that the type IIa Na/P_i cotransporter (Npt2a)in osteoclast lysates interacts with the Na/H exchanger regulatoryfactor, NHERF-1, a PDZ protein that is essential for the regulation ofvarious membrane transporters. In addition, NHERF-1 in osteoclastlysates interacts with Npt2a in spite of deletion of a putativePDZ-binding domain within the carboxy terminus of Npt2a. In contrast,deletion of the carboxy-terminal TRL amino acid motif of Npt2asignificantly reduced its interaction with NHERF-1 in kidney lysates.Studies in osteoclasts transfected with green fluorescent protein-Npt2aconstructs indicated that Npt2a colocalizes with NHERF-1 and actin ator near the plasma membrane of the osteoclast and associates withezrin, a linker protein associated with the actin cytoskeleton, likelyvia NHERF-1. Furthermore, we demonstrate by RT/PCR of osteoclast RNAand in situ hybridization that the type III Na/P_icotransporter, PiT-1, is also expressed in mouse osteoclasts. Toexamine the cellular distribution of PiT-1, we infected mouseosteoclasts with a retroviral vector encoding PiT-1 fused to an epitopetag. PiT-1 colocalizes with actin and is present on the basolateralmembrane of the polarized osteoclast, similar to that previouslyreported for Npt2a. Taken together, our data suggest that associationof Npt2a with NHERF-1, ezrin, and actin, and of PiT-1 with actin, maybe responsible for membrane sorting and regulation of theseNa/P_i cotransporters in the osteoclast.