Na+-dependent phosphate transporters in the murine osteoclast: cellular distribution and protein interactions.
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.
Khadeer
Mohammed A
MA
Department of Oral and Craniofacial Biological Sciences, University of Maryland, Baltimore, Maryland 21201, USA.
Tang
Zhihui
Z
Tenenhouse
Harriet S
HS
Eiden
Maribeth V
MV
Murer
Heini
H
Hernando
Natividad
N
Weinman
Edward J
EJ
Chellaiah
Meenakshi A
MA
Gupta
Anandarup
A
D000199:Actins / Q000378:metabolism; D000818:Animals; D016764:Cell Polarity; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins; D005260:Female; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers; D050608:Sodium-Phosphate Cotransporter Proteins; D050611:Sodium-Phosphate Cotransporter Proteins, Type III; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D027981:Symporters / Q000235:genetics / Q000378:metabolism*; D000099149:Ezrin
D000199:Actins; D003598:Cytoskeletal Proteins; D010710:Phosphates; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D012964:Sodium; D017923:Sodium-Hydrogen Exchangers; D050608:Sodium-Phosphate Cotransporter Proteins; D050611:Sodium-Phosphate Cotransporter Proteins, Type III; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D027981:Symporters; D000099149:Ezrin; C493587:Slc20a1 protein, mouse; C493555:Slc34a1 protein, mouse; C093376:sodium-hydrogen exchanger regulatory factor