Beth S. Lee

The Amino-terminal Domain of the B Subunit of Vacuolar H+-ATPase Contains a Filamentous Actin Binding Site

Vacuolar H+-ATPase (V-ATPase) binds actin filaments with high affinity (K d = 55 nm; Lee, B. S., Gluck, S. L., and Holliday, L. S. (1999) J. Biol. Chem. 274, 29164–29171). We have proposed that this interaction is an important mechanism controlling transport of V-ATPase from the cytoplasm to the plasma membrane of osteoclasts. Here we show that both the B1 (kidney) and B2 (brain) isoforms of the B subunit of V-ATPase contain a microfilament binding site in their amino-terminal domain. In pelleting assays containing actin filaments and partially disrupted V-ATPase, B subunits were found in greater abundance in actin pellets than were other V-ATPase subunits, suggesting that the B subunit contained an F-actin binding site. In overlay assays, biotinylated actin filaments also bound to the B subunit. A fusion protein containing the amino-terminal half of B1 subunit bound actin filaments tightly, but fusion proteins containing the carboxyl-terminal half of B1 subunit, or the full-length E subunit, did not bind F-actin. Fusion proteins containing the amino-terminal 106 amino acids of the B1 isoform or the amino-terminal 112 amino acids of the B2 isoform bound filamentous actin withK d values of 130 and 190 nm, respectively, and approached saturation at 1 mol of fusion protein/mol of filamentous actin. The B1 and B2 amino-terminal fusion proteins competed with V-ATPase for binding to filamentous actin. In summary, binding sites for F-actin are present in the amino-terminal domains of both isoforms of the B subunit, and likely are responsible for the interaction between V-ATPase and actin filaments in vivo.

Interaction between Vacuolar H+-ATPase and Microfilaments during Osteoclast Activation

Vacuolar H+-ATPases (V-ATPases) are multisubunit enzymes that acidify compartments of the vacuolar system of all eukaryotic cells. In osteoclasts, the cells that degrade bone, V-ATPases, are recruited from intracellular membrane compartments to the ruffled membrane, a specialized domain of the plasma membrane, where they are maintained at high densities, serving to acidify the resorption bay at the osteoclast attachment site on bone (Blair, H. C., Teitelbaum, S. L., Ghiselli, R., and Gluck, S. L. (1989) Science 249, 855–857). Here, we describe a new mechanism involved in controlling the activity of the bone-resorptive cell. V-ATPase in osteoclasts cultured in vitro was found to form a detergent-insoluble complex with actin and myosin II through direct binding of V-ATPase to actin filaments. Plating bone marrow cells onto dentine slices, a physiologic stimulus that activates osteoclast resorption, produced a profound change in the association of the V-ATPase with actin, assayed by coimmunoprecipitation and immunocytochemical colocalization of actin filaments and V-ATPase in osteoclasts. Mouse marrow and bovine kidney V-ATPase bound rabbit muscle F-actin directly with a maximum stoichiometry of 1 mol of V-ATPase per 8 mol of F-actin and an apparent affinity of 0.05 μm. Electron microscopy of negatively stained samples confirmed the binding interaction. These findings link transport of V-ATPase to reorganization of the actin cytoskeleton during osteoclast activation.

Differential Localization of Myosin II Isoforms in Resting and Activated Osteoclasts

Osteoclasts resorb bone through a cyclical process of attachment to matrix, polarization, retraction, and migration. Although this process requires major alterations in the organization of actin structures, little is known about roles that myosins play in osteoclast cytoskeletal dynamics. We performed immunolocalization of myosin II using antibodies against heavy chain isoforms IIA and IIB and found that osteoclasts expressed the isoforms in distinct subcellular locations. Myosin IIA was enriched in dynamic cytoskeletal compartments, including the sealing zones of polarized and unpolarized osteoclasts. In contrast, myosin IIB was generally absent from these regions and maintained a comparatively static distribution during different phases of the osteoclast activation cycle. Inhibition of myosin II in osteoclasts by treatment with 2,3-butanedione monoxime caused detachment of unpolarized, but not polarized, cells from the bone matrix. These results suggest that myosin IIA is critical to development of an activated osteoclast phenotype.n

Vacuolar H+-ATPase activity and expression in mouse bone marrow cultures.

We examined vacuolar H+‐ATPase (V‐ATPase) structure, enzymatic properties, and protein and mRNA expression from mouse marrow cultured in the presence or absence of 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3), which stimulates formation of bone‐resorptive osteoclasts. V‐ATPases from osteoclast‐containing cultures were similar in ion and inhibitor sensitivities to the enzyme from kidney‐derived sources. Immunopurified V‐ATPase from 1,25(OH)2D3‐stimulated cultures exhibited 20‐fold greater ATPase activity than the enzyme from unstimulated cultures, which do not contain osteoclasts. In contrast, 1,25(OH)2D3‐treated cultures contained only 2‐fold more assembled V‐ATPase, as determined by immunoprecipitation. Quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) and immunoblot analysis similarly showed ∼2‐fold increases of V‐ATPase mRNA and protein levels in 1,25(OH)2D3‐treated cultures. The bulk of the relative difference in V‐ATPase activity between the two cultures was due to a 10‐fold difference in enzyme specific activity. Quantitative RT‐PCR also revealed that expression levels of V‐ATPase mRNAs reflected the stoichiometry of enzyme subunits in the assembled complex. These data indicate that in mouse bone marrow cultures, V‐ATPase expression is controlled at the level of mRNA, and that increases in subunit expression and assembly cannot account for the 20‐fold difference in enzyme activity in osteoclast‐containing cultures. Therefore, osteoclast V‐ATPase activity may be regulated by subtle alterations in enzyme structure or associated factors.

Properties and regulation of the renal vacuolar H(+)-ATPase and H(+)-K(+)-ATPase.

Two proton-transporting ATPases participate in active proton transport in the nephron: the electrogenic vacuolar H(+)-ATPase and the electroneutral H(+)-K(+)-ATPase. The vacuolar H(+)-ATPase participates in proximal and distal hydrogen ion secretion related to acid-base homeostasis. The H(+)-K(+)-ATPase is located exclusively in the distal nephron, and its primary role may be in active potassium reabsorption. The properties, distribution, and regulation of these two enzymes are discussed.