CHARACTERIZATION OF THE OSTEOCLAST VACUOLAR H-ATPASE B-SUBUNIT()

During bone resorption, osteoclasts acidify the extracellular bone res orbing compartment via a vacuolar H+-ATPase (V-ATPase), which resides in the ruffled-border membrane. In an effort to characterize the compo sition of the osteoclast V-ATPase catalytic domain, we have isolated a cDNA clone that encodes the V-ATPase B-subunit from a cDNA library co nstructed from highly purified chicken osteoclasts. Comparison of the predicted amino-acid sequence with the published sequences of isoforms of V-ATPase B-subunits from other sources revealed that the chicken o steoclast B-subunit is brain type and not kidney type. Furthermore, on ly clones encoding the brain type isoform of subunit B could be genera ted by PCR from a cDNA library prepared from human osteoclastoma osteo clast-like cells. Northern blot analysis revealed that two B-subunit m RNAs, approx. 1.7 and 3.5 kb in length, are expressed in chicken bone marrow mononuclear cells, brain and kidney, although the relative amou nts of these two transcripts were different in each tissue. In brain, the 3.5-kb mRNA was predominantly expressed. In bone marrow cells, the levels of the 1.7-kb mRNA were higher than in other tissues and expre ssion of this message was increased by 1,25-dihydroxyvitamin D-3, sugg esting that this mRNA is specifically upregulated during osteoclast di fferentiation. These results indicate that the B-subunit isoforms pres ent in the catalytic domains of the osteoclast and kidney V-H+-ATPases are different and further suggest that selective expression of isofor ms of the B-subunit in these two tissues could provide a structural ba sis for some of the differences we have reported in the pharmacology a nd catalytic properties of these two enzymes.