Characterization of the osteoclast vacuolar H(+)-ATPase B-subunit.
During bone resorption, osteoclasts acidify the extracellular bone resorbing compartment via a vacuolar H(+)-ATPase (V-ATPase), which resides in the ruffled-border membrane. In an effort to characterize the composition of the osteoclast V-ATPase catalytic domain, we have isolated a cDNA clone that encodes the V-ATPase B-subunit from a cDNA library constructed 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 osteoclast B-subunit is brain type and not kidney type. Furthermore, only clones encoding the brain type isoform of subunit B could be generated by PCR from a cDNA library prepared from human osteoclastoma osteoclast-like cells. Northern blot analysis revealed that two B-subunit mRNAs, approx. 1.7 and 3.5 kb in length, are expressed in chicken bone marrow mono-nuclear cells, brain and kidney, although the relative amounts 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 expression of this message was increased by 1,25-dihydroxyvitamin D-3, suggesting that this mRNA is specifically upregulated during osteoclast differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)
Bartkiewicz
M
M
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520-8044, USA.
Hernando
N
N
Reddy
S V
SV
Roodman
G D
GD
Baron
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001859:Bone Neoplasms / Q000201:enzymology / Q000473:pathology; D001921:Brain / Q000201:enzymology; D002117:Calcitriol / Q000494:pharmacology; D002384:Catalysis; D002417:Cattle; D002454:Cell Differentiation / Q000187:drug effects; D002645:Chickens / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004790:Enzyme Induction / Q000187:drug effects; D005796:Genes; D018212:Giant Cell Tumor of Bone / Q000201:enzymology / Q000473:pathology; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007668:Kidney / Q000201:enzymology; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry; D009928:Organ Specificity; D010010:Osteoclasts / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000096:biosynthesis; D016415:Sequence Alignment; D014407:Tumor Cells, Cultured; D014617:Vacuoles / Q000201:enzymology*
D018076:DNA, Complementary; D007527:Isoenzymes; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D006180:Proton-Translocating ATPases; D002117:Calcitriol