Characterization and Cellular Distribution of the Osteoclast Ruffled Membrane Vacuolar H+-ATPase B-Subunit Using Isoform-Specific Antibodies

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Abstract
Acidification of the bone surface, leading to bone resorption, is accomplished by a vacuolar-type H+-ATPase present in a specialized domain of the plasma membrane of the osteoclast known as the ruffled membrane. Structure and function appears to be highly conserved within this class of multisubunit enzymes. However, cloning and sequencing of complementary DNA has shown that one of the subunits in the catalytic domain, the B-subunit, exists in at least two forms, B1 and B2. B1 messenger RNA has been found almost exclusively in the kidney, whereas messenger RNA for B2 has been found in all tissues studied, including the kidney. It has been speculated that the B1 isoform might be involved in targeting to the plasma membrane. In the present study, we have characterized the B-subunit of the chicken osteoclast H+-ATPase using antibodies directed against peptides with isoform-specific or conserved sequences of the B-subunit. Western analysis was performed on chicken osteoclast membrane vesicles and on partially purified chicken osteoclast H+-ATPase and was compared with similar analysis of H+-ATPase isolated from bovine kidney and brain. The B1-specific antibody reacted with a polypeptide of approximately 56 kD on immunoblots of the renal H+-ATPase, whereas no reaction could be detected against the osteoclast H+-ATPase or the osteoclast membrane vesicle preparation. In contrast, the antibody against a B2-specific sequence reacted with a peptide of approximately 56 kD on immunoblots of the osteoclast H+-ATPase, the renal H+-ATPase, and the clathrin-coated vesicle H+-ATPase. The antibody against a conserved region of the B-subunit did not generate any evidence for the presence of isoforms other than B2 in the osteoclast. Immunocytochemistry of rat osteoclasts on bovine bone slices using the B2 antibody showed intense polarized staining along the plasma membrane facing the bone surface in actively resorbing osteoclasts whereas nonresorbing osteoclasts were diffusely stained throughout the cytoplasm. By confocal microscopy, the B2 staining was located to the level of the ruffled membrane and appeared to be concentrated to the peripheral areas of the membrane adjacent to the sealing zone. We conclude that the osteoclast vacuolar H+-ATPase contains the B2 isoform and suggest that upon initiation of resorption the pump is translocated from the cell interior to a special domain of the ruffled membrane close to the sealing zone.