title: The V-ATPase a3 subunit mutation R740S is dominant negative and results in osteopetrosis in mice.
abstract: A mouse founder with high bone mineral density and an osteopetrotic phenotype was identified in an N-ethyl-N-nitrosourea (ENU) screen. It was found to carry a dominant missense mutation in the Tcirg1 gene that encodes the a3 subunit of the vacuolar type H(+)-ATPase (V-ATPase), resulting in replacement of a highly conserved amino acid (R740S). The +/R740S mice have normal appearance, size, and weight but exhibit high bone density. Osteoblast parameters are unaffected in bones of +/R740S mice, whereas osteoclast number and marker expression are increased, concomitant with a decrease in the number of apoptotic osteoclasts. Consistent with reduced osteoclast apoptosis, expression of Rankl and Bcl2 is elevated, whereas Casp3 is reduced. Transmission electron microscopy revealed that unlike other known mutations in the a3 subunit of V-ATPase, polarization and ruffled border formation appear normal in +/R740S osteoclasts. However, V-ATPases from +/R740S osteoclast membranes have severely reduced proton transport, whereas ATP hydrolysis is not significantly affected. We show for the first time that a point mutation within the a3 subunit, R740S, which is dominant negative for proton pumping and bone resorption, also uncouples proton pumping from ATP hydrolysis but has no effect on ruffled border formation or polarization of osteoclasts. These results suggest that the V(0) complex has proton-pumping-independent functions in mammalian cells.
authors.affiliation: Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Noelle
authors.initials: N
authors.lastname: Ochotny
authors.forename: Ann M
authors.initials: AM
authors.lastname: Flenniken
authors.forename: Celeste
authors.initials: C
authors.lastname: Owen
authors.forename: Irina
authors.initials: I
authors.lastname: Voronov
authors.forename: Ralph A
authors.initials: RA
authors.lastname: Zirngibl
authors.forename: Lucy R
authors.initials: LR
authors.lastname: Osborne
authors.forename: Janet E
authors.initials: JE
authors.lastname: Henderson
authors.forename: S Lee
authors.initials: SL
authors.lastname: Adamson
authors.forename: Janet
authors.initials: J
authors.lastname: Rossant
authors.forename: Morris F
authors.initials: MF
authors.lastname: Manolson
authors.forename: Jane E
authors.initials: JE
authors.lastname: Aubin
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D001692:Biological Transport; D002452:Cell Count; D048430:Cell Shape; D004252:DNA Mutational Analysis; D005260:Female; D005269:Femur / Q000000981:diagnostic imaging / Q000473:pathology; D005799:Genes, Dominant / Q000235:genetics*; D006579:Heterozygote; D006868:Hydrolysis; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D020125:Mutation, Missense / Q000235:genetics*; D010006:Osteoblasts / Q000378:metabolism / Q000473:pathology; D010010:Osteoclasts / Q000378:metabolism / Q000473:pathology; D010022:Osteopetrosis / Q000235:genetics* / Q000473:pathology; D021122:Protein Subunits / Q000235:genetics*; D011522:Protons; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics*; D055114:X-Ray Microtomography
chemical_list: C484226:Atp6ap1 protein, mouse; D021122:Protein Subunits; D011522:Protons; D000255:Adenosine Triphosphate; D025262:Vacuolar Proton-Translocating ATPases