Abstract
Vacuolar H(+)-ATPase (V-ATPase) binds microfilaments, and that interaction may be mediated by an actin binding domain in subunit B of the enzyme. To test for possible physiologic functions of the actin binding activity of V-ATPase, early responses of resorbing osteoclasts to inhibition of phosphatidylinositol 3-kinase activity by wortmannin and LY294002 were examined. Rapid co-localization between V-ATPase and F-actin was demonstrated by immunocytochemistry, and corresponding association between V-ATPase and F-actin in immunoprecipitations and pelleting assays was detected. This response was reversed as osteoclasts recovered resorptive activity after inhibitors were removed. By expressing and characterizing fusion proteins containing segments of the actin-binding amino-terminal regions of the B subunits of V-ATPase, we mapped the actin-binding site to a 44-amino acid domain. An 11-amino acid segment with a sequence similar to the actin-binding site of human profilin I was detected within this region. 13-Mers containing these profilin-like segments bound actin in fluorescent anisotropy studies and competed with profilin for binding to actin. Using site-directed mutagenesis, the 11-amino acid profilin-like actin-binding motifs (amino acids 49-59 of B1 and 55-65 of B2) were replaced with an 11-amino acid spacer with a sequence based on the homologous sequence from subunit B of Pyrococcus horikoshii, an organism that lacks an actin cytoskeleton. These substitutions eliminated the actin-binding activity of the B subunit fusion proteins. In summary, binding between V-ATPase and F-actin in osteoclasts occurs in response to blocking phosphatidylinositol 3-kinase activity. This response was fully reversible. The actin binding activities of the B subunits of V-ATPase required 11-amino acid actin-binding motifs that are similar in sequence to the actin-binding site of mammalian profilin I.
Highlights
From the Departments of ‡Orthodontics and ʈEndodontics, University of Florida College of Dentistry, Gainesville, Florida 32610, §The Research Service, Malcolm Randall Veterans Affairs Medical Center, Gainesville, Florida 32608, the Departments of ¶Medicine and ‡‡Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, Florida 32610, and the **Department of Physiology and Cell Biology, Ohio State University College of Medicine, Columbus, Ohio 43210
We previously found that the actin binding activity of the B subunit was located in the amino-terminal 106 amino acids of B1 and 112 amino acids of B2 [8]
We demonstrate that the binding sites are in 44-amino acid sections of the B subunits and that a portion of the binding sites, which is similar in sequence to the actin-binding site of mammalian profilin I, is required for the actin binding activities of the B subunits
Summary
From the Departments of ‡Orthodontics and ʈEndodontics, University of Florida College of Dentistry, Gainesville, Florida 32610, §The Research Service, Malcolm Randall Veterans Affairs Medical Center, Gainesville, Florida 32608, the Departments of ¶Medicine and ‡‡Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, Florida 32610, and the **Department of Physiology and Cell Biology, Ohio State University College of Medicine, Columbus, Ohio 43210. Using site-directed mutagenesis, the 11-amino acid profilin-like actin-binding motifs (amino acids 49 –59 of B1 and 55– 65 of B2) were replaced with an 11-amino acid spacer with a sequence based on the homologous sequence from subunit B of Pyrococcus horikoshii, an organism that lacks an actin cytoskeleton. These substitutions eliminated the actin-binding activity of the B subunit fusion proteins. As the enzyme primarily responsible for the acidification of these compartments, as well as the polarized secretion of protons in certain specialized cell types, such as osteoclasts and intercalated cells of the kidney, the vacuolar Hϩ-ATPase (V-ATPase) would appear a likely candidate for linkage to the cytoskeleton [7]. A PDZ-binding domain was identified in the carboxyl terminus of B1, and data were presented suggesting that B1 interacts with Naϩ/Hϩ ex-
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