Vacuolar V-ATPases (V-ATPases) have an essential role in the endocytic pathways of eukaryotic cells. In some cell types, like osteoclasts and certain renal epithelial cells, V-ATPases are expressed at high levels and are required for the specialized functions of the cells. In osteoclasts and renal epithelial cells, V-ATPases are stored in vesicles or tubules in the cytoplasm until the cell encounters an activating signal. V-ATPases are then transported to specialized domains of the plasma membrane. Although this transport is a crucial means by which V-ATPases are regulated, until recently little was known about the underlying mechanisms. A specific interaction between V-ATPases and filamentous actin (F-actin) has been identified by our lab. This represents the first example of a direct interaction between an ion pump and microfilaments. This grant will test the hypothesis that interaction between V-ATPase and F-actin accounts for the transport of V-ATPases in osteoclasts and other cells. The binding interaction between V-ATPase and F-actin is mediated by the B-subunit of V-ATPase; the B-subunit thus represents a new and unique member of the family of actin binding proteins. The interaction between actin and V-ATPase can be reconstituted using bacterially-expressed fusion proteins representing the N-terminal domains of B-subunits. This grant will continue characterization of the binding interaction between V-ATPase and actin using molecular and biochemical techniques with the goal of understanding the binding interaction in great detail. With this information, mutated molecular constructs of the B-subunit will be created which are able to integrate with other VATPase subunits to support proton pumping activity, but which lack the capacity to bind actin. These constructs will be expressed in an osteoclast cell culture model to test the physiologic importance of the binding interaction between V-ATPase and F-actin.
The specific aims are: I. To obtain detailed molecular information about the F-actin binding site on the B-subunit, and generate mutant B subunits that lack actin binding but retain catalytic activity. II. To study effects of V-ATPase-F-actin binding on F-actin organization and V-ATPase enzymatic function. III. To test the importance of the binding interaction in osteoclast cell culture models. Because V-ATPases are crucial enzymes to the lives of all cells, and have been implicated in clinical disorders including osteoporosis and renal tubular acidosis, understanding their regulation will be of great importance for both basic and clinical science.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR047959-04
Application #
6772566
Study Section
General Medicine B Study Section (GMB)
Program Officer
Sharrock, William J
Project Start
2001-09-17
Project End
2006-06-30
Budget Start
2004-07-01
Budget End
2005-06-30
Support Year
4
Fiscal Year
2004
Total Cost
$240,231
Indirect Cost
Name
University of Florida
Department
Dentistry
Type
Schools of Dentistry
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611
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Zuo, Jian; Vergara, Sandra; Kohno, Shinya et al. (2008) Biochemical and functional characterization of the actin-binding activity of the B subunit of yeast vacuolar H+-ATPase. J Exp Biol 211:1102-8
Zuo, J; Archer, L A; Cooper, A et al. (2007) Nuclear factor kappaB p65 phosphorylation in orthodontic tooth movement. J Dent Res 86:556-9
Zuo, Jian; Jiang, Jin; Chen, Shih-Hua et al. (2006) Actin binding activity of subunit B of vacuolar H+-ATPase is involved in its targeting to ruffled membranes of osteoclasts. J Bone Miner Res 21:714-21
Holliday, L Shannon; Bubb, Michael R; Jiang, Jin et al. (2005) Interactions between vacuolar H+-ATPases and microfilaments in osteoclasts. J Bioenerg Biomembr 37:419-23
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Chen, Shih-Hua; Bubb, Michael R; Yarmola, Elena G et al. (2004) Vacuolar H+-ATPase binding to microfilaments: regulation in response to phosphatidylinositol 3-kinase activity and detailed characterization of the actin-binding site in subunit B. J Biol Chem 279:7988-98
Jiang, Jin; Zuo, Jian; Hurst, I Rita et al. (2003) The synergistic effect of peptidoglycan and lipopolysaccaride on osteoclast formation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 96:738-43
Jiang, Jin; Zuo, Jian; Chen, Shih-Hua et al. (2003) Calcium hydroxide reduces lipopolysaccharide-stimulated osteoclast formation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 95:348-54
Holliday, L S; Vakani, A; Archer, L et al. (2003) Effects of matrix metalloproteinase inhibitors on bone resorption and orthodontic tooth movement. J Dent Res 82:687-91

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