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Adenosine 5'-Triphosphate-Dependent Vitamin D Sterol Binding to Heat Shock Protein-70 Chaperones

Burns and Allen Research Institute and Division of Endocrinology, Diabetes ,and Metabolism (R.C., M.A.G., J.S.A.), Cedars-Sinai Medical Center, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, California 90048; and Division of Medical Sciences (M.H.), Institute of Biomedical Research, University of Birmingham, Birmingham B15 2TT, United Kingdom

Address all correspondence and requests for reprints to: John S. Adams, Division of Endocrinology, Diabetes, and Metabolism, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Room B-131, Los Angeles, California 90048. E-mail: adamsj{at}cshs.org.

Chaperone proteins in the heat shock protein-70 family possess endogenous ATP binding and ATPase activity and interact with intracellular protein substrates in an ATP-dependent manner; the hydrolysis of ATP to ADP results in an increase in the affinity of the chaperone for protein substrates. Heat shock protein-70s can also specifically interact with 25-hydroxylated vitamin D metabolites. Using constitutively expressed heat shock protein-70 (hsc70) as chaperone, here we demonstrate that vitamin D metabolite binding to hsc70 is also ATP dependent. Transient overexpression of an hsc70-green fluorescent protein chimeric construct in primate kidney cells resulted in a 6-fold increase in specific, extractable 25-hydroxyvitamin D₃ binding. When ATPase capability of hsc70 was disabled, this increase was completely blocked. In solution, the binding of 25-hydroxylated vitamin D metabolites to hsc70 was significantly increased (P < 0.01) in the presence of ATP and a nonmetabolizable ATP analog. The ATP-directed increase in specific binding resulted from an increase in the abundance of relatively high-affinity hormone-binding sites (K_d, 0.24 nM). These results suggest that ATP hydrolysis to ADP would favor the release of vitamin D from a donor hsc70 molecule at a time when an hsc70-bound acceptor protein substrate is anchored to the chaperone with relative avidity. We theorize that the endogenous ATPase activity of hsc70 promotes the transfer of vitamin D sterols to other intracellular vitamin D binding proteins, such as the vitamin D receptor and vitamin D hydroxylases, to which hsc70 is known to bind.

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