Abstract

The immediate goal of this research proposal is to probe and critically investigate the structure/function relationship between the calciotropic hormone, 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3), and its receptor (vitamin D receptor, VDR) with a particular focus on the hormone-binding domain-structure of VDR. We propose to utilize our recently developed affinity labelling method to covalently label the hormone-binding pocket of human recombinant VDR (hVDR), fragment it proteolytically, and isolate the peptide bearing majority of the label. Sequence-determination of this peptide will delineate the binding pocket of hVDR as well as the contact points within this area. We will also develop alternate affinity analogs, with affinity probes at different part of the 1,25(OH)2D3, molecule, to determine the specificity of labelling. Functional tests for this labelling process will be provided by developing mutant hVDR proteins, point-mutated at contact points (delineated by our affinity studies), and determining the binding characteristics of 1.25(OH)2D3 and other metabolites of vitamin D3 towards these mutant proteins. Knowledge of the hormone-binding domain structure will be important to properly understand the pathophysiology of human vitamin D deficient rickets type II syndromes which involve natural mutations at hormone- and DNA-binding regions of hVDR. Information about the contact points (within the hormone-binding pocket) will also be useful in identifying the important recognition markers in the 1,25(OH)2D3 molecule, which, in turn will be useful in chemically synthesizing analogs of 1.25(OH)2D3. These analogs will be endowed with recently discovered anti-cancer properties of 1,25(OH)2D3 with less toxic side effects. The affinity labelling method will also be very useful in the identification and characterization of the putative 'membrane-bound vitamin D receptor' in tissues where 1.25(HO)2D3 is known to induce very rapid biologic effects (non-genomic effects). In summary the projects outlined in the application will provide important information regarding various physiologic properties of 1,25(OH)2D3 hormone.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
3R01DK047418-04S1
Application #
2624498
Study Section
General Medicine B Study Section (GMB)
Program Officer
Margolis, Ronald N
Project Start
1994-08-01
Project End
2000-02-29
Budget Start
1997-08-06
Budget End
2000-02-29
Support Year
4
Fiscal Year
1997
Total Cost
Indirect Cost

  Institution

Name
Boston University
Department
Miscellaneous
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118

  Publications

Kaya, Taner; Swamy, Narasimha; Persons, Kelly S et al. (2009) Covalent labeling of nuclear vitamin D receptor with affinity labeling reagents containing a cross-linking probe at three different positions of the parent ligand: structural and biochemical implications. Bioorg Chem 37:57-63
Kisker, Oliver; Onizuka, Shinya; Becker, Christian M et al. (2003) Vitamin D binding protein-macrophage activating factor (DBP-maf) inhibits angiogenesis and tumor growth in mice. Neoplasia 5:32-40
Addo, James K; Swamy, Narasimha; Ray, Rahul (2002) The C(19) position of 25-hydroxyvitamin D(3) faces outward in the vitamin D sterol-binding pocket of vitamin D-binding protein. Bioorg Med Chem Lett 12:279-81
Mohr, S C; Swamy, N; Xu, W et al. (2001) Why do we need a three-dimensional architecture of the ligand-binding domain of the nuclear 1alpha,25-dihydroxyvitamin D(3) receptor? Steroids 66:189-201
Swamy, N; Ghosh, S; Schneider, G B et al. (2001) Baculovirus-expressed vitamin D-binding protein-macrophage activating factor (DBP-maf) activates osteoclasts and binding of 25-hydroxyvitamin D(3) does not influence this activity. J Cell Biochem 81:535-46
Swamy, N; Addo, J K; Ray, R (2000) Development of an affinity-driven cross-linker: isolation of a vitamin D receptor associated factor. Bioorg Med Chem Lett 10:361-4
Nemere, I; Ray, R; McManus, W (2000) Immunochemical studies on the putative plasmalemmal receptor for 1, 25(OH)(2)D(3). I. Chick intestine. Am J Physiol Endocrinol Metab 278:E1104-14
Swamy, N; Xu, W; Paz, N et al. (2000) Molecular modeling, affinity labeling, and site-directed mutagenesis define the key points of interaction between the ligand-binding domain of the vitamin D nuclear receptor and 1 alpha,25-dihydroxyvitamin D3. Biochemistry 39:12162-71
Swamy, N; Addo, J; Vskokovic, M R et al. (2000) Probing the vitamin D sterol-binding pocket of human vitamin D-binding protein with bromoacetate affinity labeling reagents containing the affinity probe at C-3, C-6, C-11, and C-19 positions of parent vitamin D sterols. Arch Biochem Biophys 373:471-8
Chen, M L; Ray, S; Swamy, N et al. (1999) Mechanistic studies to evaluate the enhanced antiproliferation of human keratinocytes by 1alpha,25-dihydroxyvitamin D(3)-3-bromoacetate, a covalent modifier of vitamin D receptor, compared to 1alpha,25-dihydroxyvitamin D(3). Arch Biochem Biophys 370:34-44

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