The hormonally active form of vitamin D, 1,25-dihydroxyvitamin D3, and its associated intracellular receptor are members of a unique family of ligand-inducible transcription factors. The primary interest of this investigator is determining the means by which the hormone-receptor complex alters transcriptional events, with an underlying emphasis on the endocrine, metabolic and immunologic abnormalities of patients with renal failure. This includes examination of DNA binding mechanisms, modifications of the receptor or its expression, and interactions with other proteins. The present application seeks to test the hypothesis that the vitamin D receptor binds in a qualitatively different manner to representative positive and negative DNA response elements. In addition, the orientation and nature of the complex will be defined on selected response elements. To carry out this work, the electrophoretic gel mobility shift assay will be utilized to identify receptor-DNA complexes using antibody and competition experiments. Once the identity of the receptor-DNA complexes is established, relative strengths of binding will be ascertained and absolute mobilities will be compared. Interference footprinting experiments in conjunction with the mobility shift assay will be used to identify the intimate sites of DNA contact. This will include examination of the DNA phosphate backbone as well as major groove interactions with individual bases, including guanine, thymidine and adenine residues. Comparisons between both positive and negative response elements will permit more rigorous determination of those contacts critical for high affinity interactions, and delineate differences that may influence receptor function. Because of the propensity of the vitamin D receptor to heterodimerize during DNA binding, a series of bromodeoxyuridine crosslinking experiments are planned to assess the nature of the bound complexes. Crosslinking of the receptor to bromodeoxyuridine substituted, radioactively labeled DNA response elements by ultraviolet light will covalently """"""""tag"""""""" the protein components of the complex in intimate contact with the DNA. Fractionation of the resultant mix by SDS-PAGE will permit an estimation of the size of linked protein(s), and, given the appropriate strategy, their orientation with respect to the DNA binding site. This work represents the first attempt to rigorously identify the points of DNA contact made by the vitamin D receptor to its response elements and should provide insight into the functional aspects of the protein.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
1R29DK047883-01
Application #
2147800
Study Section
General Medicine B Study Section (GMB)
Project Start
1994-06-01
Project End
1999-05-31
Budget Start
1994-06-01
Budget End
1995-05-31
Support Year
1
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Kentucky
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
832127323
City
Lexington
State
KY
Country
United States
Zip Code
40506
Langub, M C; Herman, J P; Malluche, H H et al. (2001) Evidence of functional vitamin D receptors in rat hippocampus. Neuroscience 104:49-56
Koszewski, N J; Kiessling, S; Malluche, H H (2001) Isolation of genomic DNA sequences that bind vitamin D receptor complexes. Biochem Biophys Res Commun 283:188-94
Langub, M C; Monier-Faugere, M C; Qi, Q et al. (2001) Parathyroid hormone/parathyroid hormone-related peptide type 1 receptor in human bone. J Bone Miner Res 16:448-56
Koszewski, N J; Malluche, H H; Russell, J (2000) Vitamin D receptor interactions with positive and negative DNA response elements: an interference footprint comparison. J Steroid Biochem Mol Biol 72:125-32
Russell, J; Ashok, S; Koszewski, N J (1999) Vitamin D receptor interactions with the rat parathyroid hormone gene: synergistic effects between two negative vitamin D response elements. J Bone Miner Res 14:1828-37
Koszewski, N J; Ashok, S; Russell, J (1999) Turning a negative into a positive: vitamin D receptor interactions with the avian parathyroid hormone response element. Mol Endocrinol 13:455-65
Koszewski, N J; Reinhardt, T A; Horst, R L (1999) Differential effects of 20-epi vitamin D analogs on the vitamin D receptor homodimer. J Bone Miner Res 14:509-17
Koszewski, N J; Reinhardt, T A; Langub, M C et al. (1998) Selectivity of a C-terminal peptide antiserum for different DNA-binding states of the vitamin D receptor. Arch Biochem Biophys 349:388-96
Sawaya, B P; Koszewski, N J; Qi, Q et al. (1997) Secondary hyperparathyroidism and vitamin D receptor binding to vitamin D response elements in rats with incipient renal failure. J Am Soc Nephrol 8:271-8
Koszewski, N J; Reinhardt, T A; Horst, R L (1996) Vitamin D receptor interactions with the murine osteopontin response element. J Steroid Biochem Mol Biol 59:377-88

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