Differential effects of 20-epi vitamin D analogs on the vitamin D receptor homodimer.
Vitamin D analogs have received increased attention because of their possible therapeutic benefits in treating osteoporosis and various proliferative disorders. Several analogs were examined for their effects on DNA binding of the vitamin D receptor (VDR) homodimer complex with the murine osteopontin vitamin D response element. All of the tested analogs increased complex binding by recombinant human VDR in the electrophoretic mobility shift assay and notable differences in mobility of these complexes were observed. A panel of C-terminal anti-VDR antisera were screened for their ability to interact with analog-bound VDR homodimer complexes or as a heterodimer complex with recombinant human retinoid X receptor alpha (rhRXR alpha). Like calcitriol, analog-bound heterodimer complexes were largely resistant to interaction with these antisera; however, striking differences were observed with the various antisera in an analogous homodimer binding experiment. KH1060 and CB1093, analogs with 20-epi conformations, produced homodimer complexes that were 3- to 6-fold more resistant to supershifting with Ab180 compared with the hormone or EB1089. Chymotrypsin digestion in combination with Western blotting using a C-terminal anti-VDR antiserum revealed similar digestion patterns for all ligands. However, KH1060- and CB1093-bound VDR complexes were more resistant to digestion than either calcitriol or EB1089. Finally, the ability of these compounds to yield stable homodimer complexes was assessed by challenging preformed homodimer with the exogenous addition of rhRXR alpha extracts. Although new heterodimer complexes appeared in a time-dependent fashion, the preformed homodimer complexes exhibited stable binding throughout the time course of the experiment. The results indicate that VDR homodimers are targets of vitamin D analogs with differential effects on C-terminal protein conformation that may partially explain the varied biological responses of these compounds.
Koszewski
N J
NJ
University of Kentucky Medical Center, Department of Internal Medicine, Lexington, USA.
Reinhardt
T A
TA
Horst
R L
RL
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001665:Binding Sites; D019281:Dimerization; D006801:Humans; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D051381:Rats; D018167:Receptors, Calcitriol / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000378:metabolism; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology
D000906:Antibodies; D008024:Ligands; D018167:Receptors, Calcitriol; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D014807:Vitamin D