Development of Novel Bioluminescent Sensor to Detect and Discriminate between Vitamin D Receptor Agonists and Antagonists in Living Cells.
Active forms of vitamin D regulate the expression of multiple genes that play essential roles in calcium and phosphate homeostasis, cell differentiation, and the immune system via the vitamin D receptor (VDR). Many vitamin D analogs have been synthesized for clinical use in the treatment of type I rickets, osteoporosis, renal osteodystrophy, psoriasis, leukemia, and breast cancer. We have constructed two fusion proteins containing split-luciferase and the ligand binding domain (LBD) of the VDR designated as LucN-LBD-LucC and LucC-LBD-LucN. Remarkably, the LucC-LBD-LucN, which has the C-terminal domain of luciferase at the N-terminus of the fusion protein, was a significantly better biosensor than LucN-LBD-LucC. Addition of the VDR agonists to COS-7 cells expressing LucC-LBD-LucN dramatically reduced luciferase activity. In contrast, the VDR antagonist significantly increased the chimeric luciferase activity in a dose- and time-dependent manner. Our results on chimeric luciferases containing the LBDs of mutant VDRs derived from patients with vitamin D-dependent type II rickets indicated that our system could detect a conformational change of the LBD of the VDR likely based on a positional change of the helix 12, which occurs upon ligand binding. This novel system to detect and discriminate between VDR agonists and antagonists could be useful for the screening and identification of chemical compounds that bind to normal or mutant VDRs with high affinity.
Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University , 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
Hiroki
H
Mano
Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University , 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.|Imizu Institute, TOPU BIO RESEARCH Co., Ltd , 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
Miyu
M
Nishikawa
Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University , 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
Kaori
K
Yasuda
Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University , 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
Shinichi
S
Ikushiro
Faculty of Pharmaceutical Sciences, Teikyo University , Itabashi, Tokyo 173-8605, Japan.
Nozomi
N
Saito
Faculty of Pharmaceutical Sciences, Teikyo University , Itabashi, Tokyo 173-8605, Japan.
Masashi
M
Takano
Faculty of Pharmaceutical Sciences, Teikyo University , Itabashi, Tokyo 173-8605, Japan.
Atsushi
A
Kittaka
Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University , 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.|Imizu Institute, TOPU BIO RESEARCH Co., Ltd , 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
Toshiyuki
T
Sakaki
D000818:Animals; D001665:Binding Sites; D015374:Biosensing Techniques; D019556:COS Cells; D002117:Calcitriol / Q000494:pharmacology; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D008024:Ligands; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods*; D018167:Receptors, Calcitriol / Q000819:agonists* / Q000037:antagonists & inhibitors*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014807:Vitamin D / Q000031:analogs & derivatives / Q000494:pharmacology
C000609688:25-dehydro-1alpha-hydroxy-24,24-dimethylvitamin D3-26,23-lactone; D008024:Ligands; D018167:Receptors, Calcitriol; D011993:Recombinant Fusion Proteins; D014807:Vitamin D; D008156:Luciferases; D002117:Calcitriol