Portions of this work were presented at the 19th Annual Meeting of the American Society for Bone and Mineral Research, Cincinnati, Ohio, U.S.A., 1997.
The Vitamin D Receptor Gene Start Codon Polymorphism: A Functional Analysis of FokI Variants†
Article first published online: 1 NOV 1998
Copyright © 1998 ASBMR
Journal of Bone and Mineral Research
Volume 13, Issue 11, pages 1691–1699, November 1998
How to Cite
Gross, C., Krishnan, A. V., Malloy, P. J., Eccleshall, T. R., Zhao, X.-Y. and Feldman, D. (1998), The Vitamin D Receptor Gene Start Codon Polymorphism: A Functional Analysis of FokI Variants. J Bone Miner Res, 13: 1691–1699. doi: 10.1359/jbmr.1922.214.171.1241
- Issue published online: 4 DEC 2009
- Article first published online: 1 NOV 1998
- Manuscript Accepted: 27 JUL 1998
- Manuscript Revised: 22 JUN 1998
- Manuscript Received: 6 MAR 1998
The vitamin D receptor (VDR) gene contains a start codon polymorphism (SCP) which is three codons upstream of a second start site (ATG). The SCP genotype can be determined with the restriction enzyme FokI, where “f” indicates the presence of the restriction site and the first ATG, while “F” indicates its absence. Recent evidence suggests that the ff genotype is correlated with lower bone mineral density (BMD) in some populations. The SCP results in alternate VDRs that differ structurally, with the F variant (F-VDR) being three amino acids shorter than the f variant (f-VDR). To determine whether there are functional differences between the f-VDR and the F-VDR, we studied the two VDR forms expressed in COS-7 cells. The proteins were distinguishable from one another on Western blots by their different mobilities, confirming the larger size of f-VDR. Ligand binding studies showed no significant differences between the affinities of the two VDR forms for [3H]-1,25-dihydroxyvitamin D3 ([3H]-1,25(OH)2D3) (Kd = 131 ± 78 pM, f-VDR; Kd = 237 ± 190 pM, F-VDR; p = 0.24); however, a 2-fold difference in affinity can not be discriminated by this method. There were no differences in the abilities of the two receptor forms to bind DNA as determined by electrophoretic mobility shift assays. The ability of the two VDR forms to transactivate target genes was investigated using three different vitamin D responsive luciferase reporter constructs: 24-hydroxylase, osteocalcin, and osteopontin. In these transactivation experiments, 1,25(OH)2D3 dose-response (0.1–10 nM) curves revealed that the ED50 values for transactivation were indistinguishable between the two VDR forms. Additionally, cultured human fibroblasts with FF,Ff, and ff genotypes had similar sensitivity to 1,25(OH)2D3 with respect to the induction of 24-hydroxylase mRNA. In summary, we were unable to detect significant differences in ligand affinity, DNA binding, or transactivation activity between f-VDR and F-VDR forms. We must emphasize, however, that the sensitivity of the methods used limits our ability to detect minor differences in VDR affinity and function. In conclusion, we cannot define a mechanism whereby the SCP in the VDR might contribute to population differences in BMD.