2 MUTATIONS IN THE HORMONE-BINDING DOMAIN OF THE VITAMIN-D RECEPTOR CAUSE TISSUE RESISTANCE TO 1,25-DIHYDROXYVITAMIN-D3

We have identified and characterized two mutations in the hormone bind ing domain of the vitamin D receptor (VDR) in patients with hereditary vitamin D-resistant rickets. One patient was found to have a prematur e stop mutation (CAG to TAG) in the hinge region affecting amino acid 149 (Q149X) and the other demonstrated a missense mutation (CGC to CTC ) resulting in the substitution of arginine 271 by leucine (R27IL) in the steroid binding domain. Eukaryotic expression analyses in CV-1 cel ls showed the inability of both patients' VDR to induce transcription from the osteocalcin hormone gene response element at 10(-7) M 1,25-di hydroxyvitamin D3 (1,25(OH)2D3). Normal transcription levels could, ho wever, be elicted by the missense mutated VDR (R271L) in the presence of 1,000-fold higher 1,25-(OH)2D3 concentrations than needed for the w ild-type receptor. This shows that Arg 271 directly affects the affini ty of the VDR for its ligand and its conversion to leucine decreases i ts affinity for 1,25(OH)2D3 by a factor of 1,000. Arg 271 is located i mmediately 3-prime to a 30 amino acid segment (VDR amino acids 241-270 ) that is conserved among members of the steroid/thyroid/retinoid horm one receptor superfamily. These results represent the first missense m utation identified in the hormone binding domain of VDR and further de fine the structure-function relationship of 1,25(OH)2D3 ligand binding to its nuclear receptor.