DIFFERENTIAL USE OF TRANSCRIPTION ACTIVATION-FUNCTION-2 DOMAIN OF THEVITAMIN-D-RECEPTOR BY 1,25-DIHYDROXYVITAMIN-D-3 AND ITS A-RING-MODIFIED ANALOGS

Analogs of 1,25-dihydroxyvitamin D-3 (1,25D(3)) can be used to elucida te details of vitamin D receptor (VDR) activation. The A ring-modified analog, (TN-2) has 15-fold less affinity for VDR, but its transcripti onal activity is diminished 1000-fold. Likewise, the ability of TN-2 t o induce a protease-resistant conformation in VDR is 1/1000 that of 1, 25D(3). The stability of the VDR-TN-2 complexes is also significantly tower than VDR-1,25D(3) complexes. Mapping the VDR-binding site of TN- 2 showed that it had a significantly greater requirement for transcrip tion activation function 2 (AF-2) residues than 1,25D(3) did. These re sults suggest that the increased requirement for AF-2 residues that wa s induced by the A ring modifications is associated with diminished re ceptor activation. To determine whether restoring the potency of TN-2 by additional structural modifications would change the requirements f or AF-2 residues, we synthesized hybrid analogs with 1 beta-hydroxymet hyl-3-epi groups and with dimethyl groups at positions 26 and 27 of th e side chain, without or with a double bond between CD ring positions 16 and 17. We found that the side chain modification enhanced transcri ptional activity 150-fold, increased the ability of the receptor to fo rm a protease-resistant conformation 100-fold, and stabilized the VDR- analog complexes. The addition of the 16-ene group further reduced the analog's dissociation rate and increased its potency in the protease assays. These functional changes in the hybrid analogs were associated with a significant reduction in interaction with AF-2 residues. We co nclude that there is an inverse relationship between analogs' potencie s and their interaction with AF-2 residues of VDR.