Studies on the mode of action of vitamin D—XIV. Quantitative assessment of the structural requirements for the interaction of 1α,25-dihydroxyvitamin D 3 with its chick intestinal mucosa receptor system

Abstract

The structural requirements for the interaction of the 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2D 3] molecule with its chick intestinal mucosa receptor system have been quantitatively evaluated. This analysis was carried out using structural analogs of 1α,25-dihydroxyvitamin D 3 in a competitive binding assay with a reconstituted chromatin-cytosol system from rachitic chick intestinal mucosa. In this assay the steroid-cytosol receptor complex binds avidly to the chromatin. The results for the melabolites and analogs were expressed on a linear scale of relative competitive index (RCI) where the RCI of 1α,25(OH) 2D 3 is defined as 100. These studies demonstrated that the 1α-and 25-hydroxyl functions were the most critical groups to the binding process: their absence reduced the RCI to 0.5 and 0.4 respectively. The 3β-hydroxyl, although important, is somewhat less critical since the RCI was reduced to only 5.7. When all three of these hydroxyls are present, the receptor will tolerate alterations in the side chain at carbon-24. Thus shortening the side chain of 1α,25(OH) 2D 3 by only one methylene group reduced the RCI to 46. When the 25-hydroxylated side chain is present, modification of the A-ring at C-1, C-3, C-4, C-10 and C-19 all markedly reduce the RCI. Competitors with both A-ring and side chain modifications were found to have almost no ability to interact with the receptor system. Collectively these results support the view that the intestinal cytosol-chromatin receptor system has a very high degree of specificity for its hormonal ligand which reflect bonding-interactions at multiple (carbons 1, 3, 4, 10, 19, 24, and 25) sites on the 1α.25(OH) 2D 3 molecule. A model is presented for this interaction which emphasizes the key role of a standard length side chain with a 25-OH functional group.