SPECIFICITY OF STEROID BINDING IN NEW-WORLD PRIMATE B95-8 CELLS WITH A VITAMIN-D-RESISTANT PHENOTYPE

We recently described the existence of a competitive binding component in vitamin D-resistant New World primate cells that has a relatively low affinity (K(d), approximately 10(-8) M) but high capacity for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] compared to that possessed by the vitamin D receptor (VDR). Here we show that this binding component is capable of binding a vitamin D3 metabolite other than 1,25-(OH)2D3 as well as steroid hormones structurally disparate from vitamin D3 sterols. We studied the binding of [H-3]1,25-(OH)2D3 and [H-3]25-hydroxyvitamin D3 ([H-3]25OHD3) in extracts of the vitamin D-resistant marmoset lymphoblastic cell line B95-8 in the presence and absence of potential competitive ligands, including 25OHD3,1,25-(OH)2D3,17beta-estradiol, testosterone, and progesterone, at concentrations ranging from 1-100 nm. Compared to extracts containing the authentic VDR, extracts of B95-8 cells bound 180% more 1,25-(OH)2D3 and 12-fold more 25OHD3 on a weight basis. The affinity of this binder for 25OHD3 was 2.2 times as great as its affinity for 1,25-(OH)2D3. Further, at concentrations approaching the K(d) of this binder for 1,25-(OH)2D3, 25OHD, was 3 times more effective than 1,25-(OH)2D3 in competing with [H-3]1,25-(OH)2D3 for binding. This binder eluted from a Sephadex G-100 column with an apparent mol wt of 58 kilodaltons, and pooled elution fractions from the column encompassing this mol wt range were capable of inhibiting binding of 1,25-(OH)2D3 to the VDR by 65%. Competitive steroid binding analyses showed estradiol to be at least as effective as 1,25-(OH)2D3 in inhibition of [H-3] 1,25- (OH)2D3 binding; homologous binding studies with 17beta-estradiol as labeled and competitive ligand demonstrated that concentrations of the gonadal steroid that successfully displaced [H-3]1,25-(OH)2D3 also displaced 17beta-[H-3] estradiol. Using [H-3]25OHD3 as the labeled ligand and a more extensive array of competitive ligands, the rank order of steroid binding was 25OHD3 > 1,25-(OH)2D3 greater-than-or-equal-to estradiol = progesterone = testosterone. These results suggest that the phenotype of steroid hormone resistance in New World primates may result from the overexpression of an intracellular 58-kilodalton protein(s) that interferes with the steroid-receptor interaction by competing for ligand binding.