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 (Kd, 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 [3H]1,25-(OH)2D3 and [3H]25-hydroxyvitamin D3 ([3H]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, 17 beta-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 Kd of this binder for 1,25-(OH)2D3, 25OHD3 was 3 times more effective than 1,25-(OH)2D3 in competing with [3H]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 [3H]1,25-(OH)2D3 binding; homologous binding studies with 17 beta-estradiol as labeled and competitive ligand demonstrated that concentrations of the gonadal steroid that successfully displaced [3H]1,25-(OH)2D3 also displaced 17 beta-[3H] estradiol. Using [3H]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 > or = 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.
Gacad
M A
MA
Division of Endocrinology and Metabolism, Cedars-Sinai Medical Center, University of California School of Medicine, Los Angeles 90049.
Adams
J S
JS
D000818:Animals; D001667:Binding, Competitive; D002112:Calcifediol / Q000378:metabolism*; D002117:Calcitriol / Q000378:metabolism*; D002144:Callithrix; D002460:Cell Line; D002850:Chromatography, Gel; D004351:Drug Resistance; D004958:Estradiol / Q000378:metabolism; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D011374:Progesterone / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism; D014807:Vitamin D / Q000494:pharmacology*
D014807:Vitamin D; D013739:Testosterone; D011374:Progesterone; D004958:Estradiol; D002117:Calcitriol; D002112:Calcifediol