Gacad M A, Adams J S
Division of Endocrinology and Metabolism, Cedars-Sinai Medical Center, University of California School of Medicine, Los Angeles 90049.
Endocrinology. 1992 Dec;131(6):2581-7. doi: 10.1210/endo.131.6.1446602.
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.
我们最近描述了抗维生素D的新大陆灵长类动物细胞中存在一种竞争性结合成分，其亲和力相对较低（解离常数Kd约为10⁻⁸M），但与维生素D受体（VDR）相比，对1,25 - 二羟基维生素D3 [1,25-(OH)₂D3]具有高结合能力。在此我们表明，这种结合成分不仅能够结合1,25-(OH)₂D3以外的维生素D3代谢物，还能结合结构上与维生素D3甾醇不同的类固醇激素。我们研究了在存在和不存在潜在竞争性配体（包括25OHD3、1,25-(OH)₂D3、17β - 雌二醇、睾酮和孕酮，浓度范围为1 - 100 nM）的情况下，抗维生素D的狨猴淋巴母细胞系B95 - 8提取物中[³H]1,25-(OH)₂D3和[³H]25 - 羟基维生素D3（[³H]25OHD3）的结合情况。与含有真实VDR的提取物相比，B95 - 8细胞提取物以重量计结合的1,25-(OH)₂D3多180%，结合的25OHD3多12倍。这种结合剂对25OHD3的亲和力是其对1,25-(OH)₂D3亲和力的2.2倍。此外，在接近这种结合剂对1,25-(OH)₂D3的Kd浓度时，25OHD3在与[³H]1,25-(OH)₂D3竞争结合方面比1,25-(OH)₂D3有效3倍。这种结合剂从Sephadex G - 100柱上洗脱时表观分子量为58千道尔顿，汇集该柱上包含此分子量范围的洗脱级分能够抑制1,25-(OH)₂D3与VDR的结合达65%。竞争性类固醇结合分析表明，雌二醇在抑制[³H]1,25-(OH)₂D3结合方面至少与1,25-(OH)₂D3一样有效；以17β - 雌二醇作为标记和竞争性配体的同源结合研究表明，成功取代[³H]1,25-(OH)₂D3的性腺类固醇浓度也能取代17β - [³H]雌二醇。使用[³H]25OHD3作为标记配体以及更广泛的一系列竞争性配体，类固醇结合的顺序为25OHD3 > 1,25-(OH)₂D3 ≥ 雌二醇 = 孕酮 = 睾酮。这些结果表明，新大陆灵长类动物中类固醇激素抵抗的表型可能是由于一种细胞内58千道尔顿蛋白质的过度表达，该蛋白质通过竞争配体结合来干扰类固醇 - 受体相互作用。
