Regulation of ligand-induced heterodimerization and coactivator interaction by the activation function-2 domain of the vitamin D receptor.
Regulation of ligand-induced heterodimerization and coactivator interaction by the activation function-2 domain of the vitamin D receptor.
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DOI：
10.1210/mend.14.11.0560
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发表时间：
2000-11
影响因子：
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通讯作者：
Yan-Yun Liu;C. Nguyen;S. Peleg
中科院分区：
文献类型：
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作者：
Yan-Yun Liu;C. Nguyen;S. Peleg
20种环磷酰胺类似物1α，25-二羟基维生素D3(1，25D3)的转录能力是天然激素的100-1000倍。为了确定这种增强的活性是通过调节二聚化过程还是通过与辅助激活剂的相互作用而介导的，我们进行了体外翻译的维生素D受体(IVtVDR)和含有谷胱甘肽-S-转移酶(GST)和维甲酸X受体的配体结合域(RXRAlpha)、类固醇受体辅助激活剂1的核受体相互作用域(SRc-1)或糖皮质激素受体相互作用蛋白1(GRIP-1)的融合蛋白的定量蛋白质相互作用分析。我们发现RXRpha和VDR配体结合域的异源二聚主要是Deltanid依赖的，VDR与SRC-1或GRIP-1的相互作用也是如此。1，25D3诱导异二聚反应的ED50为2 nM，20-epi-1，25D3为0.05 nM。然而，1，25D3和20-epI类似物诱导VDR与SRC-1相互作用的ED50与配体介导的VDR与GRIP-1相互作用的ED50相似(ED50=0.1-0.3 nM)。七肽9的突变同时减少了1，25D3和20-epi类似物介导的二聚化，而不改变这些配体与VDR的结合。VDR激活功能2(AF-2)结构域/螺旋12残基的突变降低了1，25D3诱导异源二聚化和与SRC-1相互作用的能力。这些突变没有改变20-epi-1，25D3诱导二聚化的能力，但确实降低了它诱导与SRC-1相互作用的能力。我们假设，激素和类似物都稳定了暴露VDR功能界面的受体构象。由于AF-2结构域的参与，这两种配体暴露这些功能界面的机制不同。
Twenty-epi analogs of 1alpha,25-dihydroxyvitamin D3 (1,25D3) are 100-1000 times more potent transcriptionally than the natural hormone. To determine whether this enhanced activity is mediated through modulation of the dimerization process or through interaction with coactivators, we performed quantitative protein-protein interaction assays with in vitro translated vitamin D receptor (ivtVDR) and fusion proteins containing glutathione-S-transferase (GST) and either the ligand-binding domain of retinoid X receptor (RXRalpha), or the nuclear receptor-interacting domain of the steroid receptor coactivator 1 (SRC-1), or the glucocorticoid receptor-interacting protein 1 (GRIP-1). We found that heterodimerization of the ligand-binding domains of RXRalpha and VDR was primarily deltanoid dependent as was the interaction of VDR with the SRC-1 or with GRIP-1. The ED50 for induction of heterodimerization was 2 nM for 1,25D3 and 0.05 nM for 20-epi-1,25D3. However, the ED50 for induction of VDR interaction with SRC-1 was similar for both 1,25D3 and the 20-epi analog (ED50 = 0.7-1.0 nM) as was the ED50 for ligand-mediated interaction of VDR with GRIP-1 (ED50 = 0.1-0.3 nM). Mutations in heptad 9 diminished both 1,25D3 and the 20-epi analog-mediated dimerization, without changing binding of these ligands to VDR. Mutations in VDR's activation function 2 (AF-2) domain/helix 12 residues diminished the ability of 1,25D3 to induce heterodimerization and interaction with SRC-1. These mutations did not change the ability of 20-epi-1,25D3 to induce dimerization but did diminish its ability to induce interaction with SRC-1. We hypothesize that both the hormone and the analog stabilize receptor conformations that expose VDR's functional interfaces. The mechanisms by which the two ligands expose these functional interfaces differ with respect to participation of the AF-2 domain.