Liu Y Y, Nguyen C, Ali Gardezi S A, Schnirer I, Peleg S
Department of Medical Specialties, Section of Endocrinology, Box 15, The University of Texas M.D. Anderson Cancer Center, Texas Medical Center, Houston 77030, USA.
Steroids. 2001 Mar-May;66(3-5):203-12. doi: 10.1016/s0039-128x(00)00151-3.
Twenty-epi analogs of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) are 100-1000 times more potent transcriptionally than the natural hormone. To determine to what extent this enhanced activity is mediated through modulation of the dimerization process, we performed quantitative dimerization assays with in vitro translated vitamin D receptor (ivtVDR) and fusion proteins containing glutathione-S-transferase (GST) and either the ligand-binding domain of VDR (GST-VDR) or retinoid X receptor (RXR)alpha (GST-RXR). We found that VDR did not form homodimers in either the presence or absence of ligand, but heterodimerization of the ligand-binding domains of RXRalpha and VDR was primarily deltanoid-dependent. The ED(50) for induction of heterodimerization was 1-2 x 10(-)(9) M for 1alpha,25(OH)(2)D(3) and 0.5 x 10(-)(11) M for 20-epi 1alpha,25(OH)(2)D(3). Mutations in VDR's activation function 2 domain (AF-2) diminished the abilities of 1alpha,25(OH)(2)D(3) to induce a protease-resistant conformation and heterodimerization. These mutations changed neither the potency of 20-epi-1alpha,25(OH)(2)D(3) to induce protease-resistant conformation nor its potency to induce dimerization. Mutations in heptad 9/helix 10 abolished the ability of both 1alpha,25(OH)(2)D(3) and the 20-epi analog to induce dimerization, but not their potency to fold VDR into a protease-resistant conformation. We hypothesize that both the hormone and the analog stabilize receptor conformations that expose VDR's dimerization interface, and that interfaces exposed by these ligands are probably not significantly different. However, the mechanisms by which the two ligands expose the dimerization interface are different with respect to participation of the AF-2 domain.
1α,25 - 二羟基维生素D(3)（1α,25(OH)₂D₃）的20 - 表位类似物在转录活性上比天然激素强100 - 1000倍。为了确定这种增强的活性在多大程度上是通过二聚化过程的调节介导的，我们用体外翻译的维生素D受体（ivtVDR）以及含有谷胱甘肽 - S - 转移酶（GST）与维生素D受体（VDR）的配体结合域（GST - VDR）或视黄酸X受体（RXR）α（GST - RXR）的融合蛋白进行了定量二聚化测定。我们发现，无论有无配体，VDR均不形成同二聚体，但RXRα和VDR的配体结合域的异二聚化主要依赖于类维生素D。1α,25(OH)₂D₃诱导异二聚化的半数有效浓度（ED₅₀）为1 - 2×10⁻⁹ M，而20 - 表位1α,25(OH)₂D₃的ED₅₀为0.5×10⁻¹¹ M。VDR的激活功能2结构域（AF - 2）中的突变降低了1α,25(OH)₂D₃诱导蛋白酶抗性构象和异二聚化的能力。这些突变既未改变20 - 表位 - 1α,25(OH)₂D₃诱导蛋白酶抗性构象的效力，也未改变其诱导二聚化的效力。七肽9/螺旋10中的突变消除了1α,25(OH)₂D₃和20 - 表位类似物诱导二聚化的能力，但未改变它们将VDR折叠成蛋白酶抗性构象的效力。我们推测，激素和类似物均稳定了暴露VDR二聚化界面的受体构象，并且这些配体暴露的界面可能没有显著差异。然而，就AF - 2结构域的参与而言，两种配体暴露二聚化界面的机制不同。
