Venepally P, Reddy L G, Sani B P
Kettering-Meyer Laboratories, Southern Research Institute, Birmingham, Alabama 35255, USA.
Arch Biochem Biophys. 1997 Jul 15;343(2):234-42. doi: 10.1006/abbi.1997.0158.
To characterize the dimerization of retinoid receptors in solution, RAR alpha homodimers and RAR alpha-RXR alpha heterodimers, formed in the absence or the presence of a naturally occurring RA response element (betaRARE) under different ionic conditions, were analyzed by size-exclusion fast protein liquid chromatography and sucrose density gradient sedimentation. In the presence of [3H]RA both RAR alpha and RXR alpha existed primarily as monomers of 50 kDa in solutions containing 80 mM KCl. However, when betaRARE was included in these incubations, a 40-fold increase in the occurrence of both the RAR alpha homodimers and the RAR alpha-RXR alpha heterodimers (125 kDa) was observed. The presence of RAR alpha and RXR alpha in the betaRARE-associated homo- and heterodimers was confirmed by the positive interaction of the receptors with the specific antibodies. Both RAR alpha homodimers and RAR alpha-RXR alpha heterodimers bound betaRARE even in the absence of the ligand RA with the heterodimer showing a 2- to 4-fold greater affinity than the homodimer for the DNA binding element. When the receptors were incubated in solutions of increasing ionic concentration (50-300 mM KCl), a decrease in the amount of both RAR alpha homodimers and RAR alpha-RXR alpha heterodimers was accompanied by a corresponding increase in the monomeric fraction even in the presence of betaRARE, suggesting that the high salt concentrations inhibit the surface to surface interactions between the monomers. These observations suggest that in vivo, as in solution, the formation of a stable retinoid receptor dimer complex is dependent upon both receptor-receptor and receptor-RARE interactions.
为了表征视黄酸受体在溶液中的二聚化情况，我们通过尺寸排阻快速蛋白质液相色谱法和蔗糖密度梯度沉降法，分析了在不同离子条件下，在存在或不存在天然视黄酸反应元件（βRARE）的情况下形成的视黄酸受体α同二聚体和视黄酸受体α-RXRα异二聚体。在含有80 mM KCl的溶液中，存在[3H]视黄酸时，视黄酸受体α和RXRα主要以50 kDa的单体形式存在。然而，当在这些孵育体系中加入βRARE时，观察到视黄酸受体α同二聚体和视黄酸受体α-RXRα异二聚体（125 kDa）的出现频率增加了40倍。通过受体与特异性抗体的阳性相互作用，证实了βRARE相关的同二聚体和异二聚体中存在视黄酸受体α和RXRα。即使在没有配体视黄酸的情况下，视黄酸受体α同二聚体和视黄酸受体α-RXRα异二聚体都能结合βRARE，异二聚体对DNA结合元件的亲和力比同二聚体高2至4倍。当受体在离子浓度不断增加的溶液（50 - 300 mM KCl）中孵育时，即使存在βRARE，视黄酸受体α同二聚体和视黄酸受体α-RXRα异二聚体的数量减少，同时单体部分相应增加，这表明高盐浓度抑制了单体之间的表面相互作用。这些观察结果表明，在体内，如同在溶液中一样，稳定的视黄酸受体二聚体复合物的形成依赖于受体-受体和受体-βRARE相互作用。
