Chan Lap Shu, Wells Richard A
Molecular and Cellular Biology, Sunnybrook Research Institute, 2075 Bayview Avenue, T2-058 Toronto, Ont., Canada M4N 2M5.
Biochem Biophys Res Commun. 2007 Jul 13;358(4):1080-5. doi: 10.1016/j.bbrc.2007.05.051. Epub 2007 May 22.
Heterodimerization with RXR is essential for the high-affinity specific binding of multiple nuclear receptors to their cognate DNA sequences. NR dimerization is a two-step process, initiated in solution by interaction between amino acid residues with helices 9 and 10 of the ligand binding domains of RXR and its NR partners. Studies of the orphan nuclear receptor HNF4alpha, which forms homodimers exclusively, have indicated that two charged residues in this region, HNF4alpha(K300) and HNF4alpha(E327), are key mediators of dimerization. We have analyzed the contribution of the homologous residues in RXRalpha (RXRalpha(E395), RXRalpha(K422)) and PPARgamma (PPARgamma(E405), PPARgamma(K432)) to the formation of the RXRalpha-PPARgamma heterodimer. Charge reversal mutants of RXRalpha (RXRalpha(E395K), RXRalpha(K422E)) and PPARgamma (PPARgamma(E405K), PPARgamma(K432E)) show impaired ability to form heterodimers with wild-type PPARgamma and RXRalpha, respectively. However, pairs of mutants with balanced charge changes, i.e., RXRalpha(E395K) with PPARgamma(K432E) and RXRalpha(K422E) with PPARgamma(E405K), are able to form dimers. Ligand response is preserved in the PPARgamma mutants, indicating the mutation does not result in major structural derangement of the protein. These results establish the importance of salt bridges between these residues in the heterodimerization of nuclear receptors, and offer a technical approach to generating functional NR mutants with directed heterodimerization specificity. Such mutants will be valuable tools in the genetic analysis of NR function.
与视黄酸X受体（RXR）形成异源二聚体对于多种核受体与其同源DNA序列的高亲和力特异性结合至关重要。核受体二聚化是一个两步过程，首先在溶液中由RXR及其核受体伙伴的配体结合域的螺旋9和10中的氨基酸残基之间的相互作用引发。对仅形成同型二聚体的孤儿核受体肝细胞核因子4α（HNF4α）的研究表明，该区域的两个带电残基，即HNF4α（K300）和HNF4α（E327），是二聚化的关键介质。我们分析了视黄酸X受体α（RXRα（E395）、RXRα（K422））和过氧化物酶体增殖物激活受体γ（PPARγ（E405）、PPARγ（K432））中同源残基对RXRα-PPARγ异源二聚体形成的贡献。RXRα（RXRα（E395K）、RXRα（K422E））和PPARγ（PPARγ（E405K）、PPARγ（K432E））的电荷反转突变体分别显示出与野生型PPARγ和RXRα形成异源二聚体的能力受损。然而，具有平衡电荷变化的突变体对，即RXRα（E395K）与PPARγ（K432E）以及RXRα（K422E）与PPARγ（E405K），能够形成二聚体。PPARγ突变体中保留了配体反应，表明该突变不会导致蛋白质的主要结构紊乱。这些结果确立了这些残基之间的盐桥在核受体异源二聚化中的重要性，并提供了一种产生具有定向异源二聚化特异性的功能性核受体突变体的技术方法。此类突变体将是核受体功能遗传分析中的有价值工具。
