The molecular basis of Rieger syndrome - Analysis of Pitx2 homeodomain protein activities
The molecular basis of Rieger syndrome - Analysis of Pitx2 homeodomain protein activities
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DOI：
10.1074/jbc.273.32.20066
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发表时间：
1998-08-07
影响因子：
4.8
通讯作者：
Russo, AF
中科院分区：
文献类型：
--
作者：
Amendt, BA;Sutherland, LB;Russo, AF
Rieger综合征是一种常染色体显性遗传的发育障碍，包括青光眼和人类轻度颅面畸形。Pitx 2同源盒基因的突变与Rieger综合征有关。我们的特点是野生型和突变Pitx 2活动，电泳迁移率变动分析，蛋白质结合，和瞬时转染试验。Pitx 2优先结合bicoid同源结构域结合位点，并反式激活含有该位点的报告基因。Pitx 2和另一种同源结构域蛋白Pit-1的组合在转染测定中产生催乳素启动子的协同55倍激活。在电泳迁移率变动分析中，Pit-1的加入增加了Pitx 2与bicoid元件的结合。此外，我们在体外证明了Pit-1与Pitx 2的特异性结合。因此，野生型Pitx 2 DNA结合活性受蛋白质-蛋白质相互作用调节。我们接下来研究了两个Rieger突变体。同源结构域的第二螺旋中的苏氨酸至脯氨酸突变（T68 P）保留了DNA结合活性，具有相同的表观K-D，并且B-max仅降低约a倍。然而，该突变体不反式激活含有bicoid位点的报告基因。突变Pitx 2蛋白结合Pit-1，但没有检测到催乳素启动子的协同作用。第二个突变（L54 Q）在一个高度保守的残基在螺旋1的同源结构域产生了不稳定的蛋白质。我们的研究结果提供了深入了解潜在的机制，在Rieger综合征的发育缺陷。
Rieger syndrome is an autosomal-dominant developmental disorder that includes glaucoma and mild craniofacial dysmorphism in humans. Mutations in the Pitx2 homeobox gene have been linked to Rieger syndrome. We have characterized wild type and mutant Pitx2 activities using electrophoretic mobility shift assays, protein binding, and transient transfection assays. Pitx2 preferentially binds the bicoid homeodomain binding site and transactivates reporter genes containing this site. The combination of Pitx2 and another homeodomain protein, Pit-1, yielded a synergistic 55-fold activation of the prolactin promoter in transfection assays. Addition of Pit-1 increased Pitx2 binding to the bicoid element in electrophoretic mobility shift assays. Furthermore, we demonstrate specific binding of Pit-1 to Pitx2 in vitro. Thus, wild type Pitx2 DNA binding activity is modulated by protein-protein interactions. We next studied two Rieger mutants. A threonine to proline mutation (T68P) in the second helix of the homeodomain retained DNA binding activity with the same apparent K-D and only about a a-fold reduction in the B-max. However, this mutant did not transactivate reporter genes containing the bicoid site. The mutant Pitx2 protein binds Pit-1, but there was no detectable synergism on the prolactin promoter. A second mutation (L54Q) in a highly conserved residue in helix 1 of the homeodomain yielded an unstable protein. Our results provide insights into the potential mechanisms underlying the developmental defects in Rieger syndrome.