Cresci S, Clabby M L, Kelly D P
Center for Cardiovascular Research, Washington University School of Medicine, St. Louis, Missouri 62110, USA.
J Biol Chem. 1999 Sep 3;274(36):25668-74. doi: 10.1074/jbc.274.36.25668.
Recent studies indicate that retinoid-mediated pathways play a pivotal role in cardiac morphogenesis and function. To identify proteins that serve as interacting partners of the retinoid X receptor alpha (RXRalpha) in heart, DNA-protein binding studies were performed with an RXR-responsive element (NRRE-1) derived from the medium chain acyl-CoA dehydrogenase gene promoter and nuclear protein extracts prepared from adult rat heart. NRRE-1 is a pleiotropic RXR-responsive element comprised of three potential recognition sites for class II members of the nuclear receptor superfamily. Gel mobility shift assays performed with an NRRE-1 probe in the absence or presence of bacterially overproduced RXRalpha and nuclear protein extracts prepared from adult rat heart, liver, or brain identified a cardiac-specific, RXR-dependent DNA-protein interaction. The NRRE-1-RXR.cardiac-enriched RXR-interacting protein (CERIP) complex exhibited a distinct mobility compared with NRRE-1-RXR.peroxisome proliferator-activated receptor, NRRE-1-RXR.retinoic acid receptor, or NRRE-1-RXR.thyroid receptor complexes. Mutational analysis demonstrated that two of the three potential binding half-sites of NRRE-1 (an everted repeat separated by an 8-base pair spacer) are required for the NRRE-1-RXR. CERIP interaction. Gel mobility shift assays demonstrated that CERIP interacted with RXRalpha and RXRgamma but not with RXRbeta, indicating a receptor subtypespecific binding preference and suggesting an RXR AB region-dependent interaction. The RXR.CERIP complex did not form on NRRE-1 when a mutant GST-RXRalpha fusion protein lacking the NH(2)-terminal AB region (but containing the receptor dimerization domain) of RXRalpha was added in place of the full-length RXRalpha, confirming a role for the AB region in the RXR. CERIP interaction. DNA-protein cross-linking studies demonstrated that CERIP is a DNA-binding protein of approximately 110 kDa. These results provide evidence for the existence of a cardiac-enriched DNA-binding protein that interacts with RXRalpha via the AB region and suggest a mechanism whereby cardiac retinoid signaling is controlled in an RXR subtype-specific manner.
近期研究表明，类视黄醇介导的信号通路在心脏形态发生和功能中起关键作用。为了鉴定在心脏中作为视黄醇X受体α（RXRα）相互作用蛋白的蛋白质，我们使用了从中链酰基辅酶A脱氢酶基因启动子衍生的RXR反应元件（NRRE-1）以及从成年大鼠心脏制备的核蛋白提取物进行了DNA-蛋白质结合研究。NRRE-1是一种多效性RXR反应元件，由核受体超家族II类成员的三个潜在识别位点组成。在不存在或存在细菌过量表达的RXRα以及从成年大鼠心脏、肝脏或大脑制备的核蛋白提取物的情况下，用NRRE-1探针进行凝胶迁移率变动分析，确定了一种心脏特异性的、RXR依赖性的DNA-蛋白质相互作用。与NRRE-1-RXR·过氧化物酶体增殖物激活受体、NRRE-1-RXR·视黄酸受体或NRRE-1-RXR·甲状腺受体复合物相比，NRRE-1-RXR·心脏富集的RXR相互作用蛋白（CERIP）复合物表现出独特的迁移率。突变分析表明，NRRE-1的三个潜在结合半位点中的两个（由8个碱基对间隔隔开的反向重复序列）是NRRE-1-RXR·CERIP相互作用所必需的。凝胶迁移率变动分析表明，CERIP与RXRα和RXRγ相互作用，但不与RXRβ相互作用，表明存在受体亚型特异性结合偏好，并提示存在RXR AB区域依赖性相互作用。当添加缺乏RXRα的NH(2)-末端AB区域（但包含受体二聚化结构域）的突变型GST-RXRα融合蛋白代替全长RXRα时，NRRE-1上未形成RXR·CERIP复合物，这证实了AB区域在RXR·CERIP相互作用中的作用。DNA-蛋白质交联研究表明，CERIP是一种约110 kDa的DNA结合蛋白。这些结果为存在一种通过AB区域与RXRα相互作用的心脏富集DNA结合蛋白提供了证据，并提示了一种以RXR亚型特异性方式控制心脏类视黄醇信号传导的机制。
