NUCLEAR TRANSFER AND ACTION OF RETINOIC ACID

视黄酸的核转移和作用

• 批准号: 2100044
• 负责人: BRAHMA P SANI
• 金额: $ 23.78万
• 依托单位: SOUTHERN RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-07 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2100044
• 关键词: chloramphenicol acetyltransferase; complementary DNA; gel mobility shift assay; genetic regulatory element; genetic transcription; intracellular transport; nucleoproteins; plasmids; protein structure function; protein transport; receptor binding; receptor expression; retinoid binding proteins; retinoids; tissue /cell culture; transfection /expression vector; vitamin antagonist

The cellular transport of retinoic acid (RA) and 9-cis-RA is believed to be mediated by their cellular RA-binding protein, CRABP. The biological activity of these retinoids in the control of cellular differentiation and growth is mediated by their nuclear receptors, RARs (alpha, beta, and gamma) for RA and RXRs (alpha, beta, and gamma) for 9-cis-RA. The mechanism by which the nuclear receptors accept their ligands from the cytosol and are activated is not known, and forms the central theme of this proposal. The efficacy of RAs complexed with CRABP as compared with free RAs in the ligand-nuclear receptor interactions will be determined by using purified receptor protein preparations. The specific role of RA- response elements (REs) in enhancing/stabilizing the RA binding to the receptors, and the role of CRABP/RA in producing such effects will be elucidated by using a gel mobility shift assay. Also to be established will be, by the use of intact nuclei, the mode of transfer of RAs into the nuclei for their interactions with RAR/RXR. Experiments will be devised to utilize expression plasmids harboring full-length cDNA for CRABP, RAR and RXR to elucidate the specific role of CRABP in the binding of the ligands to the transiently expressed receptors in CV-1 cells. A CRABP- mediated functional role of RAR/RXR will be delineated by using a cis- trans cotransfection assay which would utilize cDNA expression vectors for the nuclear receptors and RARE/RXRE-chloramphenicol acetyl transferase. By extending these efforts, identification of possible activator/antagonist synthetic retinoids (from a rationally selected group of retinoids) that would alter the transcriptional activation of the receptors will also be accomplished. The unifying hypothesis to be tested is that CRABP plays a role not only in the transport of RAs to their nuclear receptors but that it also has a functional role.

视黄酸（RA）和9-CIS-RA的细胞转运被认为是 通过其细胞RA结合蛋白Crabp介导。生物学 这些类类动物在控制细胞分化和 增长是由其核受体，RARS（alpha，beta和 伽玛）用于RA和RXRS（Alpha，beta和Gamma）9-CIS-RA。这 核受体接受其配体的机制 细胞质和被激活是未知的，并构成了 这个建议。与Crabp复合的RA的功效与 配体 - 核受体相互作用中的游离RA将由 使用纯化的受体蛋白制剂。 ra-的具体作用 响应元素（RES）增强/稳定RA结合到与 受体，以及Crabp/Ra在产生此类作用中的作用将是 通过使用凝胶迁移率转移测定法阐明。也要建立 通过使用完整的核，将Ras转移到 其与RAR/RXR相互作用的核。 将设计实验 为了利用具有全长cDNA的表达质粒，rar 和RXR以阐明Crabp在结合中的特定作用 在CV-1细胞中瞬时表达的受体的配体。一个蟹 RAR/RXR的介导的功能作用将通过使用顺式来划定 反式共转染测定法，该测定将利用cDNA表达载体的 核受体和稀有的/rxre-氯苯二甲酸乙酰基转移酶。经过 扩展这些努力，识别可能的激活剂/拮抗剂 合成性类维生素类似（来自理性选择的类视黄素组） 会改变受体的转录激活也将是 成就。 要测试的统一假设是Crabp扮演A 不仅在RAS运输到其核受体中的作用，而且在 它还具有功能性的作用。