Generation of High Impact Resources for Erythropoietin Receptor Research

描述（由申请人提供）：促红细胞生成素（EPO）通过其受体（EPOR）发出信号，发挥广泛的内分泌（红细胞生成）和旁分泌-自分泌（增殖、凋亡、血管生成、器官发生、细胞保护、修复和致癌）作用。EPOR研究的进展受到知识和工具方面的明显差距的阻碍：A）关于“反义EPOR多肽”存在的不确定性。B）除了EPO本身之外，缺乏与EPOR结合的分子（激动剂或拮抗剂）。C）通过蛋白质-蛋白质相互作用对EPOR的结合配偶体的不完全了解。我们建议通过采用集中的，短期的，密集的努力来推动EPOR领域的发展，这些努力使用传统和新颖的方法来缩小这些差距，以产生适用于多个学科的高影响力试剂和基础数据。具体目标是：1）通过确定天然互补反义EPOR（asEPOR）转录物是否在体内被翻译成“反义多肽”，对协同增强由有义EPOR mRNA诱导的EPOR蛋白表达的天然互补反义EPOR（asEPOR）转录物的发现进行跟踪。2)使用现有的重组类肽文库鉴定结合、激活或抑制EPOR的类肽;类肽激动剂和拮抗剂可以作为发现治疗剂的先导化合物。3)使用全长EPOR作为诱饵的新方法鉴定EPOR结合膜和细胞质蛋白。识别EPOR结合伴侣将为操纵EPOR信号通路开辟新的视野。这些目标涉及3名研究人员和2个具有不同专业知识和资源的合作实验室。通过2年的紧张工作，我们的目标是提供新的试剂和知识，并为整个EPOR研究社区创造资源。试剂包括：1）如果在体内证实了asEPOR多肽，则可获得特异性抗血清。2)EPOR修饰类肽，以激发治疗药物的发现，并在动物模型中操纵EPOR信号传导。知识包括1）体内asEPOR多肽的证明或反证。asEPOR多肽的证据将启动一个新的研究领域，而反证将集中在未来的工作asEPOR转录的调控机制。2)一个完整的跨膜和细胞质EPOR结合蛋白的列表将被确定和验证，以促进EPOR信号的研究。我们的方法是自然的发现，而不是假设检验。 公共卫生相关性：促红细胞生成素受体生物学影响基础生物医学以及临床学科。临床治疗开发的潜力很大;因此，将这些结果公布于众将带来明确的公共卫生益处。该项目的知识和试剂立即与其他NIH资助的研究相结合。

DESCRIPTION (provided by applicant): Erythropoietin (EPO) signals via its receptor (EPOR) to exert widespread endocrine (erythropoiesis) and paracrine-autocrine (proliferation, apoptosis, angiogenesis, organogenesis, cyto-protection, repair, and carcinogenesis) actions. Progress in EPOR research has been hampered by distinct gaps in knowledge and tools: A) Uncertainty regarding the existence of "antisense EPOR polypeptides". B) Paucity of molecules (agonists or antagonists) binding to EPOR other than EPO itself. C) Incomplete knowledge of binding partners of EPOR through protein-protein interaction. We propose to propel the EPOR field forward by employing focused, short-term, intense efforts that close these gaps using conventional and novel approaches to generate high impact reagents and fundamental data that will be applicable to multiple disciplines. Specific Aims are to: 1) Follow up on the discovery of natural complementary antisense EPOR (asEPOR) transcripts that synergistically enhance EPOR protein expression induced by the sense EPOR mRNA, by determining whether asEPOR transcripts are translated into "antisense polypeptides" in vivo. 2) Identify peptoids that bind to, activate or inactivate EPOR using existing recombinatorial peptoid libraries; peptoid agonists and antagonists can serve as lead compounds for discovery of therapeutic agents. 3) Identify EPOR- binding membrane and cytoplasmic proteins using a novel approach where full length EPOR serves as bait. Identifying EPOR binding partners will open new horizons for manipulating EPOR signaling pathways. These Aims involve 3 investigators and 2 collaborating laboratories with different expertise and resources. With intense work in 2 years, we aim to deliver new reagents and knowledge, and generate resources for the entire EPOR research community. Reagents include 1) If in vivo asEPOR polypeptide is proven, specific antisera will be available. 2) EPOR-modifying peptoids to spark therapeutic drug discovery and for manipulating EPOR signaling in animal models. Knowledge includes 1) Proof or disproof of in vivo asEPOR polypeptides. Proof of asEPOR polypeptides will jumpstart a novel area of research, while disproof will focus future work on regulatory mechanisms of asEPOR transcripts. 2) A complete list of transmembrane and cytoplasmic EPOR binding proteins will be identified and validated to facilitate the study of EPOR signaling. Our approach is discovery in nature rather than hypothesis testing. PUBLIC HEALTH RELEVANCE: Erythropoietin receptor biology impacts basic biomedical as well as clinical disciplines. There is high potential for clinical therapeutic development; hence unequivocal public health benefit to having these results in the public domain. Knowledge and reagents from this project integrate immediately with other NIH funded research.