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、器官生成、细胞保护、修复和致癌）作用。EPOR研究的进展受到知识和工具的明显差距的阻碍：A)关于“反义EPOR多肽”存在的不确定性。B)除了EPO本身外，缺乏与EPOR结合的分子（激动剂或拮抗剂）。C)通过蛋白质相互作用对EPOR结合伙伴的了解不完全。我们建议通过集中、短期、密集的努力，利用传统和新颖的方法来弥补这些差距，从而推动EPOR领域向前发展，以产生适用于多学科的高影响力试剂和基础数据。具体目的是：1)通过确定asEPOR转录本在体内是否被翻译成“反义多肽”，跟踪发现天然互补反义EPOR （asEPOR）转录本，这些转录本可以协同增强EPOR mRNA诱导的EPOR蛋白表达。2)利用现有的重组肽库识别结合、激活或灭活EPOR的肽类；肽类激动剂和拮抗剂可作为先导化合物用于治疗药物的发现。3)利用全长EPOR作为诱饵的新方法鉴定EPOR结合膜和细胞质蛋白。识别EPOR结合伙伴将为操纵EPOR信号通路开辟新的视野。这些目标涉及3名调查员和2个具有不同专业知识和资源的合作实验室。通过两年的紧张工作，我们的目标是为整个EPOR研究界提供新的试剂和知识，并产生资源。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修饰肽类在动物模型中激发治疗药物的发现和调控EPOR信号。知识包括：1)体内asEPOR多肽的证明或反证。asEPOR多肽的证明将启动一个新的研究领域，而反证将把未来的工作重点放在asEPOR转录物的调控机制上。2)完整的跨膜和细胞质EPOR结合蛋白列表将被识别和验证，以促进EPOR信号传导的研究。我们的方法是在自然界中发现，而不是假设检验。

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.