Mechanisms of FGF Receptor Regulation and Signaling

描述(申请人提供)：成纤维细胞生长因子(成纤维细胞生长因子)信号在哺乳动物的发育和代谢以及疾病中起着多方面的作用。旁分泌FGF1、FGF4、FGF7、FGF8和FGF9亚家族在精子发生、中胚层诱导、体细胞发生、器官发生和图案形成中发挥重要作用，而FGF19亚家族以内分泌方式调节主要代谢过程，包括葡萄糖、脂肪、胆固醇和胆汁酸代谢，以及磷酸盐/维生素D稳态。成纤维细胞生长因子的不同活性是由成纤维细胞生长因子受体(FGFR)受体酪氨酸激酶(RTK)亚家族传递的。干扰的成纤维细胞生长因子信号导致许多人类疾病，包括骨骼、生殖综合征、听力损失、肾脏磷酸盐消耗、神经退行性疾病和癌症。几个旁分泌FGFs和所有内分泌FGFs正被用于药物开发。这种竞争性更新的四个具体目标是：1.表征上皮细胞表达的FGF4和FGF9亚家族获得间质表达的FGFRc异构体的特异性的结构基础。II.阐明a/bKlotho共受体通过内分泌FGFs促进信号转导的结构基础。剖析A环酪氨酸磷酸化在致病功能获得突变引起的FGFR酪氨酸激酶过度激活中的作用。IV.阐明FGFR募集和磷酸化FRS2a的结构基础。重组蛋白表达和工程、X射线结晶学、表面等离子体共振(SPR)光谱、等温滴定热法(ITC)、稳态动力学分析和时间分辨质谱仪将用于实现这一提议的特定目标。所获得的结构和生物物理/生化结果也将使用基于细胞和动物的分析进行验证。根据AIM I获得的数据应能为旁分泌FGFs在胚胎发育中的作用提供分子洞察力，并有助于发现用于组织修复和生物工程、促进自我更新和用于细胞替代治疗的人类胚胎干细胞分化的药物。在AIM II下生成的数据应能加深我们对内分泌FGFs在人体新陈代谢中的作用的了解，并为人类主要疾病(包括糖尿病、肥胖症、高胆固醇血症、结肠癌和慢性肾脏疾病)的药物开发提供蓝图，这些疾病中的大多数对公众健康构成巨大负担。AIM III的结果将加深我们对FGFRs和其他RTK致病突变的作用机制以及整个RTK超家族酪氨酸激酶活性调节的理解。由于底物募集和RTK的磷酸化是RTK信号中的一个普遍事件，在AIM IV下获得的机制洞察也将直接适用于整个RTK超家族。 与公共健康相关：成纤维细胞生长因子(成纤维细胞生长因子)信号在人类发育和代谢中起着至关重要的作用，当它出错时，它会导致一系列人类疾病，包括骨骼、嗅觉生殖综合征、听力损失、磷酸盐消耗障碍、神经退行性疾病和癌症。这项应用的总体目标是阐明成纤维细胞生长因子信号在发育、代谢和疾病中的分子基础。建议的研究不仅应该加强我们对成纤维细胞生长因子信号在人体生理中的作用的理解，而且还将为发现治疗目前对公众健康构成巨大负担的许多人类重大疾病的新药奠定基础。

DESCRIPTION (provided by applicant): Fibroblast growth factor (FGF) signaling plays pleiotropic roles in mammalian development and metabolism, and disease. The paracrine FGF1, FGF4, FGF7, FGF8, and FGF9 subfamilies play essential roles in spermatogenesis, mesoderm induction, somitogenesis, organogenesis, and pattern formation, whereas the FGF19 subfamily acts in an endocrine fashion to regulate major metabolic processes including glucose, lipid, cholesterol, and bile acid metabolism, and phosphate/vitamin D homeostasis. The diverse activities of FGFs are transmitted by the FGF receptor (FGFR) subfamily of receptor tyrosine kinases (RTKs). Perturbed FGF signaling leads to numerous human diseases, including skeletal, reproductive syndromes, hearing loss, renal phosphate wasting, neurodegenerative disorders, and cancer. Several paracrine FGFs and all the endocrine FGFs are being pursued for drug development. The four specific aims of this competing renewal are: I. Characterize the structural basis by which epithelially-expressed FGF4 and FGF9 subfamilies attain their specificity towards mesenchymally-expressed FGFRc isoforms. II. Elucidate the structural basis by which a/bKlotho co-receptors promote signaling by the endocrine FGFs. III. Dissect the role of A-loop tyrosine phosphorylation in the hyperactivation of FGFR tyrosine kinase by pathogenic gain-of-function mutations. IV. Elucidate the structural basis by which FGFR recruits and phosphorylates FRS2a. Recombinant protein expression and engineering, x-ray crystallography, Surface Plasmon Resonance (SPR) spectroscopy, isothermal titration calorimetry (ITC), steady-state kinetics analysis, and time-resolved mass spectrometry will be used to accomplish the Specific Aims of this proposal. The structural and biophysical/biochemical results obtained will also be validated using cell- and animal-based assays. The data obtained under Aim I should provide molecular insights into the roles of paracrine FGFs in embryonic development and also facilitate the discovery of drugs for tissue repair and bioengineering, promotion of self- renewal and differentiation of human embryonic stem cells for cell-replacement therapy. The data generated under Aim II should enhance our understanding of the role of endocrine FGFs in human metabolism and provide blueprints for drug discovery for major human diseases including diabetes, obesity, hypercholesterolemia, colon cancer, and chronic kidney disease, most of which represent a huge burden on public health. The results of Aim III will enhance our understanding of the mechanism of action of pathogenic mutations in FGFRs and other RTKs as well as the regulation of tyrosine kinase activity of the entire RTK superfamily. Since substrate recruitment and phosphorylation by RTKs is a general event in RTK signaling, the mechanistic insights gained under Aim IV will also be directly applicable to the entire RTK superfamily. PUBLIC HEALTH RELEVANCE: Fibroblast growth factor (FGF) signaling plays essential roles in human development and metabolism, and when it goes awry it leads to a wide array of human diseases, including skeletal, olfactory reproductive syndromes, hearing loss, phosphate wasting disorders, neurodegenerative disorders, and cancer. The overall goal of this application is to elucidate the molecular basis for FGF signaling in development, metabolism, and disease. The proposed studies should not only enhance our understanding of the role of the FGF signaling in human physiology but also lay the foundations for the discovery of new drugs for the treatment of many major human diseases, which currently represent a huge burden on public health.