Mechanisms of FGF Receptor Regulation and Signaling

描述（由申请人提供）：成纤维细胞生长因子（FGF）信号在哺乳动物的发育、代谢和疾病中起着多种作用。旁分泌的FGF1、FGF4、FGF7、FGF8和FGF9亚家族在精子发生、中胚层诱导、躯体发生、器官发生和模式形成中发挥重要作用，而FGF19亚家族则以内分泌方式调节主要代谢过程，包括葡萄糖、脂质、胆固醇和胆汁酸代谢，以及磷酸盐/维生素D稳态。FGFs的多种活性是通过受体酪氨酸激酶（rtk）的FGF受体（FGFR）亚家族传递的。FGF信号紊乱导致许多人类疾病，包括骨骼、生殖综合征、听力损失、肾磷酸盐消耗、神经退行性疾病和癌症。几种旁分泌FGFs和所有内分泌FGFs正在进行药物开发。这种竞争性更新的四个具体目标是：1 .表征上皮表达的FGF4和FGF9亚家族对间质表达的FGFRc亚型获得特异性的结构基础。2。阐明a/bKlotho共受体通过内分泌fgf促进信号传导的结构基础。3。剖析a环酪氨酸磷酸化在FGFR酪氨酸激酶通过致病性功能获得突变的过度激活中的作用。阐明FGFR招募和磷酸化FRS2a的结构基础。重组蛋白表达和工程、x射线晶体学、表面等离子体共振（SPR）光谱、等温滴定量热法（ITC）、稳态动力学分析和时间分辨质谱法将被用于完成本提案的具体目标。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。在Aim I下获得的数据将提供关于旁分泌FGFs在胚胎发育中的作用的分子见解，并促进组织修复和生物工程药物的发现，促进人类胚胎干细胞的自我更新和分化，用于细胞替代治疗。Aim II下产生的数据将增强我们对内分泌FGFs在人体代谢中的作用的理解，并为主要人类疾病（包括糖尿病、肥胖、高胆固醇血症、结肠癌和慢性肾脏疾病）的药物发现提供蓝图，其中大多数疾病对公共卫生构成巨大负担。Aim III的结果将增强我们对FGFRs和其他RTK致病性突变的作用机制以及整个RTK超家族酪氨酸激酶活性的调控的理解。由于RTK的底物募集和磷酸化是RTK信号传导中的普遍事件，Aim IV下获得的机制见解也将直接适用于整个RTK超家族。

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.