Ligand-dependent and -independent actions of insulin-like growth factor binding proteins in fish

胰岛素样生长因子（IGFs）是一个肽类生长因子/激素家族，对动物的正常生长和发育至关重要。最近的研究表明，IGFs的可用性和生物活性是由几个特定的，高亲和力的IGF结合蛋白（IGFBPs）调节。IGFBPs属于在细胞外环境中与IGF结合的蛋白质的保守基因家族。由于IGFBPs对IGF的结合亲和力等于或甚至大于IGF受体的结合亲和力，因此它们可以控制呈递给其细胞表面受体的IGF的量，并且可能在确定IGF信号传导途径的生物活性中起关键作用。还有体外证据表明，一些IGFBPs具有不依赖IGF的内在生物活性。该项目旨在验证IGFBP家族的不同成员以空间和时间限制的方式表达并且它们各自通过IGF依赖性和非依赖性机制在调节组织生长和分化中发挥不同作用的假设。本项目有三个目标。在第一个目标中，将研究IGFBP-3在调节头部骨骼和内耳生长和分化中的作用的机制基础，特别关注配体结合和核靶向的作用。为此，天然和突变体IGFBP-3将在IGFBP-3敲低的胚胎中表达，用于体内机制研究。目的2：确定斑马鱼IGFBP-4的结构，定位其时空表达模式，研究其在体内的生理功能。在目标3中，将在体外和体内研究IGFBP-4作用的模式和蛋白水解在控制局部IGFBP-4作用中的可能作用。目的4研究斑马鱼IGFBP-6的结构、发育表达、体内生理功能，并阐明其作用的分子机制。这些研究的完成将为IGFBPs的功能及其生物学作用的机制基础提供新的和重要的见解。第一次，将在体内研究IGFBP的非配体依赖性作用、核定位和蛋白水解的功能意义。了解鱼类生长调节的潜在机制无疑将有助于脊椎动物生长生理学的发展。更好地了解斑马鱼生长调节的潜在机制将有助于了解人类生长生理学，并可能在水产养殖中应用，以有效生产动物蛋白。该研究项目对于了解IGFBP基因家族的结构和功能进化也很重要，并将扩大密歇根州的研究基础设施，增加本科生和研究生的研究和培训机会。

Insulin-like growth factors (IGFs) are a family of peptide growth factors/hormones that are essential for normal animal growth and development. Recent studies suggest that the availability and bioactivity of IGFs are regulated by several specific, high-affinity IGF-binding proteins (IGFBPs). IGFBPs belong to a conserved gene family of proteins that bind to IGFs in extracellular environments. Since the binding affinities of IGFBPs for IGFs are equal to or even greater than those of the IGF receptors, they can control the amount of IGFs presented to their cell surface receptors and may play key roles in determining the biological activities of the IGF signaling pathway. There is also in vitro evidence that some IGFBPs have intrinsic biological activities that are IGF-independent. This project is designed to test the hypothesis that different members of the IGFBP family are expressed in spatially and temporally restricted fashions and they each play distinct roles in regulating tissue growth and differentiation through IGF-dependent and -independent mechanisms. The current project has 3 aims. In the first aim, the mechanistic basis of IGFBP-3 action in regulating head skeleton and inner ear growth and differentiation will be investigated with special focus on the role of ligand binding and nuclear targeting. For this, native and mutant IGFBP-3 will be expressed in IGFBP-3 knocked down embryos for in vivo mechanistic studies. Aim 2 seeks to determine the structure of zebrafish IGFBP-4, map its spatial and temporal expression pattern, and investigate its physiological functions in vivo. In Aim 3, the mode of IGFBP-4 action and the possible role of proteolysis in controlling local IGFBP-4 action will be studied in vitro and in vivo. Aim 4 seeks to determine zebrafish IGFBP-6 structure, developmental expression, physiological functions in vivo, and elucidate the molecular mechanisms underlying its actions. The completion of the proposed studies will provide novel and important insights into the functions of IGFBPs and the mechanistic basis underlying their biological actions. For the first time, the functional significance of ligand-independent actions, nuclear localization, and proteolysis of an IGFBP will be examined in vivo. An understanding of the underlying mechanisms of growth regulation in fish will undoubtedly contribute to vertebrate growth physiology in general. A better understanding of the underlying mechanisms of growth regulation in zebrafish should help to understand human growth physiology and may have applications in aquaculture for efficient production of animal protein as well. This research project is also of importance for understanding the structural and functional evolution of the IGFBP gene family and will expand research infrastructure in Michigan and increase research and training opportunities for undergraduate and graduate students.