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

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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.