HEPARIN-BINDING EGF--STRUCTURE AND FUNCTION

点击翻译按钮获取中文摘要

HB-EGF is a novel heparin-binding member of the EGF family, that has recently been purified to homogeneity, sequenced and cloned (Higashiyama et al. Science, 1991, 251 936-939). The overall goal of the proposal is to analyze in-depth the structural and biological properties of HB-EGF. Preliminary structural studies indicate i) that while HB-EGF is 20- 22kDa, it contains only 85-90 amino acids suggesting that it is extensively modified post-translationally, ii) that it is originally expressed as a 208 amino acid transmembrane precursor and is processed, and iii) that it contains a heparin-binding domain that modulates its mitogenic activity by interaction with low affinity heparan sulfate proteoglycan (HSPG) receptors on cell surfaces. Preliminary biological studies indicate that HB-EGF i) is synthesized by macrophages, smooth muscle cells (SMC) and by some carcinoma cells, ii) is a potent SMC mitogen (40 times more potent than EGF and equally as potent as PDGF), and a SMC chemotactic factor, iii) is a potent mitogen for epithelial cells (e.g. keratinocytes and mesothelial cells), and iv) is a constituent of wound fluid. These biological activities of HB-EGF might have physiological and pathological consequences in vivo. HB-EGF, as a macrophage product, and mitogen for epithelial cells and fibroblasts, could participate in inflammatory wound healing processes such as granulation tissue formation and re-epithelialization. HB-EGF as a SMC mitogen produced by macrophages and SMC themselves could be in part responsible for the SMC hyperplasia associated with atherosclerosis. The Specific Aims of this proposal are: 1. To analyze HB-EGF structure including obtaining the complete primary sequence of mature HB-EGF, isolation of multiple forms of HB-EGF, analysis of post-translational modifications, analysis of the biological activity of the HB-EGF precursor, and production of polyclonal anti-HB-EGF antibodies; 2) To identify a heparin-binding domain in HB-EGF; 3) To analyze i) the regulation of HB-EGF expression by macrophages and other cells, and ii) the involvement of HB-EGF in wound healing by analysis of its mitogenic effects on epithelial cells (e.g. keratinocytes) and its presence in wound fluid; 4. To analyze the mitogenic effects of HB-EGF on SMC and the biosynthesis of HB-EGF by SMC in vitro and in vivo; 5) To characterize low (heparan sulfate proteoglycan) and high affinity HB-EGF receptors.