Insights into the structure/function of hepatocyte growth factor/scatter factor from studies with individual domains |
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Authors: | Holmes O Pillozzi S Deakin J A Carafoli F Kemp L Butler P J G Lyon M Gherardi E |
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Institution: | MRC Centre, Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK. |
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Abstract: | Hepatocyte growth factor/scatter factor (HGF/SF), the ligand for the receptor tyrosine kinase encoded by the c-Met proto-oncogene, is a multidomain protein structurally related to the pro-enzyme plasminogen and with major roles in development, tissue regeneration and cancer. We have expressed the N-terminal (N) domain, the four kringle domains (K1 to K4) and the serine proteinase homology domain (SP) of HGF/SF individually in yeast or mammalian cells and studied their ability to: (i) bind the Met receptor as well as heparan sulphate and dermatan sulphate co-receptors, (ii) activate Met in target cells and, (iii) map their binding sites onto the beta-propeller domain of Met. The N, K1 and SP domains bound Met directly with comparable affinities (K(d)=2.4, 3.3 and 1.4 microM). The same domains also bound heparin with decreasing affinities (N>K1>SP) but only the N domain bound dermatan sulphate. Three kringle domains (K1, K2 and K4) displayed agonistic activity on target cells. In contrast, the N and SP domains, although capable of Met binding, displayed no or little activity. Further, cross-linking experiments demonstrated that both the N domain and kringles 1-2 bind the beta-chain moiety (amino acid residues 308-514) of the Met beta-propeller. In summary, the K1, K2 and K4 domains of HGF/SF are sufficient for Met activation, whereas the N and SP domains are not, although the latter domains contribute additional binding sites necessary for receptor activation by full length HGF/SF. The results provide new insights into the structure/function of HGF/SF and a basis for engineering the N and K1 domains as receptor antagonists for cancer therapy. |
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Keywords: | aa amino acid AMAC 2-aminoacridone BS3 bis[sulfosuccinimidyl]suberate DS dermatan sulphate ERK extracellular signal-regulated protein kinase GAG glycosaminoglycan(s) HGF/SF hepatocyte growth factor/scatter factor HGFI/MSP hepatocyte growth factor-like/macrophage stimulating protein HS heparan sulphate |
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