Insights into the Structure/Function of Hepatocyte Growth Factor/Scatter Factor from Studies with Individual Domains

• Published in: Journal of molecular biology 2007-03, Vol.367 (2), p.395-408
• Main Authors: Holmes, O., Pillozzi, S., Deakin, J.A., Carafoli, F., Kemp, L., Butler, P.J.G., Lyon, M., Gherardi, E.
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites; c-Met; Cell Line; Cell Movement; Cricetinae; Cricetulus; Dermatan Sulfate - metabolism; Dogs; Electrophoretic Mobility Shift Assay; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases - metabolism; glycosaminoglycans; Heparitin Sulfate - metabolism; Hepatocyte Growth Factor - genetics; Hepatocyte Growth Factor - metabolism; Hepatocyte Growth Factor - physiology; HGF/SF; Humans; Kringles; Mice; Mutation; Phosphorylation; Pichia; Protein Binding; Protein Structure, Tertiary; Proto-Oncogene Mas; Proto-Oncogene Proteins c-met - metabolism; receptor tyrosine kinase; Serine Endopeptidases - genetics; Structure-Activity Relationship
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2006.12.061

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.1 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 β-propeller domain of Met. The N, K1 and SP domains bound Met directly with comparable affinities (Kd=2.4, 3.3 and 1.4 μM). 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 β-chain moiety (amino acid residues 308–514) of the Met β-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.