Insights into the structure/function of hepatocyte growth factor/scatter factor from studies with individual domains.
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.
Holmes
O
O
MRC Centre, Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK.
Pillozzi
S
S
Deakin
J A
JA
Carafoli
F
F
Kemp
L
L
Butler
P J G
PJ
Lyon
M
M
Gherardi
E
E
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002465:Cell Movement; D006224:Cricetinae; D003412:Cricetulus; D003871:Dermatan Sulfate / Q000378:metabolism*; D004285:Dogs; D024202:Electrophoretic Mobility Shift Assay; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism*; D017228:Hepatocyte Growth Factor / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D018082:Kringles; D051379:Mice; D009154:Mutation; D010766:Phosphorylation; D010843:Pichia; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D000090063:Proto-Oncogene Mas; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000235:genetics; D013329:Structure-Activity Relationship
D003871:Dermatan Sulfate; D048049:Extracellular Signal-Regulated MAP Kinases; D006497:Heparan Sulfate; D017228:Hepatocyte Growth Factor; D000090063:Proto-Oncogene Mas; D019859:Proto-Oncogene Proteins c-met; D012697:Serine Endopeptidases; C000715470:MAS1 protein, human