Structure-function analysis of hepatocyte growth factor: identification of variants that lack mitogenic activity yet retain high affinity receptor binding.
Hepatocyte growth factor (HGF) is a potent mitogen for parenchymal liver, epithelial and endothelial cells. Structurally, it has similarities to kringle-containing serine proteases, although it does not possess proteolytic activity. A structure-activity relationship study of human HGF was performed by functional analysis of HGF substitution and deletion variants. Analysis of HGF variants was accomplished by defining their ability to induce DNA synthesis on hepatocytes in primary culture and to compete with wild-type HGF for binding to a soluble form of the HGF receptor. Three groups of variants were made: (i) substitutions at the cleavage site, (ii) substitutions within the protease-like domain and (iii) deletions of the beta-chain and/or kringle domains. Our results show that: (i) single-chain HGF is a zymogen-like promitogen in that cleavage into a two-chain form is required for biological activity, however, the single chain form of HGF still retains substantial receptor binding capacity; (ii) certain mutations in the protease-like domain result in variants that are completely defective for mitogenic activity, yet exhibit apparent receptor binding affinities similar to wild-type HGF (Kd approximately 50-70 pM); and (iii) a variant containing the N-terminal 272 residues of mature HGF showed only a 4-fold increase in Kd when compared with wild-type HGF indicating that a primary receptor binding determinant is located within this sequence.
Lokker
N A
NA
Department of Cell Genetics, Genentech, Inc., South San Francisco, CA 94080.
Mark
M R
MR
Luis
E A
EA
Bennett
G L
GL
Robbins
K A
KA
Baker
J B
JB
Godowski
P J
PJ
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D003198:Computer Simulation; D004247:DNA / Q000096:biosynthesis; D010450:Endopeptidases / Q000378:metabolism; D005260:Female; D006133:Growth Substances / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017228:Hepatocyte Growth Factor; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids; D019859:Proto-Oncogene Proteins c-met; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism
D006133:Growth Substances; D011956:Receptors, Cell Surface; D014443:Tyrosine; D017228:Hepatocyte Growth Factor; D004247:DNA; D019859:Proto-Oncogene Proteins c-met; D010450:Endopeptidases