Expression and characterization of hepatocyte growth factor receptor-IgG fusion proteins. Effects of mutations in the potential proteolytic cleavage site on processing and ligand binding.
The receptor for hepatocyte growth factor (HGF) is the product of the c-met proto-oncogene, a membrane-spanning tyrosine kinase receptor. To facilitate analysis of HGF and its receptor (HGFr), we expressed and purified a chimeric protein containing the extracellular domain (ECD) of the HGFr fused to the constant region of IgG heavy chain. This soluble form of the HGFr (sHGFr) bound HGF with an affinity similar to that of the authentic, membrane-associated receptor. The sHGFr also neutralized the binding of HGF to the HGFr expressed on A549 cells. Like the mature form of the HGFr, sHGFr is a heterodimer which arises by proteolytic processing within the ECD. In order to characterize the requirements for proteolytic processing of the ECD and the effects of cleavage on ligand binding, we expressed sHGFr variants containing amino acid substitutions in the putative processing site. Replacement of the P1 or P4 arginine, but not the P3 lysine, with alanine inhibited conversion to the alpha/beta heterodimer. This suggests that maturation is mediated by furin or a furin-like protease. Finally, we showed that processing of the sHGFr into the alpha/beta form is not required for high affinity binding to either pro- or mature HGF.
Mark
M R
MR
Department of Cell Genetics, Genentech, Inc., South San Francisco, California 94080.
Lokker
N A
NA
Zioncheck
T F
TF
Luis
E A
EA
Godowski
P J
PJ
D000409:Alanine; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015723:Gene Library; D017228:Hepatocyte Growth Factor / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000302:isolation & purification; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000302:isolation & purification; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D019859:Proto-Oncogene Proteins c-met; D011519:Proto-Oncogenes; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins
D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; C000715470:MAS1 protein, human; D009838:Oligodeoxyribonucleotides; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D017228:Hepatocyte Growth Factor; D004247:DNA; D011505:Protein-Tyrosine Kinases; D019859:Proto-Oncogene Proteins c-met; D000409:Alanine