Inhibition by copper(II) binding of hepatocyte growth factor (HGF) interaction with its receptor Met and blockade of HGF/Met function.
Overexpression of hepatocyte growth factor (HGF) and its receptor Met often occurs in carcinoma cells, leading to establishment of an HGF/Met autocrine loop. Therefore, disruption of the HGF/Met autocrine loop may lead to down-regulation of tumorigenesis. To study the HGF/Met interaction, we have developed a cell-free system to detect HGF binding to a Met fusion protein, Met-IgG, using a modified enzyme-linked immunosorbent assay methodology. Since we previously showed that HGF can be purified by copper(II) affinity chromatography, we further explored the effect of copper(II) on the HGF/Met interaction. The divalent metal cations copper(II) and zinc(II) significantly inhibited HGF binding to immobilized Met-IgG with IC(50) values of 230-270 microM, respectively, whereas manganese(II) and magnesium(II) were less inhibitory with 20-60-fold higher IC(50) values. Incubation of 1 mM copper(II) with HGF resulted in nondenaturing and denaturing gel-mobility shifts, indicating that copper(II) binds directly to HGF. This interaction occurs at the N terminus of HGF, as incubation of 1 mM copper(II) with both HGF and the HGF derivative NK1 yielded similar results on SDS-PAGE. HGF-induced activation of Met and cell scattering were inhibited upon addition of HGF in the presence of 1 mM and 500 microM copper(II), respectively. Chemical protonation with diethyl pyrocarbonate of HGF histidine residues impeded the ability of 500 microM copper(II) to inhibit the binding of HGF to immobilized Met-IgG. Based on the NK1 domain structure, we propose that copper(II) may interact with HGF via the histidine residues in either N-terminal or kringle domains. The inhibition of HGF/Met interaction and subsequent downstream cellular functions may be through direct interference by copper(II), such as a change in charge or an induced local conformational change. This putative copper(II) binding domain may be the basis for developing potential inhibitors of HGF/Met binding and downstream functions and could lead to novel strategies for anti-cancer treatment.
Wright
Theodore G
TG
Cancer Research Institute, Division of Cancer Biology and Genetics, Queen's University, Kingston, Ontario, Canada.
Tsai
Julie
J
Jia
Zongchao
Z
Elliott
Bruce E
BE
D000818:Animals; D001665:Binding Sites; D002412:Cations; D002460:Cell Line; D002474:Cell-Free System; D003300:Copper / Q000737:chemistry*; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D017228:Hepatocyte Growth Factor / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry; D020128:Inhibitory Concentration 50; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D019859:Proto-Oncogene Proteins c-met; D011522:Protons; D017978:Receptors, Growth Factor; D014443:Tyrosine / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
D002412:Cations; D007074:Immunoglobulin G; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011522:Protons; D017978:Receptors, Growth Factor; D014443:Tyrosine; D006639:Histidine; D017228:Hepatocyte Growth Factor; D003300:Copper; D004492:Edetic Acid; C491743:MET protein, human; D019859:Proto-Oncogene Proteins c-met; D008274:Magnesium; D015032:Zinc