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Allosteric peptide activators of pro-hepatocyte growth factor stimulate Met signaling.

ABSTRACT: Hepatocyte growth factor (HGF) binds to its target receptor tyrosine kinase, Met, as a single-chain form (pro-HGF) or as a cleaved two-chain disulfide-linked ?/?-heterodimer. However, only two-chain HGF stimulates Met signaling. Proteolytic cleavage of the Arg(494)-Val(495) peptide bond in the zymogen-like pro-HGF results in allosteric activation of the serine protease-like ?-chain (HGF ?), which binds Met to initiate signaling. We use insights from the canonical trypsin-like serine protease activation mechanism to show that isolated peptides corresponding to the first 7-10 residues of the cleaved N terminus of the ?-chain stimulate Met phosphorylation by pro-HGF to levels that are ?25% of those stimulated by two-chain HGF. Biolayer interferometry data demonstrate that peptide VVNGIPTR (peptide V8) allosterically enhances pro-HGF ? binding to Met, resulting in a K(D)(app) of 1.6 ?m, only 8-fold weaker than the Met/HGF ?-chain affinity. Most notably, in vitro cell stimulation with peptide V8 in the presence of pro-HGF leads to Akt phosphorylation, enhances cell survival, and facilitates cell migration between 75 and 100% of that found with two-chain HGF, thus revealing a novel approach for activation of Met signaling that bypasses proteolytic processing of pro-HGF. Peptide V8 is unable to enhance Met binding or signaling with HGF proteins having a mutated activation pocket (D672N). Furthermore, Gly substitution of the N-terminal Val residue in peptide V8 results in loss of all activity. Overall, these findings identify the activation pocket of the serine protease-like ?-chain as a "hot spot" for allosteric regulation of pro-HGF and have broad implications for developing selective allosteric activators of serine proteases and pseudoproteases.

SUBMITTER: Landgraf KE

PROVIDER: S-EPMC3001016 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Allosteric peptide activators of pro-hepatocyte growth factor stimulate Met signaling.

Landgraf Kyle E KE Santell Lydia L Billeci Karen L KL Quan Clifford C Young Judy C JC Maun Henry R HR Kirchhofer Daniel D Lazarus Robert A RA

The Journal of biological chemistry 20101011 51

Hepatocyte growth factor (HGF) binds to its target receptor tyrosine kinase, Met, as a single-chain form (pro-HGF) or as a cleaved two-chain disulfide-linked α/β-heterodimer. However, only two-chain HGF stimulates Met signaling. Proteolytic cleavage of the Arg(494)-Val(495) peptide bond in the zymogen-like pro-HGF results in allosteric activation of the serine protease-like β-chain (HGF β), which binds Met to initiate signaling. We use insights from the canonical trypsin-like serine protease act ...[more]

PMID: 20937841

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Project description:UNLABELLED:Hepatocyte growth factor (HGF)/c-Met supports a pleiotrophic signal transduction pathway that controls stem cell homeostasis. Here, we directly addressed the role of c-Met in stem-cell-mediated liver regeneration by utilizing mice harboring c-met floxed alleles and Alb-Cre or Mx1-Cre transgenes. To activate oval cells, the hepatic stem cell (HSC) progeny, we used a model of liver injury induced by diet containing the porphyrinogenic agent, 3,5-diethocarbonyl-1,4-dihydrocollidine (DDC). Deletion of c-met in oval cells was confirmed in both models by polymerase chain reaction analysis of fluorescence-activated cell-sorted epithelial cell adhesion molecule (EpCam)-positive cells. Loss of c-Met receptor decreased the sphere-forming capacity of oval cells in vitro as well as reduced oval cell pool, impaired migration, and decreased hepatocytic differentiation in vivo, as demonstrated by double immunofluorescence using oval- (A6 and EpCam) and hepatocyte-specific (i.e. hepatocyte nuclear factor 4-alpha) antibodies. Furthermore, lack of c-Met had a profound effect on tissue remodeling and overall composition of HSC niche, which was associated with greatly reduced matrix metalloproteinase (MMP)9 activity and decreased expression of stromal-cell-derived factor 1. Using a combination of double immunofluorescence of cell-type-specific markers with MMP9 and gelatin zymography on the isolated cell populations, we identified macrophages as a major source of MMP9 in DDC-treated livers. The Mx1-Cre-driven c-met deletion caused the greatest phenotypic impact on HSCs response, as compared to the selective inactivation in the epithelial cell lineages achieved in c-Met(fl/fl); Alb-Cre(+/-) mice. However, in both models, genetic loss of c-met triggered a similar cascade of events, leading to the failure of HSC mobilization and death of the mice. CONCLUSION:These results establish a direct contribution of c-Met in the regulation of HSC response and support a unique role for HGF/c-Met as an essential growth-factor-signaling pathway for regeneration of diseased liver.

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Allosteric peptide activators of pro-hepatocyte growth factor stimulate Met signaling.

Publications

Allosteric peptide activators of pro-hepatocyte growth factor stimulate Met signaling.

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