The biological effects mediated by Met are controlled by concomitant activation of a number of signaling pathways. Prominent among these are the Grb2-Ras pathway eliciting transient activation of MAPK, which is required for proliferation, and the Gab1/PI3K pathways, which are required for cell scatter, morphogenesis, and cell survival (23
). Recent reports have provided evidence that sustained MAPK activated the via Gab1 route (12
) is involved in the phenomena elicited by the latter pathways (1
). In this study, we found that MUC20 decreased HGF-induced transient MAPK activation through prevention of Grb2 recruitment, inhibiting HGF-induced proliferation and MMP expression. MUC20 did not, however, affect Gab1/PI3K pathways including the sustained MAPK activation, and consequently had no effect on cell scatter, branching morphogenesis, and cell survival (Fig. ).
FIG. 7. Model of MUC20 suppression. (A) A MUC20 monomer has less ability to associate with Met. In this condition, Grb2 is recruited to HGF-activated Met, followed by activation of signaling cascades leading to proliferation. (B) MUC20 oligomerization caused (more ...)
It is likely that the MUC20 binding does not cause drastic a structural change in Met, because dimerization and subsequent autophosphorylation of Met on HGF-stimulation occurred normally under MUC20-binding conditions. Recently, it was reported that a point mutation of Asn1358
in the MDS of Met causes a specific defect in Grb2 recruitment to Met (22
). Although the defect might result from a conformational change in the MDS and thus may differ from the prevention of Grb2 recruitment by MUC20 binding, it is suggestive that the domain of Met required for MUC20 binding is near Asn1358
and may include the residue. Overproduction of MUC20 was needed to detect the Met binding; thus, the binding affinity might be rather low, even if oligomerization has made the conformation of MUC20 favorable for Met binding. Grb2 is a small protein that binds to the Y1356
VNV sequence in the MDS, while Gab1 is a larger molecule that binds Met directly via Y1349
and indirectly via a Grb2 linkage. Recently, the C-terminal lobe of the Met kinase domain also was found to be involved in direct Gab1 binding (11
), suggesting that extended regions of Met might be involved in the Gab1 binding. Thus, we propose that the selective prevention of the Grb2 recruitment is caused by low-affinity attachment of MUC20 to a restricted area possibly around Y1356
VNV in the MDS, and that the Gab1 interaction is not affected due to higher-affinity binding to Met.
To gain insight into the physiological role of Grb2 recruitment to Met, mutant mice that produce Met with a point mutation to eliminate the Grb2-binding site have been generated (7
). These mice were viable but showed abnormality of postnatal cerebellar development. MUC20-Tg mice, in contrast, had no apparent phenotypic abnormalities, although we expected hypomorphological phenotypes of the tissues, due to the reduction of HGF-dependent proliferation. These results raise the possibility that the Grb2 recruitment to Met may not be required during development. In general, the Grb2-Ras pathway is a crucial common pathway that is responsible for promoting proliferation. Therefore, the down-regulation of this pathway in Met signaling might be compensated promptly by the action of other factors, e.g., EGF and fibroblast growth factor, during development. Our proposal for the physiological role of MUC20 is not only a suppression of HGF-induced proliferation but also an emphasizing of morphological stimuli of HGF. Namely, in kidney, after HGF provides a proliferative stimulus to the dedifferentiated cells, the effect of HGF on those cells could be switched to a morphogenesis stimulus by MUC20 through suppression of the Grb2-Ras pathway, leading to successful generation of renal tubules. Indeed, we observed that in mouse renal tissues injured by cisplatin, the expression of MUC20 mRNA was increased in parallel with an increase in blood urea nitrogen, a marker of renal damage, and then was returned to the normal level until the regeneration was completed (6
), whereas HGF was markedly and rapidly produced once treatment for acute renal injury was given (8
Besides recruitment of PI3K, Gab1, and Grb2, Met is also known to interact with FAP68 and RanBPM. FAP68 binds specifically to the inactive form of Met and is released upon Met phosphorylation (5
). Paradoxically, RanBPM interacts with Met regardless of HGF stimulation, but the interaction is enhanced by HGF (27
). In this study, we demonstrated that MUC20 binding to Met is independent of HGF stimulation and that the interaction tends to be strengthened by MUC20 oligomerization. Previous reports have shown that extracellular regions of mucin-type glycoproteins are able to interact with bacteria or distinct ligands, such as L- and P-selectins, and play important roles in protective functions and cell adhesion. Thus, we further propose a novel paradigm through which endogenous factors that are not ligand molecules for a growth factor receptor may be able to regulate signal transduction of the growth factor by inducing a conformation change in its recognizing proteins, e.g., mucin-type glycoproteins.
Previous studies have demonstrated that another transmembrane mucin, MUC1, interacts with the EGF receptor, and activation of EGF receptor leads to phosphorylation of MUC1 (10
). Moreover, the MUC1 phosphorylation enhances c-Src and β-catenin recruitment and modulation of EGF-induced ERK1/2 activation (26
). Recently, HGF stimulation was found to increase phosphorylation of MUC20. This phenomenon is likely to be mediated by the Gab1/PI3K pathways, because the phosphorylation of MUC20 upon stimulation with HGF was markedly reduced by LY294002 but not by U0126 (unpublished data). Although the role of MUC20 phosphorylation has not yet been established, we propose that this modification did not affect the binding ability of MUC20 to Met within 30 min after HGF stimulation (Fig. ) and that the HGF-induced phosphorylation might serve as an important regulator of events downstream of HGF signaling.
In summary, we have carried out functional analyses of MUC20 and have demonstrated that it is a novel negative regulator of the HGF-induced Grb2-Ras pathway. The Met signaling cascade is believed to serve important roles in cell growth and differentiation, organ regeneration, and tumorigenesis. Tight regulation of this system could be required for control of essential actions of HGF. HGF is considered to be a possible therapeutic agent, as it displays a remarkable ability to ameliorate renal injury and fibrosis by enhancing cell survival and tissue regeneration in acute and chronic disease conditions. Factors that regulate MUC20 expression and/or function may be useful therapeutics for the development and progression of renal diseases.