Analysis of the ectodomain shedding mechanism of HB-EGF

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Ectodomain shedding is critical process for the action of heparin-binding EGF-like growth factor (HB-EGF). This process is regulated by a number of extracellular stimuli and intracellular signaling molecules. However, the molecular mechanism for ectodomain shedding has largely remained to be clarified. We found the following results :1)Over-expression of ADAM12 mutant form with defective metalloprotease domain induced ectodomain shedding of HB-EGF in Vero cells. Over-expression of several mutant forms of ADAM12 and co-precipitation experiments with anti-ADAM12 tag antibody suggested that ADAM12 may contribute to HB-EGF shedding as an adapter protein for signaling, rather than as a protease.2)Screening of low molecular-weight chemicals which possess the inhibitory activity for HB-EGF ectodomain shedding was performed. A few compounds showed the inhibitory activity. Further characterization is underway.3)To understand the molecules which are involved in ectodomain shedding of HB-EGF, we constructed a screening strategy which enables to identify genes necessary for the shedding. To do this, siRNA library containing 8.4 k human genome sequence was constructed and transfected in human cells by retro virus vecter system. Cell populations which did not respond to the stimuli for shedding were isolated.