Study of the activity of proHB-EGF complex.

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We have studied the membrane-anchored form of heparin-binding EGF-like growth factor (proHB-EGF) and its physiological function. Recently we demonstrated that proHB-EGF and CD9 form a complex with integrin alpha3betal at cell-cell contact sites of Vero cells. To study the function of proHB-EGF complex, in this project, we studied 1) the biological activity of proHB-EGF, 2) the mechanism of the ectodomain shedding of proHB-EGF, and 3) identification and characterization of a novel component of proHB-EGF complex.1)Analysis of the biological activity of proHB-EGF We studied the biological activity of proHB-EGF by using a model in which proHJB-EGF-expressing effector cells were co-cultured with EGFR-expressing target cells. From this experimental system, we found that proHB-EGF induces growth inhibition and subsequent apoptosis of the EGER-expressing target cells. Moreover, we found that the inhibitory signal induced by proHB-EGF is mediated via EGFR and that the cytoplasmic domain of EGFR … More is essential for proHB-EGF-induced apoptosis. From these results, we concluded that proHB-EGF has unique biological activity through cell-cell contact which is distinct from the activity of sHB-EGF (on submitting).2)Analysis of the mechanism of the ectodomain shedding of proHB-EGF The ectodomains of many proteins located at the cell surface are shed upon cell stimulation. One such protein is HB-EGF that exists in a membrane-anchored form which is converted to a soluble form upon cellstimulation with TPA, an activator of PKC.We found that PKCdelta binds in vivo and in vitro to the cytoplasmic domain of MDC9/meltrin-gamma/ADAM9, a member of the metalloprotease-disintegrin family. Furthermore, the presence of constitutively active PKCdelta or MDC9 results in the shedding of the ectodomain of proHB-EGF, whereas MDC9 mutants lacking the metalloprotease domain, as well as kinase-negative PKCdelta suppress the TPA-induced shedding of the ectodomain. These results suggest that MDC9 and PKCdelta are involved in the stimulus-coupled shedding of the ptoFLB-EGF ectodomain (EMBO J., 17, 7260-, 1998).3)Identification of novel components of proHB-EGF complex To identify the novel protein(s) associated with proHB-EGF, we preapred several monoclonal antibodies that recognize molecule which is co-precipitated with proHB-EGF by diphtheria toxin (DT). Among them, mAblC9-2 recognizes its antigen that is co-precipitated with proHB-EGF and CD9 specifically by DT, but not by anti-HB-EGF antibody, suggesting that association of proHB-EGF and 1C9-2 antigen molecule is physiological because DT can bind to only proHB-EGF which forms a complex with CD9 while anti-HB-EGF antibody can bind all population of proHB-EGF.Now we are undergoing identification and purification of the 1C9-2 antigen molecule. Less