Cell Surface Ectodomain Cleavage of Human Amphiregulin Precursor Is Sensitive to a Metalloprotease Inhibitor

Abstract

Our results demonstrate that sequential proteolytic cleavage within the ectodomain of the 50-kDa pro-AR form leads to release of a predominant N-glycosylated 43-kDa soluble AR, as well as the appearance of other cellular and soluble AR forms. Cell surface biotinylation studies using a C-terminal epitope-tagged pro-AR indicate that all cell surface forms are membrane-anchored and support that AR is released by ectodomain cleavage of pro-AR at the plasma membrane. We also show that pro-AR ectodomain cleavage is a regulated process, which can be stimulated by phorbol 12-myristate 13-acetate and inhibited by the metalloprotease inhibitor, batimastat. In addition, we provide evidence that high molecular mass AR forms may retain the full-length Nterminal pro-region, which may influence the biological activities of these forms.

Novel Role for Amphiregulin in Protection from Liver Injury

“…EPR gene expression was progressively induced, being maximal 10 h post-Jo2 administration, the latest time point tested. Interestingly, in agreement with other reports and our previous observations we noticed that AR gene expression was barely detectable in normal liver (35,36); however, it was potently induced between 2 and 5 h after Jo2 injection, preceding that of EPR. In concordance with the upregulation of AR mRNA levels, Western blot analyses performed with a biotinylated affinity-purified anti-mouse AR antibody on liver samples obtained 10 after Jo2 antibody administration allowed us to detect a set of proteins that were present in the liver of treated mice (Fig.…”

“…2). Four bands of ϳ50, 43, 28, and 19 kDa are consistent with the different forms of AR described in epithelial cells (36). The 50-and 28-kDa bands likely represent membrane-anchored forms of AR, whereas the 43-and 19-kDa bands may be proteolytically processed soluble forms of AR (36).…”

“…Four bands of ϳ50, 43, 28, and 19 kDa are consistent with the different forms of AR described in epithelial cells (36). The 50-and 28-kDa bands likely represent membrane-anchored forms of AR, whereas the 43-and 19-kDa bands may be proteolytically processed soluble forms of AR (36). The fact that AR gene expression becomes induced during the early time period following Jo2 treatment suggested that AR may be protective against Fas-mediated apoptosis.…”

Amphiregulin acts as an autocrine growth factor in two human polarizing colon cancer lines that exhibit domain selective EGF receptor mitogenesis

“…After a 2 h chase, a minor AR doublet of 19 and 21 kDa was also detected ( Figure 5B). The molecular weights of the different soluble AR forms released by HCA-7 and Caco-2 cells are in agreement with our previous findings (Brown et al, 1998). Only very low levels of AR species were detected in the apical-conditioned medium.…”

“…Sequential ectodomain cleavage of AR precursor is responsible for the multiple cellular and soluble AR forms produced by HCA-7 and Caco-2 cells (Brown et al, 1998). To determine whether AR is released in a polarized manner from HCA-7 and Caco-2 cells, cells grown on Transwells were metabolically labelled overnight.…”