FIGURE 1.

Ectodomain shedding of ADAM10 in MEFs. A, model of the domain organization of ADAM10, which consists of a pro-domain (Pro) that is proteolytically removed in the trans-Golgi network by pro-protein convertases, a zinc-binding metalloprotease (Protease) domain, a disintegrin domain (Dis), which binds to integrin cell adhesion molecules, a cystein-rich domain (Cys), which can interact with cell surface proteoglycans and in some cases also contains a fusion peptide sequence, a variable stalk region, a transmembrane (TM) domain, and a cytoplasmic (Cyto) domain. Epitopes for C (B42.1) and N terminus (MAB946) specific antibodies are indicated. Ectodomain shedding (arrow) leaves a ∼10-kDa membrane-anchored CTF and releases a ∼55-kDa soluble ectodomain (sADAM10). B, Western blot analysis of total wild-type (+/+) MEF cell extracts (CE) (50 μg/lane) shows a Pro-ADAM10 (∼85 kDa) and a mature ADAM10 (ADAM10 FL, ∼65 kDa) after prodomain removal. A third 10-kDa band is detectable (ADAM10 CTF) with B42.1, the C-terminal ADAM10 antibody, but not by MAB946, the N-terminal antibody. Additional bands (asterisk) are observed in wild-type cells, but not in ADAM10-deficient cells. These bands may represent ADAM10 splice variants (see www.genecards.org/cgi-bin/carddisp.pl?gene=ADAM10) or degradation products. A secreted ADAM10 ectodomain (sADAM10) is observed in the culture supernatant (SN) (30 μg/lane), detected by MAB946, but not B42.1. C, SN from WT MEFs was able to cleave a synthetic peptide containing the APP α-secretase cleavage site in a fluorescence resonance energy transfer assay. In MEFs lacking ADAM10 we observed a strong reduction in this cleavage compared with WT. D, ADAM10 CTFs are detected by Western blot analysis in cell lysate samples from both neuronal and glial cell cultures (30 μg/lane). E, Western blot of total tissue extracts of E16.5 CD1 mouse embryos (50 μg/lane). ADAM10 CTFs were detected to a various extent in all tested organs.