Plasmid-based EGFR Ligand Shedding Assay

Plasmids encoding AP-tagged EGFR ligands were constructed by inserting partial cDNAs for human TGFα, amphiregulin, epiregulin, EGF, betacellulin, and HB-EGF into the 3′ end of human placental AP cDNA on a pRc/CMV-based expression vector pAlPh. pAlPh contains an NH₂-terminally located HB-EGF signal sequence. In all cases, the junction between AP and the EGFR ligand was placed next to the membrane-proximal EGF repeat. Release of the AP module into the culture supernatant thus requires cleavage at the COOH-terminal cleavage site. It should be noted that the results obtained with AP-tagged EGFR ligands corroborate previous results that TGFα, HB-EGF, and amphiregulin are cleaved by ADAM17 (Peschon et al., 1998 (link); Merlos-Suarez et al., 2001 (link); Sunnarborg et al., 2002 (link); Jackson et al., 2003 (link)). This validates the use of AP tags to measure ectodomain shedding for these substrates. In addition, the use of an AP tag has been independently validated using the TNF family members TNFα and TRANCE/OPGL (Zheng et al., 2002 (link); Chesneau et al., 2003 (link)). This strongly suggests that an AP tag, which provides a sensitive and quantitative means of measuring ectodomain shedding, should not interfere with the shedding properties of the other EGFR ligands tested here.