Inhibitory role of TACE/ADAM17 cytotail in protein ectodomain shedding

Abstract

To determine if the cytotail of the principal sheddase tumor necrosis factor-α converting enzyme (TACE; ADAM17) controls protein ectodomain shedding. Site-directed mutagenesis was performed to derive TACE variants. The resulting TACE expression plasmids with amino acid substitutions in the extracellular, cysteine-rich disintegrin domain (CRD) and/or deleted cytotail, along with an expression vector for the enhanced green fluorescence protein were transfected into shedding-defective M1 mutants stably expressing transmembrane L-selectin or transforming growth factor (TGF)-α. The expression levels of the TACE substrates at the cell surface were determined by flow cytometry. Consistent with published data, a single point mutation (C600Y) in the CRD led to shedding deficiency. However, removal of the cytotail from the C600Y TACE variant partially restored ectodomain cleavage of TGF-α and L-selectin. Cytotail-deleted mutants with any other substituting amino acid residues in place of Cys600 displayed similar function compared with tail-less C600Y TACE. The cytotail plays an inhibitory role, which becomes evident when it is removed from an enzyme with another mutation that affects the enzyme function.